BACKGROUND

The CRISPR type II system is a recent development that has been efficiently utilized in a broad spectrum of species. See Friedland, A. E., et al., Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods, 2013. 10(8): p. 741-3, Mali, P., et al., RNA-guided human genome engineering via Cas9. Science, 2013. 339(6121): p. 823-6, Hwang, W. Y., et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol, 2013, Jiang, W., et al., RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol, 2013, Jinek, M., et al., RNA-programmed genome editing in human cells. elife, 2013. 2: p. e00471, Cong, L., et al., Multiplex genome engineering using CRISPR/Cas systems. Science, 2013. 339(6121): p. 819-23, Yin, H., et al., Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol, 2014. 32(6): p. 551-3. CRISPR is particularly customizable because the active form consists of an invariant Cas9 protein and an easily programmable guide RNA (gRNA). See Jinek, M., et al., A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012. 337(6096): p. 816-21. Of the various CRISPR orthologs, theStreptococcus pyogenes(Sp) CRISPR is the most well-characterized and widely used. The Cas9-gRNA complex first probes DNA for the protospacer-adjacent motif (PAM) sequence (-NGG for Sp Cas9), after which Watson-Crick base-pairing between the gRNA and target DNA proceeds in a ratchet mechanism to form an R-loop. Following formation of a ternary complex of Cas9, gRNA, and target DNA, the Cas9 protein generates two nicks in the target DNA, creating a blunt double-strand break (DSB) that is predominantly repaired by the non-homologous end joining (NHEJ) pathway or, to a lesser extent, template-directed homologous recombination (HR). CRISPR methods are disclosed in U.S. Pat. Nos. 9,023,649 and 8,697,359. See also, Fu et al., Nature Biotechnology, Vol. 32, Number 3, pp. 279-284 (2014). Additional references describing CRISPR-Cas9 systems including nuclease null variants (dCas9) and nuclease null variants functionalized with effector domains such as transcriptional activation domains or repression domains include J. D. Sander and J. K. Joung,Nature biotechnology32 (4), 347 (2014); P. D. Hsu, E. S. Lander, and F. Zhang,Cell157 (6), 1262 (2014); L. S. Qi, M. H. Larson, L. A. Gilbert et al.,Cell152 (5), 1173 (2013); P. Mali, J. Aach, P. B. Stranges et al.,Nature biotechnology31 (9), 833 (2013); M. L. Maeder, S. J. Linder, V. M. Cascio et al.,Nature methods10 (10), 977 (2013); P. Perez-Pinera, D. D. Kocak, C. M. Vockley et al.,Nature methods10 (10), 973 (2013); L. A. Gilbert, M. H. Larson, L. Morsut et al.,Cell154 (2), 442 (2013); P. Mali, K. M. Esvelt, and G. M. Church,Nature methods10 (10), 957 (2013); and K. M. Esvelt, P. Mali, J. L. Braff et al.,Nature methods10 (11), 1116 (2013).

SUMMARY

Embodiments of the present disclosure are directed to methods of making a plurality of guide RNA sequences using a vector including a plurality of guide RNA sequences. A guide RNA sequence as described herein includes a spacer nucleic acid sequence and a tail or scaffold nucleic acid sequence. According to one aspect, a library is provided of the plurality of nucleic acid sequences that encode for a plurality of guide RNA spacer sequences with a restriction enzyme cut site in between the spacer sequences. A primer can be provided at one or both ends of the nucleic acid so that the nucleic acid can be amplified, if desired. According to one aspect, a library of nucleic acids with each encoding a single guide RNA or multiple guide RNAs is provided wherein the nucleic acid includes one or more of a restriction endonuclease recognition site positioned at or within the nucleic acid encoding the guide RNA such that a nucleic acid encoding functional groups can be inserted into the sequence of the single guide RNA using the methods described herein. According to this method, one or more functional groups can be added to a guide RNA using a restriction endonuclease approach. According to this method different functional groups can be added to the same guide RNA in the library depending on the particular desired function.

The nucleic acid with the spacer sequences separated by the restriction enzyme cut sites is introduced into a plasmid. Each restriction enzyme cut site can be cut and a nucleic acid encoding a guide RNA scaffold sequence can be inserted therein. Promoter sequences and other regulatory sequences required for expression can also be included so that a nucleic acid is created that encodes a series of guide RNAs that can be expressed. In addition, the guide RNA scaffold sequence can be modified or can include insertion sequences that alter or provide certain Cas9 functionality. In addition, the guide RNA scaffold sequence can include one or more functional groups as described herein using methods described herein where a restriction endonuclease is used to cut a nucleic acid encoding a guide RNA sequence and a nucleic acid sequence encoding the functional group is inserted at the cut site. Such functional groups include an RNA aptamer sequence, an antibody sequence, an antibody binding sequence, a non-coding RNA sequence, a fluorescent protein sequence, a dye-binding aptamer, a donor nucleic acid sequence, a transcriptional activator sequence or a transcriptional repressor sequence.

Further features and advantages of certain embodiments of the present invention will become more fully apparent in the following description of embodiments and drawings thereof, and from the claims.

