CAS exonuclease fusion proteins for excision, inversion and site-specific integration and related methods

Abstract

Provided herein are fusion proteins and related methods and systems for increasing the efficiency of genome editing using site-specific nuclease enzymes. These fusion proteins, systems, and methods may selectively increase desired editing outcomes (e.g., inversion, excision, and homologous directed repair). Also provided are various useful compositions for producing and using these fusion proteins and practicing these methods.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Chinese patent application No. 202210718723.X filed on June 23, 2022, which is incorporated by reference. Technical Field

[0002] The present disclosure relates to methods for increasing excision, inversion, and site-specific integration. The methods presented herein are applicable to both non-homologous end joining (NHEJ) and homology-dependent repair (HDR) mechanisms. Sequence Listing

[0003] Attached to this application is a sequence listing named 82439-ST26.xml, which is approximately 252 kilobytes in size. This sequence listing is incorporated herein by reference in its entirety. Background Art

[0004] Site-directed nucleases (SDNs) (e.g., zinc finger nucleases, transcription activator-like effector nucleases, CRISPR-associated nucleases) are becoming more and more popular in gene editing space. These SDNs act as endonucleases and usually produce double-strand breaks (DSBs) in specific DNA sequences, thereby activating the intrinsic repair mechanisms (e.g., homologous recombination) of cells. During the repair process, site-directed modification of the specific DNA sequence can be achieved. CRISPR (clustered regularly spaced short palindromic repeats) / Cas (CRISPR-associated) systems have evolved into adaptive immune systems in bacteria and archaea to defend against viral attacks. In recent years, CRISPR / Cas systems have attracted special attention as tools for genome editing. The CRISPR / Cas systems producing site-specific double-strand breaks (DSBs) can be used, for example, to edit DNA in eukaryotic cells by producing deletions, insertions, and / or changes in nucleotide sequences.

[0005] Site-directed modifications induced by SDN often lack precision (e.g., off-target editing may occur), and they often occur at low frequencies. For example, in the case where the CRISPR / Cas system is configured to cause deletions by generating one or more DSBs, the size of the deletions may vary, and the frequency of desired deletion events may be relatively low. Thus, methods for increasing the efficiency of targeted genome editing using SDN are needed. Summary of the invention

[0006] This Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to help limit the scope of the claimed subject matter.

[0007] On the one hand, fusion proteins are provided herein, which include a site-directed nuclease connected to a non-specific end processing enzyme. In some embodiments, the site-directed nuclease includes a CRISPR-associated nuclease. In some embodiments, the CRISPR-associated nuclease is selected from the group consisting of: Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14 and its nickase or inactivated form. In some embodiments, the CRISPR-associated nuclease is a Cas9 enzyme. In some embodiments, the CRISPR-associated nuclease is a Cas12a enzyme.

[0008] In some embodiments of the fusion proteins provided herein, the non-specific end processing enzyme is a non-specific exonuclease. In some embodiments, the non-specific exonuclease is T5Exo, Trex2, Escherichia coli exonuclease I, exonuclease III, exonuclease T, exonuclease IX, exonuclease X, RecJ, Pol II, Pol IIIε, WRN, MRE11, APE1, VDJP, RAD1, RAD9, p53 or Trex1. In some embodiments, the non-specific end processing enzyme comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 4, 5, 18, 19, 20, 22 or 58-74. In some embodiments, the non-specific end processing enzyme is a monomer of a dimerized protein.

[0009] In some embodiments of the fusion proteins provided herein, the fusion protein comprises a linker between the site-directed nuclease and the non-specific end processing enzyme. In some embodiments, the linker comprises SEQ ID NO: 7. In some embodiments, the fusion protein comprises a nuclear localization signal. In some embodiments, the fusion protein comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 50-57.

[0010] Also provided herein are recombinant nucleic acids, which encode any fusion protein described herein. Also provided are DNA constructs, which include promoters operably connected to recombinant nucleic acids described herein. In certain embodiments, the promoter is at least one of an inducible promoter, a constitutive promoter, an egg cell-specific promoter, a pollen-specific promoter, or an apical meristem-specific promoter. In certain embodiments, the promoter is an ubiquitin 4 promoter, an actin promoter, a tubulin promoter, a MADS box promoter, or a plant virus promoter. Also provided herein are vectors, which include recombinant nucleic acids or DNA constructs described herein. Also provided herein are cells, which include recombinant nucleic acids, DNA constructs, or vectors described herein. In certain embodiments, the cells are plant cells. In certain embodiments, the plant cells are corn plant cells, soybean plant cells, rice plant cells, wheat plant cells, and / or sunflower plant cells.

[0011] Also provided herein are methods for editing nucleic acids, the methods comprising providing at least one fusion protein described herein; providing the nucleic acid, wherein the nucleic acid comprises a first binding site, a second binding site, and a target region containing a portion of the nucleic acid, wherein the first binding site is adjacent to the 5' end of the target region and the second binding site is adjacent to the 3' end of the target region; and contacting the nucleic acid with the at least one fusion protein, wherein the at least one fusion protein specifically binds to the first binding site and the second binding site, thereby resulting in editing of the target region of the nucleic acid. In some embodiments, the site-directed nuclease of the at least one fusion protein comprises a CRISPR-associated nuclease, and the method further comprises providing at least one first guide RNA and at least one second guide RNA, wherein the at least one first guide RNA comprises a nucleotide sequence having complementarity with the first binding site and the at least one second guide RNA comprises a nucleotide sequence having complementarity with the second binding site. In some embodiments, the first binding site and the second binding site are on the same strand. In some embodiments, the first binding site and the second binding site are on opposite strands. In some embodiments, at least one of the first binding site or the second binding site is within the target region. In some embodiments, both the first binding site and the second binding site are within the target region. In some embodiments, neither the first binding site nor the second binding site is within the first target region.

