SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 18, 2021, is named CRTN-002CN_Sequence_Listing.txt and is 35416 bytes in size.

BACKGROUND

Genome editing therapies using programmable and/or engineered nucleases, combined with designed exogenous DNA repair template molecules, have been developed to treat intractable disease such as, for example, viral infection (Lin et al., (2014) Mol Ther Nucleic Acids 3:e186), enzymatic deficiency (Yin et al., (2016) Nat Biotechnol 34(3):328-333), and hereditary myopathies (Long et al., (2014) Science 345:1184-1188; Long et al., (2016) Science 351:400-403; Tabebordbar et al., (2016) Science 351:407-411). Approaches to therapeutically target the genome often rely on the homology-directed repair (HDR) pathway, which enables accurate genome repair of introduced double-stranded breaks (DSBs) using exogenous single- or double-stranded DNA repair templates (e.g., donor polynucleotides), but is often highly suppressed in non-dividing cells (e.g., G1phase cells) (Orthwein et al., (2015) Nature 528(7582):422-6). Despite rapid advances in this area of biomedical research and the potential for clinical applications, targeted integration of transgenes or other polynucleotides for therapeutic purposes in vivo remains challenging because current methods are inefficient, particularly for non-dividing cells, which compose most adult tissues.

DSBs induced into the genome by exogenous sources (e.g., an engineered nuclease) may be repaired in cells by double-strand break repair (DSBR) mechanisms, such as the HDR pathway and the non-homologous end joining (NHEJ) DNA repair pathway. The canonical HDR pathway is known to operate in dividing cells (e.g., cells in S phase), as it requires a homologous sister chromatid for execution, whereas the NHEJ pathway can function in both dividing and non-dividing cells and independent of the cell cycle (Iyama & Wilson (2013) DNA Repair 12(8), 620-636). In contrast to the HDR pathway, the NHEJ repair pathway is often used in genome editing approaches that do not employ an exogenous DNA repair template (e.g., a donor polynucleotide) and that are directed toward the formation of an stochastic insertion or deletion (‘indel’) of one or more nucleotides at the DSB site, resulting in, for example, disruption of the translational reading frame of a coding sequence or the binding sites of trans-acting factors in promoters or enhancers.

As genetic abnormalities are widely recognized as a major etiological basis of numerous diseases, the inability or inefficiency of therapeutic targeted genome editing poses a technical barrier for developing treatments for a broad range of genetic disorders. Thus, new approaches to therapeutically target the genome, particularly in non-dividing cells, are needed.

SUMMARY OF THE DISCLOSURE

Accurate pre-messenger RNA (pre-mRNA) splicing is critical for correct protein expression. Vertebrate gene architecture often consists of relatively long introns and short internal exons. Without being bound by theory, the model of exon definition posits splice sites paired across an exon as opposed to across an intron and that in pre-mRNAs with large introns, the splicing machinery searches for a pair of closely spaced splice sites in an exonic polarity (i.e., 3′ splice site upstream and 5′ splice site downstream) (Berget (1995) J Biol Chem 270:2411-2414). Recognition of a pair of splice sites in a relatively short (50 to 300 nucleotides) region surrounding an exon by the splicing machinery reduces the frequency of recognizing cryptic intronic splice sites that are randomly distributed within introns, thus increasing the accuracy of splicing. Numerous disease-causing mutations are known to be located within exons (e.g. protein-coding mutations) or within introns (e.g., splicing signal mutations) that can lead to an aberrancy in protein production or function, ultimately resulting or contributing to a disease.

The present disclosure is based, at least in part, on the discovery that a mutation (e.g., a deleterious or disease-causing mutation) can be corrected or induced in a genomic DNA molecule (gDNA) by genome editing compositions and methods, in particular by use of a donor polynucleotide as described herein, that results in a desired alteration in the nucleotide sequence of the gDNA and modulates exon definition, thereby resulting in the inclusion of a desired alteration in an RNA transcript (e.g., a pre-mRNA) transcribed from the edited gDNA. Accordingly, the disclosure provides donor polynucleotides that, when used to repair a double-strand break (DSB) introduced into a gDNA by a site-directed nuclease (e.g., a Cas nuclease), correct or induce a mutation and modulate exon definition by the incorporation of one or more splicing signals proximal to the corrected or induced mutation. In some embodiments, the donor polynucleotides provided by the disclosure are designed to comprise a nucleotide sequence comprising a desired alteration correct a mutation in a target nucleic acid (e.g., a genomic DNA) and one or more splicing signals that function to modulate splice site recognition. In some embodiments, the donor polynucleotides provided by the disclosure are designed to comprise a nucleotide sequence comprising a desired alteration and one or more splicing signals that function to modulate exon definition, directing the splicing machinery of the cell to incorporate the desired alteration into a transcription product (e.g., a pre-mRNA).

The disclosure also provides methods for correcting or inducing a mutation (e.g., a disease-causing mutation) in gDNA, including cellular, ex vivo and in vivo methods, by the insertion of a linear, double-stranded DNA donor polynucleotide into a CRISPR/Cas9-mediated DSB generated proximal to the mutation. While actively dividing cells can repair DNA damage using both HDR and NHEJ pathways, non-dividing cells predominately use the NHEJ pathway. The use of CRISPR/Cas9 allows for the introduction of site-specific DNA breaks that can be repaired by the NHEJ pathway such that exogenous DNA polynucleotides are ligated, or inserted, into the genomic DNA of dividing or non-dividing cells. In some embodiments, the donor polynucleotides of the disclosure comprise nucleotide sequences that both correct or induce a mutation and comprise one or more splicing signals, thereby promoting a desired RNA processing event to occur by establishing exon definition for a desired exon (e.g., an exon comprising a corrected mutation) and/or destroying exon definition for an undesired exon (e.g., an exon comprising a disease-causing mutation). Also provided herein are compositions, systems, and kits for performing such methods. Also provided are cells produced by such methods.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the mutation is a substitution, missense, nonsense, insertion, deletion or frameshift mutation. In some embodiments, the mutation is a substitution mutation. In some embodiments, the mutation is a missense mutation. In some embodiments, the mutation is a nonsense mutation. In some embodiments, the mutation is an insertion mutation. In some embodiments, the mutation is a deletion mutation. In some embodiments, the mutation is a frameshift mutation.

In some embodiments, the mutation is in an exon. In some embodiments, the mutation is a substitution, insertion or deletion, and the mutation is in an intron. In some embodiments, the mutation is a substitution mutation located in an intron. In some embodiments, the mutation is an insertion mutation located in an intron. In some embodiments, the mutation is a deletion mutation located in an intron.

In some embodiments, the mutation is proximal to a splicing signal in a gDNA. In some embodiments, the mutation is proximal to a 3′ splice site in a gDNA. In some embodiments, the mutation is proximal to a 5′ splice site in a gDNA. In some embodiments, the mutation is in a splicing signal in a gDNA. In some embodiments, the mutation is in a 3′ splice site in a gDNA. In some embodiments, the mutation is in a 5′ splice site in a gDNA. In some embodiments, the mutation is in a polypyrimidine tract. In some embodiments, the mutation is in a branch point sequence. In some embodiments, the mutation is a protein-coding mutation. In some embodiments, the mutation is associated with or causes a disease.

In some embodiments, the donor polynucleotides provided by the disclosure comprise an intronic sequence. In some embodiments, the intronic sequence corrects the mutation.

In some embodiments, the donor polynucleotides provided by the disclosure comprise an exonic sequence. In some embodiments, the exonic sequence corrects the mutation.

In some embodiments, the donor polynucleotides provided by the disclosure comprise one or more splicing signals is selected from the group consisting of:

(a) a natural or enhanced 3′ splice site;

(b) a natural or enhanced 5′ splice site;

(c) a polypyrimidine tract;

(d) a branch point;

(e) an exon splicing enhancer (ESE);

(f) an intron splicing enhancer (ISE);

(g) an exon splicing silencer (ESS);

(h) an intron splicing silencer (ISS); and

(i) a combination of any of (a)-(h).

In some embodiments, the one or more splicing signals is a natural or enhanced 3′ splice site. In some embodiments, the one or more splicing signals is a natural 3′ splice site. In some embodiments, the one or more splicing signals is an enhanced 3′ splice site. In some embodiments, the one or more splicing signals is a natural or enhanced 5′ splice site. In some embodiments, the one or more splicing signals is a natural 5′ splice site. In some embodiments, the one or more splicing signals is an enhanced 3′ splice site. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is a nucleotide sequence comprising a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the one or more splicing signals is a combination comprising a natural or enhanced 3′ splice site and a polypyrimidine tract. In some embodiments, the one or more splicing signals is a combination comprising a natural or enhanced 3′ splice site, a polypyrimidine tract, and a branch point.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) which corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide comprises a first splicing signal comprising a branch point sequence, wherein the donor polynucleotide comprises:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence comprising a first branch point sequence and a first nucleotide sequence which corrects the mutation in the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a nucleotide sequence comprising a second branch point sequence and a second nucleotide sequence which corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand, wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the nucleotide sequence comprising the first splicing signal conforms to a branch point consensus sequence on either strand, wherein the nucleotide sequences of the first branch point sequence and second branch point sequence are complementary.

