SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 262232001100SUBSEQLIST.txt, date recorded: Aug. 15, 2019, size: 3,509 KB).

FIELD

The present disclosure relates to the use of recombinant proteins for inducing epigenetic modifications at specific loci, as well as to methods of using these recombinant proteins for reducing the expression of genes in plants.

BACKGROUND

Epigenetic marks are enzyme-mediated chemical modifications of DNA and of its associated chromatin proteins. Although epigenetic marks do not alter the primary sequence of DNA, they do contain heritable information and play key roles in regulating genome function. Such modifications, including cytosine methylation, posttranslational modifications of histone tails and the histone core, and the positioning of nucleosomes (histone octamers wrapped with DNA), influence the transcriptional state and other functional aspects of chromatin. For example, methylation of DNA and certain residues on the histone H3 N-terminal tail, such as H3 lysine 9 (H3K9), are important for transcriptional gene silencing and the formation of heterochromatin. Such marks are essential for the silencing of nongenic sequences, including transposons, pseudogenes, repetitive sequences, and integrated viruses, that become deleterious to cells if expressed and hence activated. Epigenetic gene silencing is also important in developmental phenomena such as imprinting in both plants and mammals, as well as in cell differentiation and reprogramming. Having the ability to specifically control target gene silencing is thus of great interest.

Different pathways involved in epigenetic silencing have been previously described, and include histone deacetylation, H3K27 and H3K9 methylation, H3K4 demethylation, and DNA methylation of promoters. An avenue to achieve DNA methylation is via a phenomenon known as RNA-directed DNA methylation, where non-coding RNAs act to direct methylation of a DNA sequence. In plants, proteins generally do not link the recognition of a specific DNA sequence with the establishment of an epigenetic state. Thus, endogenous plant epigenetic regulators generally cannot be used for epigenetic silencing of specific genes or transgenes in plants.

Accordingly, a need exists for improved epigenetic regulators that are capable of being targeted to specific loci to induce epigenetic gene silencing activity in plants.

BRIEF SUMMARY

The present disclosure relates to a method for reducing expression of one or more target nucleic acids in a plant, the method including: (a) providing a plant including a recombinant nucleic acid encoding a recombinant polypeptide including a nuclease-deficient CAS9 polypeptide (dCAS9) or fragment thereof and a polypeptide selected from an SHH1 polypeptide or fragment thereof, an SHH2 polypeptide or fragment thereof, an AGO4 polypeptide or fragment thereof, an HDA6 polypeptide or fragment thereof, an NRPD1 polypeptide or fragment thereof, an NRPE1 polypeptide or fragment thereof, a JMJ14 polypeptide or fragment thereof, an RDR2 polypeptide or fragment thereof, an NRPD2A/NRPE2 polypeptide or fragment thereof, an NRPB3/NRPD3/NRPE3A polypeptide or fragment thereof, an NRPE3B polypeptide or fragment thereof, an NRPB11/NRPD11/NRPE11 polypeptide or fragment thereof, an NRPB10/NRPD10/NRPE10 polypeptide or fragment thereof, an NRPB12/NRPD12/NRPE12 polypeptide or fragment thereof, an NRPB6A/NRPD6A/NRPE6A polypeptide or fragment thereof, an NRPB6B/NRPD6B/NRPE6B polypeptide or fragment thereof, an NRPB8A/NRPE8A polypeptide or fragment thereof, an NRPB8B/NRPD8B/NRPE8B polypeptide or fragment thereof, an NRPE5 polypeptide or fragment thereof, an NRPD4/NRPE4 polypeptide or fragment thereof, an NRPE7 polypeptide or fragment thereof, an NRPD7 polypeptide or fragment thereof, an NRPB5/NRPD5 polypeptide or fragment thereof, an NRPB9A/NRPD9A/NRPE9A polypeptide or fragment thereof, an NRPB9B/NRPD9B/NRPE9B polypeptide or fragment thereof, an ATRX polypeptide or fragment thereof, a MOM1 polypeptide or fragment thereof, a MORC1 polypeptide or fragment thereof, an SssI polypeptide or fragment thereof, a DRM2-MTase polypeptide or fragment thereof, a DNMT3A polypeptide or fragment thereof, a DNMT3L polypeptide or fragment thereof, an MBD9 polypeptide or fragment thereof, a SUVH2 polypeptide or a fragment thereof, a SUVH9 polypeptide or a fragment thereof, a DMS3 polypeptide or a fragment thereof, a MORC6 polypeptide or a fragment thereof, a SUVR2 polypeptide or a fragment thereof, a DRD1 polypeptide or a fragment thereof, an RDM1 polypeptide or a fragment thereof, a DRM3 polypeptide or a fragment thereof, a DRM2 polypeptide or a fragment thereof, and an FRG polypeptide or a fragment thereof; and a crRNA and tracrRNA, or fusions thereof; and (b) growing the plant under conditions where the recombinant nucleic acid is expressed and where the recombinant polypeptide is targeted to the one or more target nucleic acids, thereby reducing expression of the one or more target nucleic acids. In some embodiments, the recombinant polypeptide interacts with an RNA polymerase. In some embodiments, the RNA polymerase is RNA polymerase IV or RNA polymerase V. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polypeptide induces RNA-directed DNA methylation. In some embodiments that may be combined with any of the preceding embodiments, the one or more target nucleic acids are endogenous nucleic acids. In some embodiments that may be combined with any of the preceding embodiments, the one or more target nucleic acids are heterologous nucleic acids. In some embodiments that may be combined with any of the preceding embodiments, expression of the one or more target nucleic acids is silenced.