[0012] In some embodiments of the methods of editing nucleic acids provided herein, the methods further include providing a donor nucleic acid, wherein the donor nucleic acid comprises a third binding site, a fourth binding site, and a donor nucleotide region, wherein the third binding site is adjacent to the 5' end of the donor nucleotide region and the fourth binding site is adjacent to the 3' end of the donor nucleotide region, and wherein the at least one fusion protein specifically binds to the third binding site and the fourth binding site. In some embodiments, the site-directed nuclease of the at least one fusion protein includes a CRISPR-associated nuclease, and the method further includes providing at least one third guide RNA and at least one fourth guide RNA, wherein the at least one third guide RNA comprises a nucleotide sequence having complementarity with the third binding site and the at least one fourth guide RNA comprises a nucleotide sequence having complementarity with the fourth binding site. In some embodiments, the third binding site and the fourth binding site are on the same chain. In some embodiments, the third binding site and the fourth binding site are on opposite chains. In some embodiments, at least one of the third binding site or the fourth binding site is within the donor nucleotide region. In some embodiments, both the third binding site and the fourth binding site are within the donor nucleotide region. In some embodiments, neither the third binding site nor the fourth binding site is within the donor nucleotide region.

[0013] In some embodiments of the methods of editing nucleic acids provided herein, the nucleic acid is a portion of a first chromosome. In some embodiments, the donor nucleic acid is a portion of a donor template. In some embodiments, the donor template is a portion of a plasmid or a linear nucleic acid.

[0014] In some embodiments of the methods of editing nucleic acids provided herein, the editing is an excision, inversion, or replacement of at least a portion of the target region.

[0015] In some embodiments of the methods of editing nucleic acids provided herein, the donor nucleic acid is a portion of a second chromosome. In some embodiments, the first chromosome and the second chromosome are homologous chromosomes or non-homologous chromosomes. In some embodiments, the editing is a chromosome rearrangement or a replacement of at least a portion of the target region. In some embodiments, the chromosome rearrangement is a reciprocal translocation or a non-reciprocal translocation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present application includes the following figures. These figures are intended to illustrate certain embodiments and / or features of the compositions and methods and to supplement any one or more descriptions of the compositions and methods. These figures do not limit the scope of the compositions and methods unless the written description clearly indicates that this is the case.

[0017] Figure 1The stepwise cutting and inversion of the target polynucleotide sequence according to the aspects of the present disclosure are shown. Two fusion proteins are bound to the first binding site and the second binding site on the same chain of the target DNA sequence. The first binding site is adjacent to the 5' end of the target region (that is, the region is located between the nuclease cleavage sites), and the second binding site is adjacent to the 3' end of the target region. For illustrative purposes, the fusion protein in this embodiment includes LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. In the target DNA sequence, for example, the gene sequence further including a promoter, a first exon, a first intron, etc. is a first binding site, which includes a PAM sequence located in the promoter of the gene, and the second binding site includes a PAM sequence located in intron 1 of the gene. LbCas12a (that is, the SDN of the fusion protein) cleaves DNA from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA 1") is upstream of the cleavage site (that is, outside the target region), the fusion protein combined with the first binding site remains combined with the cleavage polynucleotide ends outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA 2") is upstream of the cleavage site (that is, within the target region), the fusion protein bound to the second binding site remains combined with the downstream end of the target region. Not subject to any particular theory, the dimerization of the fusion protein combined with the first binding site and the second binding site (that is, by the dimerization of Trex2) will make the downstream end of the target region and the polynucleotide ends upstream of the target region closely approach, wherein they will be connected by DNA repair mechanisms (such as NHEJ or MMEJ). The connection of the remaining polynucleotide ends (that is, those not bound by the fusion protein) by DNA repair mechanisms therefore results in inversion of the target region.

[0018] Figure 2The stepwise cutting and excision of the target polynucleotide sequence according to the aspects of the present disclosure are shown. Two fusion proteins are bound to the first binding site and the second binding site on the opposite strands of the DNA polynucleotide. The first binding site is adjacent to the 5' end of the target region, and the second binding site is adjacent to the 3' end of the target region. For illustrative purposes, the fusion protein in this embodiment comprises LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. In this diagram, the DNA polynucleotide is a gene; the first binding site comprises a PAM sequence located in the promoter of the gene, and the second binding site comprises a PAM sequence located in intron 1 of the gene. In this diagram, LbCas12a (i.e., the SDN of the fusion protein) cleaves the polynucleotide from PAM in the 3' direction, and the fusion protein remains bound to the binding site comprising PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA 1") is upstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the first binding site remains bound to the end of the cleavage polynucleotide outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA 2") is downstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the second binding site remains bound to the cleavage polynucleotide end outside and downstream of the target region. Without being bound by any particular theory, dimerization of the fusion protein bound to the first binding site and the second binding site will bring the cleavage polynucleotide ends upstream and downstream of the target region into close proximity, where they will be connected by DNA repair mechanisms (such as NHEJ or MMEJ), resulting in excision of the target region.