In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) which corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide comprises a first splicing signal comprising a branch point sequence, wherein the donor polynucleotide comprises:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence comprising a first branch point sequence and a first nucleotide sequence which corrects the mutation in the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a nucleotide sequence comprising a second branch point sequence and a second nucleotide sequence which corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the nucleotide sequence comprising the first splicing signal conforms to a branch point consensus sequence on either strand, wherein the nucleotide sequences of the first branch point sequence and second branch point sequence are complementary.

In some embodiments, the branch point consensus sequence is YTNAY (SEQ ID NO: 49), wherein Y is a nucleotide comprising either a cytosine (C) or thymine (T) nucleobase, and wherein N is a nucleotide comprising a nucleobase selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C). In some embodiments, Y comprises a cytosine (C). In some embodiments, Y comprises a thymine (T). In some embodiments, N comprises an adenine (A). In some embodiments, N comprises a guanine (G). In some embodiments, N comprises a thymine (T). In some embodiments, N comprises a cytosine (C).

In some embodiments, the first branch point sequence is TATTAAC (SEQ ID NO: 50).

In some embodiments, the second branch point sequence is GTTAATA (SEQ ID NO: 51).

In some embodiments, the second branch point sequence is TACTGAC (SEQ ID NO: 52).

In some embodiments, the donor polynucleotide comprises a second splicing signal comprising a polypyrimidine tract, wherein the first strand comprises a first polypyrimidine tract located downstream of the first branch point sequence; and the second strand comprises a second polypyrimidine tract located downstream of the second branch point sequence. In some embodiments, the nucleotide sequence comprising the first and second polypyrimidine tracts each comprise nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), and wherein the nucleotide sequence is about 100%, about 90%-100%, or about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence is about 80%-90% pyrimidine nucleobases.

In some embodiments, the nucleotide sequence comprising the polypyrimidine tract is TTTTTTTCT (SEQ ID NO: 53). In some embodiments, the nucleotide sequence comprising the polypyrimidine tract is TTTTTTTCTTTTT (SEQ ID NO: 54). In some embodiments, the nucleotide sequence comprising the polypyrimidine tract is CTTCTTCTCTTCTTCC (SEQ ID NO: 55).

In some embodiments, the first branch point sequence and the first polypyrimidine tract are adjacent to each other. In some embodiments, the second branch point sequence and the second polypyrimidine tract are adjacent to each other.

In some embodiments, the donor polynucleotide comprises a third splicing signal, wherein the third splicing signal comprises a 3′ splice site, wherein the first strand comprises a nucleotide sequence comprising a first 3′ splice site located downstream of the first polypyrimidine tract; and wherein second strand comprises a nucleotide sequence comprising a second 3′ splice site located downstream of the second polypyrimidine tract. In some embodiments, the first and second 3′ splice sites comprise the nucleotide sequence YAG, and wherein Y is a nucleotide comprising a nucleobase selected from the group consisting of: thymine (T) and cytosine (C). In some embodiments, Y comprises a thymine (T). In some embodiments, Y comprises a cytosine (C).

In some embodiments, the donor polynucleotide comprises a coding sequence, wherein the first strand comprises a first coding sequence, wherein the second strand comprises a second coding sequence, wherein the first nucleotide sequence that corrects the mutation in the gDNA comprises the first coding sequence, wherein the second nucleotide sequence that corrects the mutation in the gDNA comprises the second coding sequence, wherein the first coding sequence is located downstream of the first 3′ splice site, and wherein the second coding sequence is located downstream of the second 3′ splice site. In some embodiments, the nucleotide sequence comprising the first and second coding sequences comprise nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C). In some embodiments, the coding sequence comprising (i) and (ii) are not identical or complementary to reduce self-annealing.

In some embodiments, the donor polynucleotide comprises one or more delimiter sequences comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or 1 nucleotide(s) in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-40 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-30 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-20 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-15 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1-10 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 30 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 20 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 10 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 9 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 8 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 7 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 6 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 5 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 4 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 3 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 2 nucleotides in length. In some embodiments, the nucleotide sequence comprising one or more delimiter sequences is about 1 nucleotide in length.

In some embodiments, the one or more delimiter sequences is located between the first branch point sequence and the second branch point sequence. In some embodiments, the one or more delimiter sequences is located between the first branch point sequence and the first polypyrimidine tract. In some embodiments, the one or more delimiter sequences is located between the second branch point and the second polypyrimidine tract.

In some embodiments, the disclosure provides donor polynucleotides configured for bi-directional insertion into the DSB, wherein, when a donor polynucleotide is inserted into the DSB in either orientation, the first splicing signal and second splicing signal, optionally, the third splicing signal and coding sequence comprise a sense strand, thereby correcting the mutation and providing one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) which corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide comprises:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence comprising a first 5′ splice site and a first nucleotide sequence which corrects the mutation in the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a nucleotide sequence comprising a second 5′ splice site and a second nucleotide sequence which corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the first strand comprises a first coding sequence, wherein the second strand comprises a second coding sequence, wherein the first coding sequence is located upstream of the first 5′ splice site, and wherein the second coding sequence is located upstream of the second 5′ splice site, and wherein the coding sequences in the first and second strand are not complementary (or comprise one, two, three, four or more mismatches) to reduce self-annealing. In some embodiments, the donor polynucleotide comprises a delimiter sequence comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or 1 nucleotide(s) in length located between the first and second 5′ splice sites. In some embodiments, the donor polynucleotide is configured for bi-directional insertion into the DSB, wherein when the donor polynucleotide is inserted into the DSB in a first orientation, the first 5′ splice site and first coding sequence comprise a sense strand, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, and wherein when the donor polynucleotide is inserted into the DSB in a second orientation, the second 5′ splice site and second coding sequence comprise a sense strand, thereby correcting the mutation and providing one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) which corrects or induces a mutation in a genomic DNA (gDNA) molecule in a cell and one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the donor polynucleotide comprises:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence comprising a first 5′ splice site and a first nucleotide sequence which corrects the mutation in the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a nucleotide sequence comprising a second 5′ splice site and a second nucleotide sequence which corrects the mutation in the gDNA, wherein the second strand is complementary to the first strand,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the first strand comprises a first coding sequence, wherein the second strand comprises a second coding sequence, wherein the first coding sequence is located upstream of the first 5′ splice site, and wherein the second coding sequence is located upstream of the second 5′ splice site, and wherein the coding sequences in the first and second strand are not complementary (or comprise one, two, three, four or more mismatches) to reduce self-annealing. In some embodiments, the donor polynucleotide comprises a delimiter sequence comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein the nucleotide sequence is about 1-40, about 1-30, about 1-20, about 1-15, about 1-10, about 30, about 20, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2 or 1 nucleotide(s) in length located between the first and second 5′ splice sites. In some embodiments, the donor polynucleotide is configured for bi-directional insertion into the DSB, wherein when the donor polynucleotide is inserted into the DSB in a first orientation, the first 5′ splice site and first coding sequence comprise a sense strand, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, and wherein when the donor polynucleotide is inserted into the DSB in a second orientation, the second 5′ splice site and second coding sequence comprise a sense strand, thereby correcting the mutation and providing one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals is selected from the group consisting of:(a) a natural or enhanced 3′ splice site;(b) a polypyrimidine tract;(c) a branch point;(d) an exon splicing enhancer (ESE);(e) an intron splicing enhancer (ISE);(f) an exon splicing silencer (ESS);(g) an intron splicing silencer (ISS); and(h) a combination of any of (a)-(g),

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is a natural or enhanced 3′ splice site. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals is selected from the group consisting of:(a) a natural or enhanced 3′ splice site;(b) a polypyrimidine tract;(c) a branch point;(d) an exon splicing enhancer (ESE);(e) an intron splicing enhancer (ISE);(f) an exon splicing silencer (ESS);(g) an intron splicing silencer (ISS); and(h) a combination of any of (a)-(g),