The present disclosure also relates to a recombinant nucleic acid which encodes a recombinant polypeptide including a nuclease-deficient CAS9 polypeptide (dCAS9) or fragment thereof and a polypeptide selected from an SHH1 polypeptide or fragment thereof, an SHH2 polypeptide or fragment thereof, an AGO4 polypeptide or fragment thereof, an HDA6 polypeptide or fragment thereof, an NRPD1 polypeptide or fragment thereof, an NRPE1 polypeptide or fragment thereof, a JMJ14 polypeptide or fragment thereof, an RDR2 polypeptide or fragment thereof, an NRPD2A/NRPE2 polypeptide or fragment thereof, an NRPB3/NRPD3/NRPE3A polypeptide or fragment thereof, an NRPE3B polypeptide or fragment thereof, an NRPB11/NRPD11/NRPE11 polypeptide or fragment thereof, an NRPB10/NRPD10/NRPE10 polypeptide or fragment thereof, an NRPB12/NRPD12/NRPE12 polypeptide or fragment thereof, an NRPB6A/NRPD6A/NRPE6A polypeptide or fragment thereof, an NRPB6B/NRPD6B/NRPE6B polypeptide or fragment thereof, an NRPB8A/NRPE8A polypeptide or fragment thereof, an NRPB8B/NRPD8B/NRPE8B polypeptide or fragment thereof, an NRPE5 polypeptide or fragment thereof, an NRPD4/NRPE4 polypeptide or fragment thereof, an NRPE7 polypeptide or fragment thereof, an NRPD7 polypeptide or fragment thereof, an NRPB5/NRPD5 polypeptide or fragment thereof, an NRPB9A/NRPD9A/NRPE9A polypeptide or fragment thereof, an NRPB9B/NRPD9B/NRPE9B polypeptide or fragment thereof, an ATRX polypeptide or fragment thereof, a MOM1 polypeptide or fragment thereof, a MORC1 polypeptide or fragment thereof, an SssI polypeptide or fragment thereof, a DRM2-MTase polypeptide or fragment thereof, a DNMT3A polypeptide or fragment thereof, a DNMT3L polypeptide or fragment thereof, an MBD9 polypeptide or fragment thereof, a SUVH2 polypeptide or a fragment thereof, a SUVH9 polypeptide or a fragment thereof, a DMS3 polypeptide or a fragment thereof, a MORC6 polypeptide or a fragment thereof, a SUVR2 polypeptide or a fragment thereof, a DRD1 polypeptide or a fragment thereof, an RDM1 polypeptide or a fragment thereof, a DRM3 polypeptide or a fragment thereof, a DRM2 polypeptide or a fragment thereof, and an FRG polypeptide or a fragment thereof. The present disclosure further relates to expression vectors including the recombinant nucleic acid of the preceding embodiment, and a host cell including the expression vector of the preceding embodiment. In some embodiments, the host cell is a plant cell. The present disclosure also relates to a recombinant plant including the recombinant nucleic acids of the preceding embodiments.

Other aspects of the present disclosure relate to a plant having reduced expression of one or more target nucleic acids according to the method of any one of the preceding embodiments, as well as a progeny plant of the plant of the preceding embodiment. In some embodiments, the progeny plant has reduced expression of the one or more target nucleic acids and does not include the recombinant nucleic acid.