[0019] Figure 3A and 3BThe stepwise cutting, excision and insertion of polynucleotide fragments at the target polynucleotide sequence according to aspects of the present disclosure are shown. Two fusion proteins including the first binding site and the second binding site on the opposite strand of a DNA polynucleotide (i.e., nucleotides to be edited, shown here as "genomic DNA") and the method of two fusion proteins of the third binding site and the fourth binding site on the opposite strand of a donor nucleic acid (shown here as "donor DNA") are shown. In two embodiments, the first binding site is adjacent to the 5' end of the target region (i.e., the region is between the nuclease cleavage sites), the second binding site is adjacent to the 3' end of the target region, the third binding site is adjacent to the 5' end of the donor nucleotide region (i.e., the region of the donor nucleotide is between the nuclease cleavage sites), and the fourth binding site is adjacent to the 3' end of the donor nucleotide region. For illustrative purposes, the fusion protein in these embodiments includes LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. LbCas12a (i.e., the SDN of the fusion protein) cleaves polynucleotides (i.e., both nucleotides to be edited and donor nucleic acids) from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA-a") is upstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the first binding site remains bound to the cleavage polynucleotide end outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA-b") is downstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the second binding site remains bound to the cleavage polynucleotide end outside the target region and downstream thereof. Because the third binding site (indicated by "gRNA-a / c") is downstream of the cleavage site (i.e., in the donor nucleotide region), the fusion protein bound to the third binding site remains bound to the cleavage polynucleotide end of the donor nucleotide region (i.e., the upstream end of the donor nucleotide region). Because the fourth binding site (indicated by "gRNA-b / d") is upstream of the cleavage site (i.e., in the donor nucleotide region), the fusion protein bound to the fourth binding site remains bound to the cleavage polynucleotide end of the donor nucleotide region (i.e., the downstream end of the donor nucleotide region). Without being bound by any particular theory, the dimerization of the fusion protein bound to the first and third binding sites (e.g., by the dimerization of Trex2) will bring the cleavage polynucleotide end upstream of the target region into close proximity with the upstream end of the donor nucleotide region, and the dimerization of the fusion protein bound to the second and fourth binding sites will bring the cleavage polynucleotide end downstream of the target region into close proximity with the downstream end of the donor nucleotide region. Connection of the closely approached polynucleotide ends by DNA repair mechanisms (such as NHEJ or MMEJ) will result in replacement of the target region with the donor nucleotide region.The donor nucleotide region can also be inserted in the reverse orientation (ie, if the fusion protein that binds to the first and fourth binding sites dimerizes and the fusion protein that binds to the second and third binding sites dimerizes). Figure 3A In , the donor nucleotide region does not contain homology arms, so NHEJ or MMEJ repair is more likely than HDR. Figure 3B In the embodiment, the donor nucleotide region comprises homology arms. Without being bound by any particular theory, dimerization of the fusion protein bound to the first and third binding sites and dimerization of the fusion protein bound to the second and fourth binding sites will promote HDR-mediated repair by bringing the donor nucleotide region homology arms into close proximity with homologous sequences in the nucleotide to be edited (e.g., genomic DNA).

[0020] Figure 4Shown is the stepwise cutting and translocation of chromosomes according to aspects of the present disclosure.Two fusion proteins are bound to the first binding site and the second binding site on the opposite strands of DNA polynucleotides (i.e., nucleotides to be edited, shown here as "acceptor chromosomes"), and two fusion proteins are bound to the third binding site and the fourth binding site on the opposite strands of donor nucleic acids (shown here as "donor chromosomes").The first binding site is adjacent to the 5' end of the target region (i.e., the region is between nuclease cleavage sites), the second binding site is adjacent to the 3' end of the target region, the third binding site is adjacent to the 5' end of the donor nucleotide region (i.e., the region of the donor nucleotide is between nuclease cleavage sites), and the fourth binding site is adjacent to the 3' end of the donor nucleotide region.For illustrative purposes, the fusion proteins in these embodiments include LbCas12a as SDN connected to Trex2 exonucleases as non-specific end processing enzymes. LbCas12a (i.e., the SDN of the fusion protein) cleaves polynucleotides (i.e., both nucleotides to be edited and donor nucleic acids) from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA-a") is upstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the first binding site remains bound to the cleavage polynucleotide end outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA-b") is downstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the second binding site remains bound to the cleavage polynucleotide end outside the target region and downstream thereof. Because the third binding site (indicated by "gRNA-c") is upstream of the cleavage site (i.e., outside the donor nucleotide region), the fusion protein bound to the third binding site remains bound to the cleavage donor nucleotide end outside the donor nucleotide region and upstream thereof. Because the fourth binding site (indicated by "gRNA-d") is downstream of the cleavage site (i.e., outside the donor nucleotide region), the fusion protein bound to the fourth binding site remains bound to the cleavage donor nucleotide end outside the donor nucleotide region and downstream thereof. Without being bound by any particular theory, the dimerization of the fusion protein bound to the first binding site and the fourth binding site (e.g., by the dimerization of Trex2) will bring the cleavage polynucleotide end upstream of the target region into close proximity with the cleavage polynucleotide end downstream of the donor nucleotide region, and the dimerization of the fusion protein bound to the second binding site and the third binding site will bring the cleavage polynucleotide end downstream of the target region into close proximity with the cleavage polynucleotide end upstream of the donor nucleotide region. The connection of the closely approached polynucleotide ends by DNA repair mechanisms (such as NHEJ or MMEJ) will result in the translocation of the polynucleotide (and the excision of the target region and the donor nucleotide region).

[0021] Figure 5The expected outcome of editing according to aspects of the present disclosure is shown, wherein the guide RNA molecule ("gRNA") is designed to bind on the same strand of the target DNA polynucleotide sequence and in the same orientation. In this figure, in the first row, the gRNA targets the sequence in the promoter and the first intron of the target gene to be edited. In the second row, the excision of the intervening sequence between the gRNA binding sites is illustrated, thereby destroying the gene. In the third row, an alternative outcome is illustrated, wherein the intervening sequence is inverted and connected in the reverse orientation, thereby destroying the gene.

[0022] Figure 6 A comparison of Cas9 and Cas12a cutting according to aspects of the present disclosure is shown. Compared with Cas9, it is more difficult to use Cas12a to excise or invert the genomic sequence between the paired guide RNA targeting sites. This can be explained by the following phenomenon: Cas12a produces double-strand breaks with sticky ends, which may be more difficult to separate from each other than the blunt ends produced by Cas9. In order to improve the frequency of fragment excision and inversion, various DNA exonucleases are fused to LbCas12a to trim and detach the sticky ends immediately after LbCas12a produces double-strand breaks.