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is a natural or enhanced 3′ splice site. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA (gDNA) molecule in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 5′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals is selected from the group consisting of:(a) a natural or enhanced 5′ splice site;(b) an exon splicing enhancer (ESE);(c) an intron splicing enhancer (ISE);(d) an exon splicing silencer (ESS);(e) an intron splicing silencer (ISS); and(f) a combination of any of (a)-(e),

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is a natural or enhanced 5′ splice site. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA (gDNA) molecule in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 5′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals is selected from the group consisting of:(a) a natural or enhanced 5′ splice site;(b) an exon splicing enhancer (ESE);(c) an intron splicing enhancer (ISE);(d) an exon splicing silencer (ESS);(e) an intron splicing silencer (ISS); and(f) a combination of any of (a)-(e),

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is a natural or enhanced 5′ splice site. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a natural or enhanced 3′ splice site; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a natural or enhanced 3′ splice site; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals is a combination of a natural or enhanced 3′ splice site and a polypyrimidine tract; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals is a combination of a natural or enhanced 3′ splice site and a polypyrimidine tract; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals is a combination of a natural or enhanced 3′ splice site, a polypyrimidine tract, and a branch point; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals is a combination of a natural or enhanced 3′ splice site, a polypyrimidine tract, and a branch point; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is in a 3′ splice site, wherein the first strand comprises an intronic sequence, optionally, an exonic sequence, wherein the intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the one or more splicing signals is selected from the group consisting of:(a) a polypyrimidine tract;(b) a branch point;(c) an exon splicing enhancer (ESE);(d) an intron splicing enhancer (ISE);(e) an exon splicing silencer (ESS);(f) an intron splicing silencer (ISS); and(g) a combination of any of (a)-(f),

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is in a 3′ splice site, wherein the first strand comprises an intronic sequence, optionally, an exonic sequence, wherein the intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the one or more splicing signals is selected from the group consisting of:(a) a polypyrimidine tract;(b) a branch point;(c) an exon splicing enhancer (ESE);(d) an intron splicing enhancer (ISE);(e) an exon splicing silencer (ESS);(f) an intron splicing silencer (ISS); and(g) a combination of any of (a)-(f),

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is a polypyrimidine tract. In some embodiments, the one or more splicing signals is a branch point. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 5′ splice site, wherein the donor polynucleotide comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a natural or enhanced 5′ splice site; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 5′ splice site, wherein the donor polynucleotide comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein at least one splicing signal is a natural or enhanced 5′ splice site; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is in a 5′ splice site, wherein the first strand comprises an intronic sequence, optionally, an exonic sequence, wherein the intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals is selected from the group consisting of:(a) an exon splicing enhancer (ESE);(b) an intron splicing enhancer (ISE);(c) an exon splicing silencer (ESS);(d) an intron splicing silencer (ISS); and(e) a combination of any of (a)-(d),

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is in a 5′ splice site, wherein the first strand comprises an intronic sequence, optionally, an exonic sequence, wherein the intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the one or more splicing signals is selected from the group consisting of:(a) an exon splicing enhancer (ESE);(b) an intron splicing enhancer (ISE);(c) an exon splicing silencer (ESS);(d) an intron splicing silencer (ISS); and(e) a combination of any of (a)-(d),

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the one or more splicing signals is an exon splicing enhancer (ESE). In some embodiments, the one or more splicing signals is an intron splicing enhancer (ISE). In some embodiments, the one or more splicing signals is an exon splicing silencer (ESS). In some embodiments, the one or more splicing signals is an intron splicing silencer (ISS). In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, insertion of the donor polynucleotide into the DSB results in the formation of an exon in the gDNA comprising the exonic sequence. In some embodiments, the one or more splicing signals directs the inclusion of the exon comprising the exonic sequence which corrects the mutation into an mRNA.

In some embodiments, the insertion of the donor polynucleotide results in the formation of an intron comprising the intronic sequence, wherein the intronic sequence corrects the mutation.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the first strand comprises the formula:
5′-[B]a-[S1]b-[P]c-[S2]d-X-E-3′, wherein

(i) B, if present, is a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a is an integer whose value indicates the number of nucleotides comprising B, wherein a=0 or 5-7;

(ii) P is a polypyrimidine tract comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprising P, wherein c=9-20, wherein the nucleotide sequence comprising P is about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases;

(iii) E is an exonic sequence comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C);

(iv) X is a nucleotide sequence comprising a 3′ splice site; and

(v) S1 and S2, if either is present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein b and d=0-20,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, B, if present, P, X, if present, and E, if present, comprise a sense strand, wherein B, if present, P, and X, if present, comprise the one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, B is present. In some embodiments, B is absent. In some embodiments, a=0. In some embodiments, a=5-7. In some embodiments, a=5. In some embodiments, a=6. In some embodiments, a=7. In some embodiments, the nucleotide sequence comprising P is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P is about 80%-90% pyrimidine nucleobases. In some embodiments, c=9. In some embodiments, c=10. In some embodiments, c=11. In some embodiments, c=12. In some embodiments, c=13. In some embodiments, c=14. In some embodiments, c=15. In some embodiments, c=16. In some embodiments, c=17. In some embodiments, c=18. In some embodiments, c=19. In some embodiments, c=20. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments b=20. In some embodiments, d=0. In some embodiments, d=1. In some embodiments, d=2. In some embodiments, d=3. In some embodiments, d=4. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, d=8. In some embodiments, d=9. In some embodiments, d=10. In some embodiments, d=11. In some embodiments, d=12. In some embodiments, d=13. In some embodiments, d=14. In some embodiments, d=15. In some embodiments, d=16. In some embodiments, d=17. In some embodiments, d=18. In some embodiments, d=19. In some embodiments d=20. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand comprising from 5′ to 3′ a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the first strand comprises the formula:
5′-[B]a-[S1]b-[P]c-[S2]d-X-E-3′, wherein

(i) B, if present, is a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a is an integer whose value indicates the number of nucleotides comprising B, wherein a=0 or 5-7;

(ii) P is a polypyrimidine tract comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprising P, wherein c=9-20, wherein the nucleotide sequence comprising P is about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases;

(iii) E is an exonic sequence comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C);

(iv) X is a nucleotide sequence comprising a 3′ splice site; and

(v) S1 and S2, if either is present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein b and d=0-20,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, B, if present, P, X, if present, and E, if present, comprise a sense strand, wherein B, if present, P, and X, if present, comprise the one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into the DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, B is present. In some embodiments, B is absent. In some embodiments, a=0. In some embodiments, a=5-7. In some embodiments, a=5. In some embodiments, a=6. In some embodiments, a=7. In some embodiments, the nucleotide sequence comprising P is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P is about 90%400% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P is about 80%-90% pyrimidine nucleobases. In some embodiments, c=9. In some embodiments, c=10. In some embodiments, c=11. In some embodiments, c=12. In some embodiments, c=13. In some embodiments, c=14. In some embodiments, c=15. In some embodiments, c=16. In some embodiments, c=17. In some embodiments, c=18. In some embodiments, c=19. In some embodiments, c=20. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments b=20. In some embodiments, d=0. In some embodiments, d=1. In some embodiments, d=2. In some embodiments, d=3. In some embodiments, d=4. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, d=8. In some embodiments, d=9. In some embodiments, d=10. In some embodiments, d=11. In some embodiments, d=12. In some embodiments, d=13. In some embodiments, d=14. In some embodiments, d=15. In some embodiments, d=16. In some embodiments, d=17. In some embodiments, d=18. In some embodiments, d=19. In some embodiments d=20. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand from 5′ to 3′ comprises a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 5′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA,

wherein the first strand comprises the formula:
5′-E-Y-I-3′, wherein

(i) E is an exonic sequence comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C);

(ii) Y is a nucleotide sequence comprising a 5′ splice site; and

(iii) I, if present, comprises an intronic sequence comprising nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C),

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, E, if present, Y, and I, if present, comprise a sense strand, wherein Y comprises the one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, E is present. In some embodiments E is absent. In some embodiments, I is present. In some embodiments, I is absent. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising a first strand and a second strand, wherein the second strand is complementary to the first strand, wherein the first strand from 5′ to 3′ comprises a nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 5′ splice site, wherein the first strand comprises an intronic sequence and an exonic sequence, wherein the exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA,

wherein the first strand comprises the formula:
5′-E-Y-I-3′, wherein

(i) E is an exonic sequence comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C);

(ii) Y is a nucleotide sequence comprising a 5′ splice site; and

(iii) I, if present, comprises an intronic sequence comprising nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C),

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB, E, if present, Y, and I, if present, comprise a sense strand, wherein Y comprises the one or more splicing signals, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, E is present. In some embodiments E is absent. In some embodiments, I is present. In some embodiments, I is absent. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence, wherein the first intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence, wherein the second intronic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[P1]a-[S1]b-[B]c-[S2]d-[P2]e-3′, wherein