The present disclosure also relates to a method for reducing expression of one or more target nucleic acids in a plant, the method including: (a) providing a plant including: a recombinant nucleic acid encoding a recombinant polypeptide selected from an SHH1-like protein, an SHH2-like protein, an AGO4-like protein, an HDA6-like protein, an NRPD1-like protein, an NRPE1-like protein, a JMJ14-like protein, an RDR2-like protein, an NRPD2A/NRPE2-like protein, an NRPB3/NRPD3/NRPE3A-like protein, an NRPE3B-like protein, an NRPB11/NRPD11/NRPE11-like protein, an NRPB10/NRPD10/NRPE10-like protein, an NRPB12/NRPD12/NRPE12-like protein, an NRPB6A/NRPD6A/NRPE6A-like protein, an NRPB6B/NRPD6B/NRPE6B-like protein, an NRPB8A/NRPE8A-like protein, an NRPB8B/NRPD8B/NRPE8B-like protein, an NRPE5-like protein, an NRPD4/NRPE4-like protein, an NRPE7-like protein, an NRPD7-like protein, an NRPB5/NRPD5-like protein, an NRPB9A/NRPD9A/NRPE9A-like protein, an NRPB9B/NRPD9B/NRPE9B-like protein, an ATRX-like protein, a MOM1-like protein, a MORC1-like protein, an SssI-like protein, a DRM2-MTase-like protein, a DNMT3A-like protein, a DNMT3L-like protein, a MBD9-like protein, an SUVH2-like protein, a SUVH9-like protein, a DMS3-like protein, a MORC6-like protein, a SUVR2-like protein, a DRD1-like protein, an RDM1-like protein, a DRM3-like protein, a DRM2-like protein, and an FRG-like protein; and a crRNA and tracrRNA, or fusions thereof, and where the plant expresses a dCAS9 protein; and (b) growing the plant under conditions where the recombinant nucleic acid is expressed and where the recombinant polypeptide is targeted to the one or more target nucleic acids, thereby reducing expression of the one or more target nucleic acids. In some embodiments, the recombinant polypeptide includes a dCAS9 protein or fragment thereof. In some embodiments, the recombinant polypeptide includes an MS2 coat protein or fragment thereof. In some embodiments, the recombinant polypeptide includes an scFV antibody or fragment thereof. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polypeptide interacts with an RNA polymerase. In some embodiments, the RNA polymerase is RNA polymerase IV or RNA polymerase V. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polypeptide induces RNA-directed DNA methylation. In some embodiments that may be combined with any of the preceding embodiments, the one or more target nucleic acids are endogenous nucleic acids. In some embodiments that may be combined with any of the preceding embodiments, the one or more target nucleic acids are heterologous nucleic acids. In some embodiments that may be combined with any of the preceding embodiments, expression of the one or more target nucleic acids is silenced.

The present disclosure also relates to a recombinant nucleic acid which encodes a recombinant polypeptide selected from an SHH1-like protein, an SHH2-like protein, an AGO4-like protein, an HDA6-like protein, an NRPD1-like protein, an NRPE1-like protein, a JMJ14-like protein, an RDR2-like protein, an NRPD2A/NRPE2-like protein, an NRPB3/NRPD3/NRPE3A-like protein, an NRPE3B-like protein, an NRPB11/NRPD11/NRPE11-like protein, an NRPB10/NRPD10/NRPE10-like protein, an NRPB12/NRPD12/NRPE12-like protein, an NRPB6A/NRPD6A/NRPE6A-like protein, an NRPB6B/NRPD6B/NRPE6B-like protein, an NRPB8A/NRPE8A-like protein, an NRPB8B/NRPD8B/NRPE8B-like protein, an NRPE5-like protein, an NRPD4/NRPE4-like protein, an NRPE7-like protein, an NRPD7-like protein, an NRPB5/NRPD5-like protein, an NRPB9A/NRPD9A/NRPE9A-like protein, an NRPB9B/NRPD9B/NRPE9B-like protein, an ATRX-like protein, a MOM1-like protein, a MORC1-like protein, an SssI-like protein, a DRM2-MTase-like protein, a DNMT3A-like protein, a DNMT3L-like protein, a MBD9-like protein, a SUVH2-like protein, a SUVH9-like protein, a DMS3-like protein, a MORC6-like protein, a SUVR2-like protein, a DRD1-like protein, an RDM1-like protein, a DRM3-like protein, a DRM2-like protein, and an FRG-like protein. The present disclosure further relates to expression vectors including the recombinant nucleic acid of the preceding embodiment, and a host cell including the expression vector of the preceding embodiment. In some embodiments, the host cell is a plant cell. The present disclosure also relates to a recombinant plant including the recombinant nucleic acids of the preceding embodiments.