[0023] Fig. 7A and 7B An example according to aspects of the present disclosure is shown, in which two gRNAs targeting opposite strands are selected to excise a genomic fragment between the two targeting sites. Fig. 7A In, two gRNA pairs were selected to test the prediction that the binding site on the opposite strand outside the target region can further increase the excision frequency induced by LbCas12a-Trex2. In order to design such gRNA pairs, ZmDMR6-crRNA1 was retained and paired with two gRNAs (ZmDMR6-crRNA3 (SEQ ID NO: 23) and ZmDMR6-crRNA4 (SEQ ID NO: 24)) whose targets are downstream of the target of ZmDMR6-crRNA2 and on complementary strands. The expected excision in the case of gRNA pairing ZmDMR6-crRNA1 and ZmDMR6-crRNA3 is 1233bp. The expected excision in the case of gRNA pairing ZmDMR6-crRNA1 and ZmDMR6-crRNA4 is 1387bp. In Figure 7BIn the present invention, a gRNA pair was selected to test whether a binding site on the opposite strand within the target region could significantly increase the frequency of excision. ZmWaxy1-crRNA1 (SEQ ID NO: 25) was designed to target a sequence adjacent to the TTTG PAM in exon 4 on the coding strand, while ZmWaxy1-crRNA5 (SEQ ID NO: 26) was designed to target a sequence adjacent to the TTTA PAM in the promoter region on the complementary strand. The expected excision in the case of the gRNA pairing ZmWaxy1-crRNA1 and ZmWaxy1-crRNA5 was 1.1 kb.

[0024] Figure 8 The gRNA design for editing genes in soybean according to aspects of the present disclosure is shown. The first gRNA, GmFAD2A-crRNA1 (SEQ ID NO: 28) is designed to target the sequence adjacent to the TTTG PAM in the FAD2-1A promoter region on the coding strand. The second gRNA, GmFAD2A-crRNA2 (SEQ ID NO: 29) is designed to target the sequence adjacent to the TTTG PAM near the 3' splice site of the first intron on the coding strand. The distance between crRNA1 and crRNA2 target sites is about 1.1kb. The third gRNA, GmFAD2A-crRNA3 (SEQ ID NO: 30) is designed to target the sequence adjacent to the TTTG in the second exon on the complementary strand. The distance between crRNA1 and crRNA3 target sites is about 1.2kb.

[0025] Fig. 9 The design of the donor sequence according to aspects of the present disclosure is shown, and the donor design includes a left homology arm and a right homology arm to promote HDR. The 458-bp genomic sequence (SEQ ID NO:48) upstream of the PAM of the ZmSH1-crRNA2 target is selected as a left homology arm (LHA) to mediate HDR, and is added to the 5' end of the cargo sequence. The 509-bp genomic sequence (SEQ ID NO:49) downstream of the PAM of the ZmSH1-crRNA2 target is selected as a right homology arm (RHA), and is added to the 3' end of the cargo sequence. The target sequence of ZmSH1-crRNA1 and ZmSH1-crRNA2 (both have the TTTGPAM added to the 5' end) is added to the 5' end of the left homology arm and the 3' end of the right homology arm respectively and added in such a way that the crRNA binding site is on the opposite strand, but in the donor nucleotide region, to produce donor DNA. DETAILED DESCRIPTION

[0026] The following description lists various aspects and embodiments of the compositions and methods of the present invention. The specific embodiments are not intended to limit the scope of the compositions and methods. Instead, the embodiments merely provide non-limiting examples of various compositions and methods at least within the scope of the disclosed compositions and methods. The description should be read from the perspective of a person of ordinary skill in the art; therefore, information familiar to a skilled person is not necessarily included. I. Terminology

[0027] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. Reference to the technology employed herein is intended to refer to the technology commonly understood in the art, including variations of those technologies and / or substitutes for equivalent technologies that are clear to those of ordinary skill in the art. Although it is believed that the following terms may be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the interpretation of the subject matter disclosed in the present invention.

[0028] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an enzyme" optionally includes combinations of two or more such molecules, etc.

[0029] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0030] As used herein, the term "about" refers to the common error range of the corresponding value that is readily known to those skilled in the art, for example, ±20%, ±10% or ±5% within the intended meaning of the recited value.

[0031] As used herein, the term "comprising" or "comprises" is open ended. When used in conjunction with a subject nucleic acid (or amino acid sequence), it refers to a nucleic acid sequence (or amino acid sequence) that includes the subject sequence as a part or as its entire sequence.

[0032] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is interpreted to include the specified materials or steps recited in the claim and those materials or steps that do not materially affect one or more basic and novel characteristics of the claimed subject matter. Therefore, when used in the claims of the present disclosure, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0033] The term "plurality" refers to more than one entity. Thus, a "majority of individuals" refers to at least two individuals. In some embodiments, the term majority refers to more than half of a whole. For example, in some embodiments, a "majority of a group" refers to more than half of the members of that group.

[0034] As used herein, the term "plant" refers to any plant, particularly seed plants, in any developmental stage. As used herein, the term "plant cell" is the structural and physiological unit of a plant, including protoplasts and cell walls. Plant cells can be in the form of isolated single cells or cultured cells, or as a part of a higher organized unit (such as, for example, plant tissue, plant organ or whole plant). Plant cells can be derived from angiosperms or gymnosperms or be a part of them. Plant cells can be monocotyledonous plant cells (for example, corn cells, rice cells, sorghum cells, sugarcane cells, barley cells, wheat cells, oat cells, turf grass cells or ornamental grass cells) or dicotyledonous plant cells (for example, tobacco cells, pepper cells, eggplant cells, sunflower cells, cruciferous plant cells, flax cells, potato cells, cotton cells, soybean cells, sugar beet cells or oilseed rape cells). As used herein, the term "plant cell culture" means a culture of plant units (such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at different developmental stages). As used herein, the term "plant tissue" refers to a group of plant cells organized into structural and functional units. Including any plant tissue in a plant or in culture. This term includes but is not limited to whole plants, plant organs, plant seeds, tissue cultures, and any plant cell groups organized into structural and / or functional units. This term is combined with any specific type of plant tissue listed above (or when it does not exist) or the use of other methods covered by this definition is not intended to exclude any other type of plant tissue. As used herein, the term "plant part" refers to a part of a plant, including single cells and cell tissues (such as complete plant cells in plants), cell masses, and tissue cultures that can regenerate plants. Examples of plant parts include, but are not limited to, single cells and tissues from: pollen, ovules, zygotes, leaves, embryos, roots, root tips, anthers, flowers, floral parts, fruits, stems, buds, cuttings, and seeds; as well as pollen, ovules, egg cells, zygotes, leaves, embryos, roots, root tips, anthers, flowers, floral parts, fruits, stems, buds, cuttings, scions, rhizomes, seeds, protoplasts, callus, etc.