(a) B comprises a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprising B, wherein c=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a and e are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein a=9-20 and e=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, and wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2; and

(c) S1 and S2, if either is present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein b and d=0-20, wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in the first orientation, B and P2 comprise a sense strand and B and P2 comprise the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein, when the donor polynucleotide is inserted into the DSB in the second orientation, B and P1 comprise a sense strand and B and P2 comprise the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, a=9. In some embodiments, a=10. In some embodiments, a=11. In some embodiments, a=12. In some embodiments, a=13. In some embodiments, a=14. In some embodiments, a=15. In some embodiments, a=16. In some embodiments, a=17. In some embodiments, a=18. In some embodiments, a=19. In some embodiments, a=20. In some embodiments, e=9. In some embodiments, e=10. In some embodiments, e=11. In some embodiments, e=12. In some embodiments, e=13. In some embodiments, e=14. In some embodiments, e=15. In some embodiments, e=16. In some embodiments, e=17. In some embodiments, e=18. In some embodiments, e=19. In some embodiments, e=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments b=20. In some embodiments, d=0. In some embodiments, d=1. In some embodiments, d=2. In some embodiments, d=3. In some embodiments, d=4. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, d=8. In some embodiments, d=9. In some embodiments, d=10. In some embodiments, d=11. In some embodiments, d=12. In some embodiments, d=13. In some embodiments, d=14. In some embodiments, d=15. In some embodiments, d=16. In some embodiments, d=17. In some embodiments, d=18. In some embodiments, d=19. In some embodiments d=20. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence, wherein the first intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence, wherein the second intronic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[P1]a-[S1]b-[B]c-[S2]d-[P2]e-3′, wherein

(a) B comprises a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c is an integer whose value indicates the number of nucleotides comprising B, wherein c=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a and e are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein a=9-20 and e=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, and wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2; and

(c) S1 and S2, if either is present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and d are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein b and d=0-20,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in the first orientation, B and P2 comprise a sense strand and B and P2 comprise the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein, when the donor polynucleotide is inserted into the DSB in the second orientation, B and P1 comprise a sense strand and B and P2 comprise the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, a=9. In some embodiments, a=10. In some embodiments, a=11. In some embodiments, a=12. In some embodiments, a=13. In some embodiments, a=14. In some embodiments, a=15. In some embodiments, a=16. In some embodiments, a=17. In some embodiments, a=18. In some embodiments, a=19. In some embodiments, a=20. In some embodiments, e=9. In some embodiments, e=10. In some embodiments, e=11. In some embodiments, e=12. In some embodiments, e=13. In some embodiments, e=14. In some embodiments, e=15. In some embodiments, e=16. In some embodiments, e=17. In some embodiments, e=18. In some embodiments, e=19. In some embodiments, e=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments b=20. In some embodiments, d=0. In some embodiments, d=1. In some embodiments, d=2. In some embodiments, d=3. In some embodiments, d=4. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, d=8. In some embodiments, d=9. In some embodiments, d=10. In some embodiments, d=11. In some embodiments, d=12. In some embodiments, d=13. In some embodiments, d=14. In some embodiments, d=15. In some embodiments, d=16. In some embodiments, d=17. In some embodiments, d=18. In some embodiments, d=19. In some embodiments d=20. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence, wherein the first intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence, wherein the second intronic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[P1]a-[S1]b-[B1]c-[S2]d-[B2]e-[S3]f-[P2]g-3′, wherein

(a) B1, if present, and B2 are each branch point sequences, wherein B2 comprises a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein the branch point sequence comprising B1, if present, is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the branch point sequence comprising B2, wherein B1 and B2 each comprise a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c and e are integers whose value indicates the number of nucleotides comprising B1 and B2, respectively, wherein c=0 or 5-7, wherein e=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein a and g are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein a=9-20 and g=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2; and

(c) S1, S2 and S3, if any are present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b, d and f are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein b and f=0 or 1-20, wherein d=0 or 1-40,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in the first orientation, B2 and P2 comprise a sense strand and B2 and P2 comprise the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in the second orientation, B1 and P1 comprise a sense strand and B1 and P1 provide the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, c=0. In some embodiments, c=5-7. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, e=5. In some embodiments, e=6. In some embodiments, e=7. In some embodiments, a=9. In some embodiments, a=10. In some embodiments, a=11. In some embodiments, a=12. In some embodiments, a=13. In some embodiments, a=14. In some embodiments, a=15. In some embodiments, a=16. In some embodiments, a=17. In some embodiments, a=18. In some embodiments, a=19. In some embodiments, a=20. In some embodiments, g=9. In some embodiments, g=10. In some embodiments, g=11. In some embodiments, g=12. In some embodiments, g=13. In some embodiments, g=14. In some embodiments, g=15. In some embodiments, g=16. In some embodiments, g=17. In some embodiments, g=18. In some embodiments, g=19. In some embodiments, g=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, f=0. In some embodiments, f=1. In some embodiments, f=2. In some embodiments, f=3. In some embodiments, f=4. In some embodiments, f=5. In some embodiments, f=6. In some embodiments, f=7. In some embodiments, f=8. In some embodiments, f=9. In some embodiments, f=10. In some embodiments, f=11. In some embodiments, f=12. In some embodiments, f=13. In some embodiments, f=14. In some embodiments, f=15. In some embodiments, f=16. In some embodiments, f=17. In some embodiments, f=18. In some embodiments, f=19. In some embodiments, f=20. In some embodiments, d=0. In some embodiments, d=1. In some embodiments, d=2. In some embodiments, d=3. In some embodiments, d=4. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, d=8. In some embodiments, d=9. In some embodiments, d=10. In some embodiments, d=11. In some embodiments, d=12. In some embodiments, d=13. In some embodiments, d=14. In some embodiments, d=15. In some embodiments, d=16. In some embodiments, d=17. In some embodiments, d=18. In some embodiments, d=19. In some embodiments, d=20. In some embodiments, d=21. In some embodiments, d=22. In some embodiments, d=23. In some embodiments, d=24. In some embodiments, d=25. In some embodiments, d=26. In some embodiments, d=27. In some embodiments, d=28. In some embodiments, d=29. In some embodiments, d=30. In some embodiments, d=31. In some embodiments, d=32. In some embodiments, d=33. In some embodiments, d=34. In some embodiments, d=35. In some embodiments, d=36. In some embodiments, d=37. In some embodiments, d=38. In some embodiments, d=39. In some embodiments, d=40. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence, wherein the first intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence, wherein the second intronic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[P1]a-[S1]b-[B1]c-[S2]d-[B2]e-[S3]f-[P2]g-3′, wherein

(a) B1, if present, and B2 are each branch point sequences, wherein B2 comprises a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein the branch point sequence comprising B1, if present, is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the branch point sequence comprising B2, wherein B1 and B2 each comprise a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c and e are integers whose value indicates the number of nucleotides comprising B1 and B2, respectively, wherein c=0 or 5-7, wherein e=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein a and g are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein a=9-20 and g=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2; and

(c) S1, S2 and S3, if any are present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b, d and f are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein b and f=0 or 1-20, wherein d=0 or 1-40,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in the first orientation, B2 and P2 comprise a sense strand and B2 and P2 comprise the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in the second orientation, B1 and P1 comprise a sense strand and B1 and P1 provide the first and second splicing signals, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, c=0. In some embodiments, c=5-7. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, e=5. In some embodiments, e=6. In some embodiments, e=7. In some embodiments, a=9. In some embodiments, a=10. In some embodiments, a=11. In some embodiments, a=12. In some embodiments, a=13. In some embodiments, a=14. In some embodiments, a=15. In some embodiments, a=16. In some embodiments, a=17. In some embodiments, a=18. In some embodiments, a=19. In some embodiments, a=20. In some embodiments, g=9. In some embodiments, g=10. In some embodiments, g=11. In some embodiments, g=12. In some embodiments, g=13. In some embodiments, g=14. In some embodiments, g=15. In some embodiments, g=16. In some embodiments, g=17. In some embodiments, g=18. In some embodiments, g=19. In some embodiments, g=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, f=0. In some embodiments, f=1. In some embodiments, f=2. In some embodiments, f=3. In some embodiments, f=4. In some embodiments, f=5. In some embodiments, f=6. In some embodiments, f=7. In some embodiments, f=8. In some embodiments, f=9. In some embodiments, f=10. In some embodiments, f=11. In some embodiments, f=12. In some embodiments, f=13. In some embodiments, f=14. In some embodiments, f=15. In some embodiments, f=16. In some embodiments, f=17. In some embodiments, f=18. In some embodiments, f=19. In some embodiments, f=20. In some embodiments, d=0. In some embodiments, d=1. In some embodiments, d=2. In some embodiments, d=3. In some embodiments, d=4. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, d=8. In some embodiments, d=9. In some embodiments, d=10. In some embodiments, d=11. In some embodiments, d=12. In some embodiments, d=13. In some embodiments, d=14. In some embodiments, d=15. In some embodiments, d=16. In some embodiments, d=17. In some embodiments, d=18. In some embodiments, d=19. In some embodiments, d=20. In some embodiments, d=21. In some embodiments, d=22. In some embodiments, d=23. In some embodiments, d=24. In some embodiments, d=25. In some embodiments, d=26. In some embodiments, d=27. In some embodiments, d=28. In some embodiments, d=29. In some embodiments, d=30. In some embodiments, d=31. In some embodiments, d=32. In some embodiments, d=33. In some embodiments, d=34. In some embodiments, d=35. In some embodiments, d=36. In some embodiments, d=37. In some embodiments, d=38. In some embodiments, d=39. In some embodiments, d=40. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence and a first exonic sequence, wherein the first exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence and a second exonic sequence, wherein the second exonic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[E1]a-X1-[P1]b-[S1]c-[B]d-[S2]e-[P2]f-X2-[E2]g-3′, wherein