Other aspects of the present disclosure relate to a plant having reduced expression of one or more target nucleic acids according to the method of any one of the preceding embodiments, as well as a progeny plant of the plant of the preceding embodiment. In some embodiments, the progeny plant has reduced expression of the one or more target nucleic acids and does not include the recombinant nucleic acid.

The present disclosure also relates to a method for reducing expression of one or more target nucleic acids in a plant, including: (a) providing a plant including a recombinant nucleic acid, where the recombinant nucleic acid encodes a recombinant polypeptide including a first amino acid sequence including a DNA-binding domain and a second amino acid sequence including a polypeptide selected from the group of an SHH1 polypeptide or fragment thereof, an SHH2 polypeptide or fragment thereof, an AGO4 polypeptide or fragment thereof, an HDA6 polypeptide or fragment thereof, an NRPD1 polypeptide or fragment thereof, an NRPE1 polypeptide or fragment thereof, a JMJ14 polypeptide or fragment thereof, an RDR2 polypeptide or fragment thereof, an NRPD2A/NRPE2 polypeptide or fragment thereof, an NRPB3/NRPD3/NRPE3A polypeptide or fragment thereof, an NRPE3B polypeptide or fragment thereof, an NRPB11/NRPD11/NRPE11 polypeptide or fragment thereof, an NRPB10/NRPD10/NRPE10 polypeptide or fragment thereof, an NRPB12/NRPD12/NRPE12 polypeptide or fragment thereof, an NRPB6A/NRPD6A/NRPE6A polypeptide or fragment thereof, an NRPB6B/NRPD6B/NRPE6B polypeptide or fragment thereof, an NRPB8A/NRPE8A polypeptide or fragment thereof, an NRPB8B/NRPD8B/NRPE8B polypeptide or fragment thereof, an NRPE5 polypeptide or fragment thereof, an NRPD4/NRPE4 polypeptide or fragment thereof, an NRPE7 polypeptide or fragment thereof, an NRPD7 polypeptide or fragment thereof, an NRPB5/NRPD5 polypeptide or fragment thereof, an NRPB9A/NRPD9A/NRPE9A polypeptide or fragment thereof, an NRPB9B/NRPD9B/NRPE9B polypeptide or fragment thereof, an ATRX polypeptide or fragment thereof, a MOM1 polypeptide or fragment thereof, a MORC1 polypeptide or fragment thereof, an SssI polypeptide or fragment thereof, a DRM2-MTase polypeptide or fragment thereof, a DNMT3A polypeptide or fragment thereof, a DNMT3L polypeptide or fragment thereof, an MBD9 polypeptide or fragment thereof, a SUVH2 polypeptide or a fragment thereof, a SUVH9 polypeptide or a fragment thereof, a DMS3 polypeptide or a fragment thereof, a MORC6 polypeptide or a fragment thereof, a SUVR2 polypeptide or a fragment thereof, a DRD1 polypeptide or a fragment thereof, an RDM1 polypeptide or a fragment thereof, a DRM3 polypeptide or a fragment thereof, a DRM2 polypeptide or a fragment thereof, and an FRG polypeptide or a fragment thereof, and (b) growing the plant under conditions where the recombinant polypeptide encoded by the recombinant nucleic acid is expressed and binds to the one or more target nucleic acids, thereby reducing expression of the one or more target nucleic acids. In some embodiments, the DNA-binding domain includes a zinc finger domain. In some embodiments, the zinc finger domain includes two, three, four, five, six, seven, eight, or nine zinc fingers. In some embodiments, the zinc finger domain is a zinc finger array. In some embodiments, the zinc finger domain is selected from the group of a Cys2His2 (C2H2) zinc finger domain, a CCCH zinc finger domain, a multi-cysteine zinc finger domain, and a zinc binuclear cluster domain. In some embodiments, the DNA-binding domain is selected from the group of a TAL effector targeting domain, a helix-turn-helix family DNA-binding domain, a basic domain, a ribbon-helix-helix domain, a TBP domain, a barrel dimer domain, a real homology domain, a BAH domain, a SANT domain, a Chromodomain, a Tudor domain, a Bromodomain, a PHD domain, a WD40 domain, and a MBD domain. In some embodiments, the DNA-binding domain includes a TAL effector targeting domain. In some embodiments, the DNA-binding domain includes three C2H2 zinc finger domains. In some embodiments, the recombinant polypeptide interacts with an RNA polymerase. In some embodiments, the RNA polymerase is RNA polymerase IV or RNA polymerase V. In some embodiments that may be combined with any of the preceding embodiments, the recombinant polypeptide induces RNA-directed DNA methylation. In some embodiments that may be combined with any of the preceding embodiments, the one or more target nucleic acids are endogenous nucleic acids. In some embodiments that may be combined with any of the preceding embodiments, the one or more target nucleic acids are heterologous nucleic acids. In some embodiments that may be combined with any of the preceding embodiments, expression of the one or more target nucleic acids is silenced.