[0035] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0036] The terms "nucleic acid" and "polynucleotide" are used interchangeably and as used herein, refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and polymers thereof, and refer to both the sense and antisense strands of RNA, cDNA, genomic DNA, mitochondrial DNA, and synthetic forms and mixed polymers of the above items. In higher plants, DNA is the genetic material, and RNA is involved in the transfer of information contained in DNA into proteins. "Genome" is the entirety of the genetic material contained in each cell of an organism. It should be understood that when RNA is described, its corresponding cDNA is also described, wherein uridine is represented as thymidine. In specific embodiments, nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotides and combinations thereof. In addition, the polynucleotides disclosed herein may include either or both of naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, tags, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side groups (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformations, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin-shaped, circular, and padlock-shaped conformations. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complement. Therefore, reference to a nucleic acid molecule with a specific sequence is understood to encompass its complementary chain with its complementary sequence. When nucleotide sequences specifically hybridize in a solution, these nucleotide sequences are "complementary" (e.g., according to Watson-Crick base pairing principles). The term also includes codon-optimized nucleic acids encoding the same polypeptide sequence. It is also understood that the nucleic acid can be unpurified, purified, or attached to, for example, a synthetic material such as a bead or column matrix.

[0037] In the context of nucleic acid sequences, the term "corresponding to" means that when the nucleic acid sequences of certain sequences are aligned with each other, the nucleic acids "corresponding to" certain enumerated positions in the present invention are those aligned with these positions in the reference sequence, but are not necessarily located in these precise numerical positions relative to the specific nucleic acid sequence of the present invention. The best alignment of sequences for comparison can be performed by computerized implementations of known algorithms or by visual inspection. Easily available sequence comparison and multiple sequence alignment algorithms are the basic local alignment search tool (BLAST) and ClustalW / ClustalW2 / Clustal Omega programs available on the Internet (e.g., the website of EMBL-EBI), respectively. Other suitable programs include, but are not limited to, GAP, BestFit, Plot Similarity and FASTA, which are part of the Accelrys GCG software package available from Accelrys (San Diego, California, USA). See also Smith and Waterman, 1981; Needleman and Wunsch, 1970; Pearson and Lipman, 1988; Ausubel et al., 1988; and Sambrook and Russell, 2001.

[0038] Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly specified. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. See Batzer et al., Nucleic Acid Res. [Nucleic Acids Research] 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. [Journal of Biological Chemistry] 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes [Molecular Cell Probes] 8: 91-98 (1994).

[0039] As used in the context of polynucleotide or polypeptide sequences described herein, the term "identity" or "substantial identity" refers to a sequence having at least 60% sequence identity with a reference sequence. Alternatively, the percent identity can be any integer from 60% to 100%. Exemplary embodiments include: using the programs described herein, preferably BLAST using standard parameters as described below, such as at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence. Those skilled in the art will recognize that these values ​​can be appropriately adjusted by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc., in order to determine the corresponding identity of proteins encoded by two nucleotide sequences.

[0040] For sequence comparison, typically, a kind of sequence serves as the reference sequence compared with the test sequence.When using the sequence comparison algorithm, the test sequence and the reference sequence are input into the computer, and the subsequence coordinates are specified if necessary, and the sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified.Then, the sequence comparison algorithm will calculate the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0041] As used herein, "comparison window" includes reference to a segment having any one of the number of consecutive positions selected from the group consisting of from 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Waterman Add. APL. Math. [Advances in Applied Mathematics] 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. [Journal of Molecular Biology] 48:443 (1970); by the search by similarity method of Pearson and Lipman Proc. Natl. Acad. Sci. [Proceedings of the National Academy of Sciences] (USA) 85:2444 (1988); by computer implementations of these algorithms (e.g., BLAST); or by manual alignment and visual inspection.

[0042] Algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI). The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initial searches to find longer HSPs containing them. These word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues; always>0) and N (penalty score for mismatched residues; always<0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction terminates when the following situations occur: the cumulative alignment score drops by the amount X from its maximum obtained value; the cumulative score moves to zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of any sequence is reached. The parameters W, T, and X of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 28, an expectation value (E) of 10, M=1, N=-2, and a comparison of two chains as default values. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expectation value (E) of 10, and a BLOSUM62 scoring matrix as default values. See Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989).

[0043] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences. See, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the smallest sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.01, more preferably less than about 10, -5 and most preferably less than about 10 -20 , the nucleic acid is considered similar to the reference sequence.

[0044] "Recombination" is the exchange of DNA strands to produce new nucleotide sequence arrangements. The term can refer to the homologous recombination process that occurs in the repair of double-stranded DNA breaks, in which polynucleotides are used as templates to repair homologous polynucleotides. The term can also refer to the information exchange between two homologous chromosomes during meiosis. The frequency of double recombination is the product of the frequency of single recombinants. For example, the frequency of recombinants found in a 10cM region is 10%, and the frequency of double recombinants is found to be 10% x 10% = 1% (1 centimorgan is defined as 1% of the recombinant generations in the test hybridization).