(a) B is a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein d is an integer whose value indicates the number of nucleotides comprising B, wherein d=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and f are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein b=9-20 and f=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2;

(c) E1 and E2 are each exonic sequences each comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a and g are integers whose value indicates the number of nucleotides comprising E1 and E2, respectively, wherein the exonic sequence comprising E1 is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the exonic sequence comprising E2, wherein the nucleotide sequences comprising E1 and E2 are not complementary;

(d) X1 and X2 are each nucleotide sequences comprising a 3′ splice site, wherein the nucleotide sequence comprising X1 is in the reverse orientation and on the opposite strand relative to the nucleotide sequence comprising X2; and

(e) S1 and S2, if either is present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein c and e are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein c=0 or 1-20 and e=0 or 1-20,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in the first orientation, B, P2, E2 and X2 and comprise a sense strand, wherein B, P2 and X2 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B, P1, E1 and X1 comprise a sense strand, wherein B, P1 and X1 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,

and wherein, when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, f=9. In some embodiments, f=10. In some embodiments, f=11. In some embodiments, f=12. In some embodiments, f=13. In some embodiments, f=14. In some embodiments, f=15. In some embodiments, f=16. In some embodiments, f=17. In some embodiments, f=18. In some embodiments, f=19. In some embodiments, f=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, c=0. In some embodiments, c=1. In some embodiments, c=2. In some embodiments, c=3. In some embodiments, c=4. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, c=8. In some embodiments, c=9. In some embodiments, c=10. In some embodiments, c=11. In some embodiments, c=12. In some embodiments, c=13. In some embodiments, c=14. In some embodiments, c=15. In some embodiments, c=16. In some embodiments, c=17. In some embodiments, c=18. In some embodiments, c=19. In some embodiments, c=20. In some embodiments, e=0. In some embodiments, e=1. In some embodiments, e=2. In some embodiments, e=3. In some embodiments, e=4. In some embodiments, e=5. In some embodiments, e=6. In some embodiments, e=7. In some embodiments, e=8. In some embodiments, e=9. In some embodiments, e=10. In some embodiments, e=11. In some embodiments, e=12. In some embodiments, e=13. In some embodiments, e=14. In some embodiments, e=15. In some embodiments, e=16. In some embodiments, e=17. In some embodiments, e=18. In some embodiments, e=19. In some embodiments, e=20. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence and a first exonic sequence, wherein the first exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence and a second exonic sequence, wherein the second exonic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[E1]a-X1-[P1]b-[S1]c-[B]d-[S2]e-[P2]f-X2-[E2]g-3′, wherein

(a) B is a branch point sequence comprising a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein B comprises a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein d is an integer whose value indicates the number of nucleotides comprising B, wherein d=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b and f are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein b=9-20 and f=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2;

(c) E1 and E2 are each exonic sequences each comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a and g are integers whose value indicates the number of nucleotides comprising E1 and E2, respectively, wherein the exonic sequence comprising E1 is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the exonic sequence comprising E2, wherein the nucleotide sequences comprising E1 and E2 are not complementary;

(d) X1 and X2 are each nucleotide sequences comprising a 3′ splice site, wherein the nucleotide sequence comprising X1 is in the reverse orientation and on the opposite strand relative to the nucleotide sequence comprising X2; and

(e) S1 and S2, if either is present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein c and e are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein c=0 or 1-20 and e=0 or 1-20,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in the first orientation, B, P2, E2 and X2 and comprise a sense strand, wherein B, P2 and X2 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B, P1, E1 and X1 comprise a sense strand, wherein B, P1 and X1 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,and wherein, when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, f=9. In some embodiments, f=10. In some embodiments, f=11. In some embodiments, f=12. In some embodiments, f=13. In some embodiments, f=14. In some embodiments, f=15. In some embodiments, f=16. In some embodiments, f=17. In some embodiments, f=18. In some embodiments, f=19. In some embodiments, f=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, c=0. In some embodiments, c=1. In some embodiments, c=2. In some embodiments, c=3. In some embodiments, c=4. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, c=8. In some embodiments, c=9. In some embodiments, c=10. In some embodiments, c=11. In some embodiments, c=12. In some embodiments, c=13. In some embodiments, c=14. In some embodiments, c=15. In some embodiments, c=16. In some embodiments, c=17. In some embodiments, c=18. In some embodiments, c=19. In some embodiments, c=20. In some embodiments, e=0. In some embodiments, e=1. In some embodiments, e=2. In some embodiments, e=3. In some embodiments, e=4. In some embodiments, e=5. In some embodiments, e=6. In some embodiments, e=7. In some embodiments, e=8. In some embodiments, e=9. In some embodiments, e=10. In some embodiments, e=11. In some embodiments, e=12. In some embodiments, e=13. In some embodiments, e=14. In some embodiments, e=15. In some embodiments, e=16. In some embodiments, e=17. In some embodiments, e=18. In some embodiments, e=19. In some embodiments, e=20. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence and a first exonic sequence, wherein the first exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence and a second exonic sequence, wherein the second exonic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

wherein the first strand and the second strand each comprise the formula:
5′-[E1]a-X1-[P1]b-[S1]c-[B1]d-[S2]e-[B2]f-[S3]g-[P2]h-X2-[E2]i-3′, wherein

(a) B1, if present, and B2 are each branch point sequences, wherein B2 comprises a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein the branch point sequence comprising B1, if present, is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the branch point sequence comprising B2, wherein B1 and B2 each comprise a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein d and f are integers whose value indicates the number of nucleotides comprising B1 and B2, respectively, wherein d and f=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein b and h are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein b=9-20 and h=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2;

(c) E1 and E2 are each exonic sequences each comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a and i are integers whose value indicates the number of nucleotides comprising E1 and E2, respectively, wherein the exonic sequence comprising E1 is in the reverse orientation and on the opposite strand relative to the exonic sequence comprising E2, wherein the nucleotide sequences comprising E1 and E2 are not complementary;

(d) X1 and X2 each comprise a nucleotide sequence comprising a 3′ splice site, wherein the nucleotide sequence of X1 is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the nucleotide sequence of X2; and