In another aspect, the present disclosure provides a method for reducing expression of one or more target nucleic acids in a plant, including: (a) providing a plant including a recombinant polypeptide selected from an SHH1-like protein, an SHH2-like protein, an AGO4-like protein, an HDA6-like protein, an NRPD1-like protein, a JMJI4-like protein, an RDR2-like protein, an NRPD2A/NRPE2-like protein, an NRPB3/NRPD3/NRPE3A-like protein, an NRPE3B-like protein, an NRPB11/NRPD11/NRPE11-like protein, an NRPB10/NRPD10/NRPE10-like protein, an NRPB12/NRPD12/NRPE12-like protein, an NRPB6A/NRPD6A/NRPE6A-like protein, an NRPB6B/NRPD6B/NRPE6B-like protein, an NRPB8A/NRPE8A-like protein, an NRPB8B/NRPD8B/NRPE8B-like protein, an NRPE5-like protein, an NRPD4/NRPE4-like protein, an NRPE7-like protein, an NRPD7-like protein, an NRPB5/NRPD5-like protein, an NRPB9A/NRPD9A/NRPE9A-like protein, an NRPB9B/NRPD9B/NRPE9B-like protein, an ATRX-like protein, a MOM1-like protein, a MORC1-like protein, an SssI-like protein, a DRM2-MTase-like protein, a DNMT3A-like protein, a DNMT3L-like protein, a MBD9-like protein, a SUVH2-like protein, a SUVH9-like protein, a DMS3-like protein, a MORC6-like protein, a SUVR2-like protein, a DRD1-like protein, an RDM1-like protein, a DRM3-like protein, a DRM2-like protein, and an FRG-like protein; and (b) growing the plant under conditions whereby the recombinant polypeptide is targeted to the one or more target nucleic acids, thereby reducing expression of the one or more target nucleic acids.

In another aspect, the present disclosure provides a method for reducing expression of one or more target nucleic acids in a plant, including: (a) providing a plant including: a first recombinant polypeptide including a nuclease-deficient CAS9 polypeptide (dCAS9) or fragment thereof and a multimerized epitope; a second recombinant polypeptide including a DRM2-MTase polypeptide or a DNMT3A-DNMT3L fusion polypeptide, and an affinity polypeptide that specifically binds to the epitope; a crRNA and a tracrRNA, or fusions thereof; and (b) growing the plant under conditions whereby the first and second recombinant polypeptides are targeted to the one or more target nucleic acids, thereby reducing expression of the one or more target nucleic acids.

In another aspect, the present disclosure provides a recombinant vector including: a first nucleic acid sequence including a plant promoter and that encodes a recombinant polypeptide including a nuclease-deficient CAS9 polypeptide (dCAS9) or fragment thereof and a multimerized epitope; a second nucleic acid sequence including a plant promoter and that encodes a recombinant polypeptide including a DRM2-MTase polypeptide or a DNMT3A-DNMT3L fusion polypeptide, and an affinity polypeptide that specifically binds to the epitope; and a third nucleic acid sequence including a promoter and that encodes a crRNA and a tracrRNA, or fusions thereof. Also provided are host cells including the vector of the preceding embodiment, and a recombinant plant including the vector of the preceding embodiment.

In another aspect, the present disclosure provides a plant having reduced expression of one or more target nucleic acids as a consequence of the method of any of the preceding embodiments. Also provided is a progeny plant of the plant of the preceding embodiment. In some embodiments, the progeny plant has reduced expression of the one or more target nucleic acids and does not include the recombinant polypeptide targeted to the one or more target nucleic acids.