[0045] "Gene" is a defined region located within a genome, and in addition to the aforementioned coding nucleic acid sequence, it also contains other major regulatory nucleic acid sequences responsible for controlling the expression (i.e., transcription and translation) of the coding portion. A gene may include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and 5' and 3' untranslated regions). Genes typically express mRNA, functional RNA, or specific proteins, including regulatory sequences. Genes may or may not be used to produce functional proteins. In some embodiments, a gene refers only to a coding region. The term "natural gene" refers to a gene as found in nature. The term "chimeric gene" refers to any gene comprising the following: 1) a DNA sequence, including regulatory sequences and coding sequences that are not found together in nature, or 2) a sequence encoding a portion of a protein that is not naturally adjacent, or 3) a portion of a promoter that is not naturally adjacent. Therefore, a chimeric gene may include regulatory sequences and coding sequences obtained from different sources, or regulatory sequences and coding sequences obtained from the same source but arranged in a different manner from that found in nature. A gene may be "isolated," which means a nucleic acid molecule that is substantially or essentially free of components normally found associated with the nucleic acid molecule in its natural state. Such components include other cellular material, culture medium from recombinant production, and / or various chemicals used in the chemical synthesis of nucleic acid molecules.

[0046] "Target gene" or "target nucleotide sequence" refers to any gene that, when transferred to a plant, confers a desired characteristic on the plant (such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, improved nutritional value, improved performance of industrial processes, or altered breeding capacity). "Target gene" can also be a gene transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0047] "Isolated" nucleic acid molecules or nucleotide sequences or "isolated" polypeptides are nucleic acid molecules, nucleotide sequences or polypeptides that exist by artificially detaching from their natural environment and / or have different, modified, regulated and / or changed functions when compared with their functions in their natural environment and are therefore not natural products.Isolated nucleic acid molecules or isolated polypeptides can exist in purified form or can exist in non-natural environments (such as, for example, recombinant host cells).Therefore, for example, with respect to polynucleotides, the term isolated means that polynucleotides are separated from chromosomes and / or cells in which they are naturally present.If a polynucleotide is separated from chromosomes and / or cells in which it is naturally present, and then inserted into a genetic background, chromosome, chromosomal position and / or cell in which it is not naturally present, the polynucleotide is also isolated.Recombinant nucleic acid molecules and nucleotide sequences of the present invention can be considered as "isolated" as defined above.

[0048] Thus, an "isolated nucleic acid molecule" or "isolated nucleotide sequence" is a nucleic acid molecule or nucleotide sequence that is not immediately adjacent to a nucleotide sequence (either a sequence at the 5' end or a sequence at the 3' end) that is immediately adjacent to it in the naturally occurring genome of the organism from which it is derived. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences that are immediately adjacent to the coding sequence. Thus, the term includes, for example, recombinant nucleic acids that are incorporated into a vector, into a self-replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exist as a separate molecule (e.g., a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment) that is independent of other sequences. It also includes recombinant nucleic acids that are part of a hybrid nucleic acid molecule encoding another polypeptide or peptide sequence. An "isolated nucleic acid molecule" or "isolated nucleotide sequence" may also include a nucleotide sequence that is derived from and inserted into the same natural original cell type, but is present in a non-natural state, for example, in a different copy number, and / or under the control of regulatory sequences that are different from those found in the natural state of the nucleic acid molecule.

[0049] The term "isolated" may further refer to a nucleic acid molecule, nucleotide sequence, polypeptide, peptide or fragment that is substantially free of cellular material, viral material and / or culture medium (e.g., when produced by recombinant DNA techniques), or chemical precursors or other chemicals (e.g., when chemically synthesized). In addition, an "isolated fragment" is a fragment of a nucleic acid molecule, nucleotide sequence or polypeptide that does not naturally exist as a fragment and would not exist as such in nature. "Isolated" does not necessarily mean that the preparation is industrially pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form that can be used for the intended purpose.

[0050] "Homology-dependent repair" or "homologous directed repair" or "HDR" refers to a mechanism for repairing ssDNA and double-stranded DNA (dsDNA) damage in cells. This repair mechanism can be used by cells in the presence of an HDR template with a sequence that has significant identity to the site of damage. The term "perfect HDR" refers to a situation in which the genomic homology junction in the replaced allele is subjected to complete HDR, and "imperfect HDR" refers to a situation in which the genomic homology junction in the replaced allele is subjected to partial or incomplete HDR. A donor DNA molecule having homology to the cleavage target DNA sequence is used as a template for repairing the cleavage target DNA sequence, thereby generating a transfer of genetic information from the donor polynucleotide to the target DNA. In this way, new nucleic acid material can be inserted / copied into the site. In some cases, the target DNA is contacted with a donor molecule (e.g., a donor DNA molecule). In some cases, the donor DNA molecule is introduced into the cell. In some cases, at least one segment of the donor DNA molecule is integrated into the genome of the cell.

[0051] "Microhomology-mediated end joining" or "MMEJ" or "alternative non-homologous end joining" (Alt-NHEJ) refers to a form of repairing double-strand breaks in DNA. This repair mechanism uses microhomology sequences to align the broken strands. "Non-homologous end joining" or "NHEJ" refers to a form of repairing double-strand breaks in DNA. Double-strand breaks are repaired by direct ligation of the broken ends to each other. Generally, no new nucleic acid material is inserted into the site, but some nucleic acid material may be lost or added, resulting in small deletions or small insertions. II. Introduction