(e) S1, S2 and S3, if any present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c, e and g are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein c=0 or 1-20 and g=0 or 1-20, wherein e=0 or 1-40,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2, P2, E2 and X2 comprise a sense strand, wherein B2, P2 and X2 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B1, P1, E1 and X1 comprise a sense strand, wherein B1, P1 and X1 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, f=5. In some embodiments, f=6. In some embodiments, f=7. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, h=9. In some embodiments, h=10. In some embodiments, h=11. In some embodiments, h=12. In some embodiments, h=13. In some embodiments, h=14. In some embodiments, h=15. In some embodiments, h=16. In some embodiments, h=17. In some embodiments, h=18. In some embodiments, h=19. In some embodiments, h=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, c=0. In some embodiments, c=1. In some embodiments, c=2. In some embodiments, c=3. In some embodiments, c=4. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, c=8. In some embodiments, c=9. In some embodiments, c=10. In some embodiments, c=11. In some embodiments, c=12. In some embodiments, c=13. In some embodiments, c=14. In some embodiments, c=15. In some embodiments, c=16. In some embodiments, c=17. In some embodiments, c=18. In some embodiments, c=19. In some embodiments, c=20. In some embodiments, g=0. In some embodiments, g=1. In some embodiments, g=2. In some embodiments, g=3. In some embodiments, g=4. In some embodiments, g=5. In some embodiments, g=6. In some embodiments, g=7. In some embodiments, g=8. In some embodiments, g=9. In some embodiments, g=10. In some embodiments, g=11. In some embodiments, g=12. In some embodiments, g=13. In some embodiments, g=14. In some embodiments, g=15. In some embodiments, g=16. In some embodiments, g=17. In some embodiments, g=18. In some embodiments, g=19. In some embodiments, g=20. In some embodiments, e=0. In some embodiments, e=1. In some embodiments, e=2. In some embodiments, e=3. In some embodiments, e=4. In some embodiments, e=5. In some embodiments, e=6. In some embodiments, e=7. In some embodiments, e=8. In some embodiments, e=9. In some embodiments, e=10. In some embodiments, e=11. In some embodiments, e=12. In some embodiments, e=13. In some embodiments, e=14. In some embodiments, e=15. In some embodiments, e=16. In some embodiments, e=17. In some embodiments, e=18. In some embodiments, e=19. In some embodiments, e=20. In some embodiments, e=21. In some embodiments, e=22. In some embodiments, e=23. In some embodiments, e=24. In some embodiments, e=25. In some embodiments, e=26. In some embodiments, e=27. In some embodiments, e=28. In some embodiments, e=29. In some embodiments, e=30. In some embodiments, e=31. In some embodiments, e=32. In some embodiments, e=33. In some embodiments, e=34. In some embodiments, e=35. In some embodiments, e=36. In some embodiments, e=37. In some embodiments, e=38. In some embodiments, e=39. In some embodiments, e=40. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence and a first exonic sequence, wherein the first exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence and a second exonic sequence, wherein the second exonic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

wherein the first strand and the second strand each comprise the formula:
5′-[E1]a-X1-[P1]b-[S1]c-[B1]d-[S2]e-[B2]f-[S3]g-[P2]h-X2-[E2]i-3′, wherein

(a) B1, if present, and B2 are each branch point sequences, wherein B2 comprises a nucleotide sequence that conforms to a branch point consensus sequence on each strand of the donor polynucleotide, wherein the branch point sequence comprising B1, if present, is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the branch point sequence comprising B2, wherein B1 and B2 each comprise a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein d and f are integers whose value indicates the number of nucleotides comprising B1 and B2, respectively, wherein d and f=5-7;

(b) P1 and P2 are polypyrimidine tracts each comprising a nucleotide sequence comprising nucleobases selected from the group consisting of: adenine, guanine, thymine and cytosine, wherein b and h are integers whose value indicates the number of nucleotides comprising P1 and P2, respectively, wherein b=9-20 and h=9-20, wherein the nucleotide sequence comprising P1 and P2 are each about 100%, about 90%-100%, about 80%-90% pyrimidine nucleobases, wherein P1 is in the reverse orientation and on the opposite strand of the donor polynucleotide relative to P2;

(c) E1 and E2 are each exonic sequences each comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein a and i are integers whose value indicates the number of nucleotides comprising E1 and E2, respectively, wherein the exonic sequence comprising E1 is in the reverse orientation and on the opposite strand relative to the exonic sequence comprising E2, wherein the nucleotide sequences comprising E1 and E2 are not complementary;

(d) X1 and X2 each comprise a nucleotide sequence comprising a 3′ splice site, wherein the nucleotide sequence of X1 is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the nucleotide sequence of X2; and

(e) S1, S2 and S3, if any present, are each delimiter sequences comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein c, e and g are each integers whose value indicates the number of nucleotides comprising the delimiter sequence, respectively, wherein c=0 or 1-20 and g=0 or 1-20, wherein e=0 or 1-40,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, B2, P2, E2 and X2 comprise a sense strand, wherein B2, P2 and X2 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, B1, P1, E1 and X1 comprise a sense strand, wherein B1, P1 and X1 comprise the first, second and third splicing signal, respectively, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA,and wherein, when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, B1 is present. In some embodiments, B1 is absent. In some embodiments, d=5. In some embodiments, d=6. In some embodiments, d=7. In some embodiments, f=5. In some embodiments, f=6. In some embodiments, f=7. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, h=9. In some embodiments, h=10. In some embodiments, h=11. In some embodiments, h=12. In some embodiments, h=13. In some embodiments, h=14. In some embodiments, h=15. In some embodiments, h=16. In some embodiments, h=17. In some embodiments, h=18. In some embodiments, h=19. In some embodiments, h=20. In some embodiments, the nucleotide sequence comprising P1 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P1 is about 80%-90% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 90%-100% pyrimidine nucleobases. In some embodiments, the nucleotide sequence comprising P2 is about 80%-90% pyrimidine nucleobases. In some embodiments, S1 is present. In some embodiments, 51 is absent. In some embodiments, S2 is present. In some embodiments, S2 is absent. In some embodiments, S3 is present. In some embodiments, S3 is absent. In some embodiments, c=0. In some embodiments, c=1. In some embodiments, c=2. In some embodiments, c=3. In some embodiments, c=4. In some embodiments, c=5. In some embodiments, c=6. In some embodiments, c=7. In some embodiments, c=8. In some embodiments, c=9. In some embodiments, c=10. In some embodiments, c=11. In some embodiments, c=12. In some embodiments, c=13. In some embodiments, c=14. In some embodiments, c=15. In some embodiments, c=16. In some embodiments, c=17. In some embodiments, c=18. In some embodiments, c=19. In some embodiments, c=20. In some embodiments, g=0. In some embodiments, g=1. In some embodiments, g=2. In some embodiments, g=3. In some embodiments, g=4. In some embodiments, g=5. In some embodiments, g=6. In some embodiments, g=7. In some embodiments, g=8. In some embodiments, g=9. In some embodiments, g=10. In some embodiments, g=11. In some embodiments, g=12. In some embodiments, g=13. In some embodiments, g=14. In some embodiments, g=15. In some embodiments, g=16. In some embodiments, g=17. In some embodiments, g=18. In some embodiments, g=19. In some embodiments, g=20. In some embodiments, e=0. In some embodiments, e=1. In some embodiments, e=2. In some embodiments, e=3. In some embodiments, e=4. In some embodiments, e=5. In some embodiments, e=6. In some embodiments, e=7. In some embodiments, e=8. In some embodiments, e=9. In some embodiments, e=10. In some embodiments, e=11. In some embodiments, e=12. In some embodiments, e=13. In some embodiments, e=14. In some embodiments, e=15. In some embodiments, e=16. In some embodiments, e=17. In some embodiments, e=18. In some embodiments, e=19. In some embodiments, e=20. In some embodiments, e=21. In some embodiments, e=22. In some embodiments, e=23. In some embodiments, e=24. In some embodiments, e=25. In some embodiments, e=26. In some embodiments, e=27. In some embodiments, e=28. In some embodiments, e=29. In some embodiments, e=30. In some embodiments, e=31. In some embodiments, e=32. In some embodiments, e=33. In some embodiments, e=34. In some embodiments, e=35. In some embodiments, e=36. In some embodiments, e=37. In some embodiments, e=38. In some embodiments, e=39. In some embodiments, e=40. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence and a first exonic sequence, wherein the first exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence and a second exonic sequence, wherein the second exonic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and wherein the first strand and the second strand each comprise the formula:
5′-[E1]a-Y1-[S1]b-Y2-[E2]c-3′, wherein

(a) E1 and E2 each are exonic sequences each comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (T), wherein a and c are integers whose value indicates the number of nucleotides comprising E1 and E2, respectively, wherein the exonic sequence comprising E1 is in the reverse orientation and on the opposite strand relative to the exonic sequence comprising E2, wherein the nucleotide sequences comprising E1 and E2 are not complementary;

(b) Y1 and Y2 each comprise a nucleotide sequence comprising a 5′ splice site, wherein the nucleotide sequence of Y1 is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the nucleotide sequence of Y2; and

(c) S1, if present, is a delimiter sequence comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b is an integers whose value indicates the number of nucleotides comprising the delimiter sequence, wherein b=0 or 1-50,

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, E2 and Y2 comprise a sense strand and E2 comprises the splicing signal, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, E1 and Y1 comprise a sense strand and E1 comprises the splicing signal, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, b=21. In some embodiments, b=22. In some embodiments, b=23. In some embodiments, b=24. In some embodiments, b=25. In some embodiments, b=26. In some embodiments, b=27. In some embodiments, b=28. In some embodiments, b=29. In some embodiments, b=30. In some embodiments, b=31. In some embodiments, b=32. In some embodiments, b=33. In some embodiments, b=34. In some embodiments, b=35. In some embodiments, b=36. In some embodiments, b=37. In some embodiments, b=38. In some embodiments, b=39. In some embodiments, b=40. In some embodiments, b=41. In some embodiments, b=42. In some embodiments, b=43. In some embodiments, b=44. In some embodiments, b=45. In some embodiments, b=46. In some embodiments, b=47. In some embodiments, b=48. In some embodiments, b=49. In some embodiments, b=50. In some embodiments, the donor polynucleotide is about 10-400, about 10-300, or about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-500 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-400 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-300 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-200 nucleotides in length. In some embodiments, the donor polynucleotide is about 10-100 nucleotides in length. In some embodiments, the donor polynucleotide is about 20-80 nucleotides in length. In some embodiments, the donor polynucleotide is about 30-70 nucleotides in length. In some embodiments, the donor polynucleotide is about 40-60 nucleotides in length.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects a disease-causing mutation in a genomic DNA molecule (gDNA) in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the first strand comprises a first intronic sequence and a first exonic sequence, wherein the first exonic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA; and

(ii) a second strand comprising from 5′ to 3′ a second nucleotide sequence which corrects a disease-causing mutation in a gDNA in a cell, wherein the disease-causing mutation is a protein-coding mutation proximal to a 3′ splice site, wherein the second strand comprises a second intronic sequence and a second exonic sequence, wherein the second exonic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the second strand is complementary to the first strand, and

wherein the first strand and the second strand each comprise the formula:
5′-[E1]a-Y1-[S1]b-Y2-[E2]c-3′, wherein

(a) E1 and E2 each are exonic sequences each comprising a nucleotide sequence which corrects the mutation, wherein the nucleotide sequence comprises nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (T), wherein a and c are integers whose value indicates the number of nucleotides comprising E1 and E2, respectively, wherein the exonic sequence comprising E1 is in the reverse orientation and on the opposite strand relative to the exonic sequence comprising E2, wherein the nucleotide sequences comprising E1 and E2 are not complementary;

(b) Y1 and Y2 each comprise a nucleotide sequence comprising a 5′ splice site, wherein the nucleotide sequence of Y1 is in a reverse orientation and on the opposite strand of the donor polynucleotide relative to the nucleotide sequence of Y2; and

(c) S1, if present, is a delimiter sequence comprising one or more nucleotides comprising nucleobases selected from the group consisting of: adenine (A), guanine (G), thymine (T) and cytosine (C), wherein b is an integers whose value indicates the number of nucleotides comprising the delimiter sequence, wherein b=0 or 1-50,

wherein the donor polynucleotide is about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length, wherein the donor polynucleotide is configured for bi-directional insertion into a double-stranded DNA break (DSB), wherein when the donor polynucleotide is inserted into the DSB in a first orientation, E2 and Y2 comprise a sense strand and E2 comprises the splicing signal, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein when the donor polynucleotide is inserted into the DSB in a second orientation, E1 and Y1 comprise a sense strand and E1 comprises the splicing signal, thereby correcting the mutation and providing splicing signals to control processing of a pre-mRNA transcribed from the gDNA

and wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, S1 is present. In some embodiments, S1 is absent. In some embodiments, b=0. In some embodiments, b=1. In some embodiments, b=2. In some embodiments, b=3. In some embodiments, b=4. In some embodiments, b=5. In some embodiments, b=6. In some embodiments, b=7. In some embodiments, b=8. In some embodiments, b=9. In some embodiments, b=10. In some embodiments, b=11. In some embodiments, b=12. In some embodiments, b=13. In some embodiments, b=14. In some embodiments, b=15. In some embodiments, b=16. In some embodiments, b=17. In some embodiments, b=18. In some embodiments, b=19. In some embodiments, b=20. In some embodiments, b=21. In some embodiments, b=22. In some embodiments, b=23. In some embodiments, b=24. In some embodiments, b=25. In some embodiments, b=26. In some embodiments, b=27. In some embodiments, b=28. In some embodiments, b=29. In some embodiments, b=30. In some embodiments, b=31. In some embodiments, b=32. In some embodiments, b=33. In some embodiments, b=34. In some embodiments, b=35. In some embodiments, b=36. In some embodiments, b=37. In some embodiments, b=38. In some embodiments, b=39. In some embodiments, b=40. In some embodiments, b=41. In some embodiments, b=42. In some embodiments, b=43. In some embodiments, b=44. In some embodiments, b=45. In some embodiments, b=46. In some embodiments, b=47. In some embodiments, b=48. In some embodiments, b=49. In some embodiments, b=50.

In some embodiments, the exonic sequence comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising a 3′ splice site comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising a 5′ splice site comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising a polypyrimidine tract comprising the sense strand corrects the mutation. In some embodiments, the nucleotide sequence comprising a branch point sequence comprising the sense strand corrects the mutation.

In some embodiments, the 5′ most nucleotide of the donor polynucleotide comprises a 5′ phosphate group. In some embodiments, the donor polynucleotide is about 40-70 nucleotides or about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is about 50-60 nucleotides in length. In some embodiments, the donor polynucleotide is 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 nucleotides in length. In some embodiments, the donor polynucleotide is 40 nucleotides in length. In some embodiments, the donor polynucleotide is 41 nucleotides in length. In some embodiments, the donor polynucleotide is 42 nucleotides in length. In some embodiments, the donor polynucleotide is 43 nucleotides in length. In some embodiments, the donor polynucleotide is 44 nucleotides in length. In some embodiments, the donor polynucleotide is 45 nucleotides in length. In some embodiments, the donor polynucleotide is 46 nucleotides in length. In some embodiments, the donor polynucleotide is 47 nucleotides in length. In some embodiments, the donor polynucleotide is 49 nucleotides in length. In some embodiments, the donor polynucleotide is 42 nucleotides in length. In some embodiments, the donor polynucleotide is 50 nucleotides in length. In some embodiments, the donor polynucleotide is 51 nucleotides in length. In some embodiments, the donor polynucleotide is 52 nucleotides in length. In some embodiments, the donor polynucleotide is 53 nucleotides in length. In some embodiments, the donor polynucleotide is 54 nucleotides in length. In some embodiments, the donor polynucleotide is 55 nucleotides in length. In some embodiments, the donor polynucleotide is 56 nucleotides in length. In some embodiments, the donor polynucleotide is 57 nucleotides in length. In some embodiments, the donor polynucleotide is 58 nucleotides in length. In some embodiments, the donor polynucleotide is 59 nucleotides in length. In some embodiments, the donor polynucleotide is 60 nucleotides in length.

In some embodiments, the donor polynucleotide comprises natural nucleotides. In some embodiments, the donor polynucleotide comprises naturally occurring nucleotides. In some embodiments, the donor polynucleotide comprises one or more non-natural and/or modified nucleotides. In some embodiments, the one or more non-natural and/or modified nucleotides is a 2′-O-methyl nucleotide. In some embodiments, the donor polynucleotide comprises one or more backbone modifications. In some embodiments, the one or more backbone modification is a phosphorothioate. In some embodiments, the donor polynucleotide comprises two blunt ends. IN some embodiments, the donor polynucleotide comprises one blunt end and comprises one end comprising an overhang (e.g. a 5′ or 3′ overhang). In some embodiments, the nucleotide sequence of the donor polynucleotide comprises one or more nucleotides that prevent the site-directed nuclease from recognizing and cleaving the donor polynucleotide.

In some embodiments, the disease is Glycogen Storage Disease 1a (GSD1a). In some embodiments, the mutation is located in the human G6PC gene on human chromosome 17q21. In some embodiments, the mutation in the G6PC gene results in an R83C, an R83H, or an E110K amino acid substitution in the human G6PC protein. In some embodiments, the mutation in the G6PC gene results in an R83C amino acid substitution in the human G6PC protein. In some embodiments, the mutation in the G6PC gene results in an R83H amino acid substitution in the human G6PC protein. In some embodiments, the mutation in the G6PC gene results in an E110K amino acid substitution in the human G6PC protein.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative double-stranded DNA molecule (dsDNA) comprising a nucleotide sequence which corrects a mutation that causes Glycogen Storage Disease 1a in a genomic DNA molecule (gDNA) in a cell, wherein the mutation is located in the human G6PC gene on human chromosome 17q21 and results in the amino acid substitution R83C or R83H, the donor polynucleotide comprising:

(i) a first strand comprising from 5′ to 3′ a nucleotide sequence comprising an exonic sequence which corrects the mutation, wherein the exonic sequence comprises a codon encoding arginine (R) corresponding to the codon at position 83 in the G6PC gene, and a nucleotide sequence comprising one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the genomic DNA molecule, wherein the one or more splicing signals is a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

(ii) a second strand comprising a nucleotide sequence complementary to the first strand, wherein the donor polynucleotide is about 40-70 nucleotides in length and comprises two blunt ends, wherein the 5′ most nucleotide of each strand of the donor polynucleotide comprises a 5′ phosphate moiety, wherein when the donor polynucleotide is introduced into the cell in combination with an site-directed nuclease a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, wherein the insertion of the donor polynucleotide forms an exon in the gDNA comprising the exonic sequence that corrects the mutation, wherein the splicing signals direct the inclusion of the exon into an mRNA, thereby correcting the mutation. In some embodiments, the branch point sequence comprises the nucleotide sequence TTCAT, wherein the polypyrimidine tract comprises the nucleotide sequence CTTGTTCTGTTTTTTT (SEQ ID NO: 109), wherein the 3′ splice site comprises the nucleotide sequence TAG, and wherein the exonic sequence comprises the nucleotide sequence GATTCTCTTTGGACAGCGCCCTTACT (SEQ ID NO: 110).