[0052] Provided herein are fusion proteins and related recombinant nucleic acids, systems and methods for increasing the efficiency of genome editing using SDN by inversion, excision and HDR performed using fusion proteins and donor DNA ligation methods. This disclosure is based in part on the following findings of the inventors: the fusion of SDN with a non-specific end processing enzyme (e.g., a non-specific exonuclease) results in an increase in the frequency of desired editing outcomes (such as inversion of genomic fragments between two targeted SDN-induced double-strand breaks (DSBs)), as demonstrated in Example 1 herein. It is generally believed that the specific cellular mechanism for DSB repair depends on the properties of the DNA ends generated by the DSB (e.g., blunt ends vs. sticky ends) and / or the level of end processing that occurs (e.g., whether a chain is excised). DSBs where end excision does not occur are usually repaired by classical non-homologous end joining (C-NHEJ). C-NHEJ is considered an "error-prone" approach because it leads to the formation of small insertions and small deletions in some cases. However, if end resection does occur, the end of the DSB may include one or more overhangs (e.g., 3' overhangs or 5' overhangs), which may interact with nearby homologous sequences. The mechanism of repairing DSBs can vary depending on the extent of processing. When the ends of the DSB are subjected to relatively limited end resection, the DSB is usually processed by alternative non-homologous end joining (ALT-NHEJ). ALT-NHEJ refers to a class of pathways including blunt-end joining (blunt EJ) and microhomology-mediated end joining (MMEJ) that tend to cause deletions, as well as synthesis-dependent microhomology-mediated end joining (SD-MMEJ) that tend to cause insertions. However, when end resection is extensive, the resulting overhangs can be subjected to chain invasion of highly homologous sequences (which may be endogenous sequences or heterologous sequences), followed by DSB repair by homology-dependent recombination (HDR) pathway. Without being bound by any particular theory, the fusion proteins provided herein may increase the frequency of desired editing outcomes by coupling DSB formation to the desired type and / or level of end processing to bias DSB repair toward a particular pathway.

[0053] The present disclosure is also based in part on the following findings of the inventors: the use of fusion proteins capable of dimerization can increase the frequency of desired editing outcomes. As further discussed herein, the fusion protein can remain bound to its nucleic acid target after DSB formation. In addition, the fusion protein can be targeted to remain bound to a portion of the nucleic acid target upstream or downstream of the DSB cleavage site. When two or more fusion proteins capable of forming dimers are used, the polynucleotide ends to which the fusion proteins are bound are brought into close proximity. Without being bound by any particular theory, this close proximity may affect the likelihood that a specific DSB repair pathway will be used. In addition, the inventors have shown that by adjusting the targeting of the fusion protein, DSB repair can be biased toward different outcomes (e.g., excision of the target fragment, inversion of the target fragment, or HDR using a donor template). III. Fusion Protein

[0054] On the one hand, fusion proteins are provided herein, which include site-directed nucleases connected to non-specific terminal processing enzymes. As used herein, "fusion protein" is a protein comprising two different polypeptide sequences connected (join) or connected (link) to form a single polypeptide, i.e., a site-directed nuclease polypeptide sequence and a non-specific terminal processing enzyme polypeptide sequence. In some embodiments, the two amino acid sequences are encoded by separate nucleic acid sequences, and these separate nucleic acid sequences have been connected so that they are transcribed and translated to produce a single polypeptide. Site-directed nucleases and non-specific terminal processing enzymes can be connected in any order and orientation relative to each other. For example, the C' end of the site-directed nuclease can be connected to the N' end or C' end of the non-specific terminal processing enzyme. Site-directed nucleases and non-specific terminal processing enzymes can be separated by one or more additional fusion protein domains, as described below. A. Site-directed nuclease

[0055] The fusion proteins provided herein include site-directed polypeptides. "Site-directed modifying polypeptides" modify target DNA (e.g., via cleavage or methylation of target DNA) and / or polypeptides associated with target DNA (e.g., methylation or acetylation of histone tails). In some embodiments, due to the association of the site-directed modifying polypeptide with the guide RNA, the site-directed modifying polypeptide interacts with the guide RNA (the guide RNA is a single RNA molecule or an RNA duplex of at least two RNA molecules) and is guided to a DNA sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, such as an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.). In some embodiments, the site-directed polypeptide is a site-directed nuclease that is capable of cleaving one or both chains of DNA at a specified target sequence.

[0056] The term "cleavage" or "cleaving" refers to the break of the covalent phosphodiester linkage in the ribosylphosphodiester backbone of a polynucleotide, and encompasses both single-strand breaks and double-strand breaks. As a result of two different single-strand cleavage events, double-strand cleavage can occur. Cleavage can result in the production of blunt ends or staggered ends (also referred to as sticky ends). "Nuclease cleavage site" or "genomic nuclease cleavage site" is a region of nucleotides where a site-directed nuclease cleaves (e.g., when combined with a proximal binding site). When a polynucleotide is DNA (e.g., genomic DNA), one or two chains can be cleaved at the nuclease cleavage site. This cleavage performed by a nuclease triggers a DNA repair mechanism in the cell, which establishes an environment in which homologous recombination occurs.

[0057] A variety of site-directed nucleases can be used in the fusion proteins, systems and methods disclosed herein. Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); meganucleases; RNA binding proteins (RBPs); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), eukaryotic Argonaute (eAgo), and Natronobacterium gregoryi Argonaute (NgAgo); adenosine deaminases acting on RNA (ADARs); CRISPR-Cas stimulated RNA targeting (CIRT) systems; Pumilio / fem-3 binding factors (PUFs), homing endonucleases, or any functional fragments thereof, any derivatives thereof; any variants thereof; and any fragments thereof. Exemplary site-directed nucleases suitable for use in the fusion proteins, systems, and methods disclosed herein are further described below.

[0058] In some embodiments, the site-directed nuclease is a naturally occurring site-directed nuclease. Exemplary naturally occurring site-directed nucleases are known in the art (see, e.g., Makarova et al., 2017, Cell 168:328-328.e1 and Shmakov et al., 2017, Nat Rev Microbiol 15(3):169-182, both of which are incorporated herein by reference). In some embodiments, the site-directed nuclease binds to a DNA-targeting polynucleotide (e.g., a guide RNA) and is thereby directed to a specific sequence within the target DNA and cleaves the target DNA.

[0059] In some embodiments, the site-directed nuclease is modified relative to its native sequence (e.g., via mutation of one or more amino acid residues) to change its function. For example, the site-directed nuclease can be modified to be enzymatically inactive. The term "enzymatically inactive" can mean that the site-directed nuclease can bind to the nucleic acid sequence in the polynucleotide in a sequence-specific manner, but cannot cleave the target polynucleotide. The enzymatically inactive site-directed polypeptide can include an enzymatically inactive domain (e.g., a nuclease domain). Enzymatically inactive can refer to inactivity. Enzymatically inactive can refer to substantially inactive. Enzymatically inactive can refer to substantially inactive. Enzymatically inactive can refer to an activity that is no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, or no more than 10% active compared to a wild-type exemplary activity (e.g., nucleic acid cleavage activity, wild-type Cas9 activity).