In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 30 (CH34 54-0). In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 20 (CH32 50-0).

In some embodiments, the disease is Pompe's Disease. In some embodiments, the mutation is located in the human glucosidase alpha (GAA) gene on human chromosome 17q25.3. In some embodiments, the mutation is in a splicing signal of GAA that results in mRNA transcripts of the GAA gene lacking exon2 and/or activation of one or more cryptic splice sites.

In some embodiments, the disclosure provides a donor polynucleotide comprising a non-replicative dsDNA molecule comprising a nucleotide sequence which corrects a mutation that causes Pompe's Disease in a gDNA molecule in a cell, wherein the mutation is in a splicing signal of GAA that results in mRNA transcripts of the GAA gene lacking exon2 and/or activation of one or more cryptic splice sites, the donor polynucleotide comprising:

(i) a first strand comprising from 5′ to 3′ a first nucleotide sequence which corrects the mutation, wherein the first strand comprises a first intronic sequence, wherein the first intronic sequence corrects the mutation, wherein the first strand comprises one or more splicing signals to control processing of a precursor mRNA (pre-mRNA) transcribed from the gDNA, wherein the one or more splicing signals comprises a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence; and

(ii) a second strand comprising from 5′ to 3′ a second intronic sequence, wherein the second intronic sequence corrects the mutation, wherein the second strand comprises one or more splicing signals to control processing of a pre-mRNA transcribed from the gDNA, wherein the one or more splicing signals comprises a combination of a 3′ splice site, a polypyrimidine tract, and a branch point sequence, wherein the second strand is complementary to the first strand

wherein the donor polynucleotide is about 10-500, about 10-400, about 10-300, about 10-200, about 10-100, about 20-80, about 30-70, or about 40-60 nucleotides in length and comprises two blunt ends, wherein the 5′ most nucleotide of each strand of the donor polynucleotide comprises a 5′ phosphate moiety, wherein when the donor polynucleotide is introduced into the cell in combination with a site-directed nuclease a NHEJ DNA repair pathway inserts the donor polynucleotide into a DSB introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, wherein the donor polynucleotide is configured for bi-directional insertion into a DSB break, wherein insertion in either direction forms a 3′ splice site, a polypyrimidine tract, and a branch point sequence that corrects the mutation, wherein the splicing signals direct the inclusion of exon2 into an mRNA, thereby correcting the mutation.

In some embodiments, the nucleotide sequence of the donor polynucleotide is set forth in SEQ ID NO: 63 (GAA_50-0).

In some embodiments, the disclosure provides a system to correct a mutation in a genomic DNA molecule (gDNA) in a cell, the system comprising the components:

(a) any one of the aforementioned donor polynucleotides;

(b) one or more gRNA molecules; and

(c) a site-directed nuclease,

wherein when the system is introduced into a cell, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break (DSB) introduced into the gDNA by the site-directed nuclease at a location proximal to the mutation, thereby correcting the mutation. In some embodiments, the site-directed nuclease is encoded by an mRNA. In some embodiments, the site-directed nuclease is a polypeptide. In some embodiments, the one or more gRNA molecules and the site-directed nuclease comprise a ribonucleoprotein. In some embodiments, the site-directed nuclease is a Cas nuclease. In some embodiments, the Cas nuclease isS. pyogenesCas9 (SpCas9) or a homolog, derivative or modified version thereof. In some embodiments, the Cas nuclease isS. aureusCas9 (SaCas9) or a homolog, derivative or modified version thereof.

In some embodiments, the one or more gRNA molecules comprises a modification selected from the group consisting of: a backbone modification, a sugar modification, a modified internucleoside linkage, or a modified or non-natural nucleobase. In some embodiments, the one or more gRNA molecules comprise a backbone modification. In some embodiments, the backbone modification is a phosphorothioate modification.

In some embodiments of the system provided by the disclosure, the donor polynucleotide, the gRNA molecule and the site-directed nuclease are individually formulated or co-formulated in a liposome or lipid nanoparticle. In some embodiments, the donor polynucleotide, the gRNA molecule and the site-directed nuclease are individually formulated in a liposome or lipid nanoparticle. In some embodiments, the donor polynucleotide, the gRNA molecule and the site-directed nuclease are co-formulated in a liposome or lipid nanoparticle. In some embodiments, the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 107 (CH32 Mutant-CTX1 sgRNA spacer). In some embodiments, the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 108 (CH34 Mutant-CTX1 sgRNA spacer). In some embodiments, the nucleotide sequence of the gRNA comprises the sequence set forth in SEQ ID NO: 127 (mutant GAA sgRNA spacer).

In some embodiments, the disclosure provides a cell comprising any one of the aforementioned donor polynucleotides or systems. In some embodiments, the cell is a dividing or non-dividing cell. In some embodiments, the cell is a dividing cell. In some embodiments, the cell is a non-dividing cell. In some embodiments, the cell is a patient-specific induced pluripotent stem cell (iPSC). In some embodiments, the cell is a primary hepatocyte.

In some aspects, the disclosure provides a pharmaceutical composition comprising any one of the aforementioned donor polynucleotides and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a pharmaceutical composition comprising any one of the aforementioned systems and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a pharmaceutical composition comprising any one of the aforementioned cells and a pharmaceutically acceptable carrier.

In some embodiments, the disclosure provides any one of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, for use in treating a patient with a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the treatment comprising: isolating a cell from the patient, contacting the cell with the donor polynucleotide, the system, or the pharmaceutical composition, wherein when the donor polynucleotide, the system, or the pharmaceutical composition contacts the cell, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA at a location proximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides any one of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, for use in treating a patient with a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the treatment comprising: administering to the patient an effective amount of the donor polynucleotide, the system, or the pharmaceutical composition, wherein, when the donor polynucleotide, system or composition is administered, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA at a location proximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method to correct a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: contacting the cell with any one of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, wherein when the donor polynucleotide, system or composition contacts the cell, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA at a location proximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method of treating a patient with a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: isolating a cell from the patient, contacting the cell with any one of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, wherein, when the donor polynucleotide, system or composition contacts the cell, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA at a location proximal to the mutation, thereby correcting the mutation.

In some embodiments, the disclosure provides a method of treating a patient with a disease by correcting a mutation in a genomic DNA molecule (gDNA) in a cell, the method comprising: administering to the patient an effective amount of any one of the aforementioned donor polynucleotides, systems, or pharmaceutical compositions, wherein, when the donor polynucleotide, system or composition is administered, a non-homologous end-joining (NHEJ) DNA repair pathway inserts the donor polynucleotide into a double-stranded DNA break introduced into the gDNA at a location proximal to the mutation, thereby correcting the mutation. (in vivo).

In some embodiments of the methods provided by the disclosure, the cell is a patient-specific induced pluripotent stem cell (iPSC). In some embodiments, the cell is a hepatocyte. In some embodiments, of the methods provided by the disclosure, the method further comprises differentiating the iPSC comprising the corrected mutation into a differentiated cell; and implanting the differentiated cell into a patient.

In some embodiments of the methods provided by the disclosure, treatment results in the translation of an mRNA transcribed from the genomic DNA molecule (gDNA) comprising the inserted donor polynucleotide, wherein the translation results in the formation of a translation product (protein) that alleviates the disease or that does not cause or contribute to the disease.

In some embodiments, the disclosure provides a kit comprising a container comprising any one of the aforementioned donor polynucleotides, systems, or pharmaceutical composition, for correcting a mutation in a genomic DNA molecule (gDNA) in a cell, and a package insert comprising instructions for use.