[0060] In some embodiments, a site-directed nuclease (e.g., a site-directed nuclease without enzymatic activity) is fused to one or more transcription repressor domains, activator domains, epigenetic domains, recombinase domains, transposase domains, flippase domains, nickase domains, cleavage domains, or any combination thereof. The activator domain may include one or more tandem activation domains located at the carboxyl terminus of the enzyme. In other cases, the actuator moiety includes one or more tandem repressor domains located at the carboxyl terminus of the protein. Non-limiting exemplary activation domains include GAL4, herpes simplex activation domain VP16, VP64 (tetramer of herpes simplex activation domain VP16), NF-KB p65 subunits, Epstein-Barr virus R transactivator (Rta) and are described in Chavez et al., Nat Methods [Natural Methods], 2015, 12 (4): 326-328 and U.S. Patent Application Publication No. 20140068797. Non-limiting exemplary repressor domains include the KRAB (Kruppel associated box) domain of Kox1, the Mad mSIN3 interaction domain (SID), the ERF repressor domain (ERD), and are described in Chavez et al., Nat Methods, 2015, 12(4):326-328 and U.S. Patent Application Publication No. 20140068797. Nucleases can also be fused to heterologous polypeptides to provide increased or decreased stability. The fusion domain or heterologous polypeptide can be located at the N-terminus, C-terminus, or internal to the nuclease. CRISPR / Cas nucleases

[0061] In some embodiments, the site-directed nuclease comprises a CRISPR-associated (Cas) protein or a Cas nuclease that functions in a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas system. In bacteria, this system can provide adaptive immunity against foreign DNA (Barrangou, R. et al., “CRISPR provides acquired resistance against viruses in prokaryotes,” Science (2007) 315:1709-1712; Makarova, KS et al., “Evolution and classification of the CRISPR-Cas systems,” Nat Rev Microbiol (2011) 9:467-477; Garneau, JE et al., “The CRISPR / Cas bacterial immune system cleaves bacteriophage and plasmid DNA,” Nature (2010) 468:67-71; Sapranauskas, R. et al., “The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichiacoli [Thermophilus Streptococcus CRISPR / Cas system provides immunity in Escherichia coli]," Nucleic Acids Res [Nucleic Acids Research] (2011) 39:9275-9282). CRISPR / Cas systems (e.g., modified and / or unmodified) can be used as genome engineering tools in a variety of organisms (including different mammals, animals, plants, microorganisms and yeast). CRISPR / Cas systems can include guide nucleic acids such as guide RNA (gRNA), which are complexed with Cas proteins for targeted regulation of gene expression and / or activity or nucleic acid editing. RNA-guided Cas proteins (e.g., Cas nucleases such as Cas9 nucleases) can specifically bind to target polynucleotides (e.g., DNA) in a sequence-dependent manner.Cas proteins can cleave DNA if they have nuclease activity (Gasiunas, G. et al., "Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria," Proc Natl Acad Sci USA (2012) 109: E2579-E286; Jinek, M. et al. "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity," Science (2012) 337: 816-821; Sternberg, SH et al. "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 [DNA interrogation by CRISPR RNA-guided endonuclease Cas9]," Nature (2014) 507:62; Deltcheva, E. et al., "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III," Nature (2011) 471:602-607). DNA cleavage (e.g., double-strand break) can result in DNA break repair, thereby allowing the introduction of one or more genetic modifications (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR). In some embodiments, donor nucleic acids are used to promote HDR, as described in detail below in the "System" section.CRISPR-Cas systems have been widely used for programmable genome editing in a variety of organisms and model systems (Cong, L. et al. "Multiplex genome engineering using CRISPR Cas systems," Science (2013) 339:819-823; Jiang, W. et al. "RNA-guided editing of bacterial genomes using CRISPR-Cas systems," Nat. Biotechnol. (2013) 31:233-239; Sander, JD and Joung, JK, "CRISPR-Cas systems for editing, regulating and targeting genomes," Nature Biotechnol. (2014) 32:347-355).

[0062] In some embodiments, the site-directed nuclease described herein includes a Cas protein that forms a complex with a guide nucleic acid (such as a guide RNA) (further described below in the "system" section). In some embodiments, the site-directed nuclease includes a Cas protein that forms a complex with a single guide nucleic acid (such as a single guide RNA (sgRNA)). In some embodiments, the site-directed nuclease includes an RNA binding protein (RBP) that is optionally compounded with a guide nucleic acid (such as a guide RNA (e.g., sgRNA)), and the RNA binding protein can form a complex with the Cas protein. In some cases, the RNA-guided Cas protein recognizes a DNA target that is complementary to a portion of a gRNA (referred to as a CRISPR RNA (crRNA) sequence). The target sequence is often referred to as a protospacer, and the crRNA sequence portion that is complementary to the protospacer is often referred to as a spacer. In order to work (e.g., to cleave DNA), many Cas nucleases also require a specific protospacer adjacent motif (PAM) (a DNA sequence of about 2 to 6 base pairs) immediately following the protospacer sequence.

[0063] Various site-directed Cas nucleases (e.g., Cas proteins from different species) can be used in the fusion proteins, systems and methods provided herein based on various enzyme features of different Cas proteins (e.g., different protospacer adjacent motif (PAM) sequence preferences; increased or decreased enzyme activity; increased or decreased cytotoxicity levels; a tendency to cause one or more of NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.). Cas proteins from various species (e.g., those disclosed in Shmakov et al., 2017, or polypeptides derived therefrom) may require different PAM sequences in the target DNA. Therefore, for a specific Cas enzyme selected, the PAM sequence requirement may be different from the 5'-NGG-3' sequence (wherein N is A, T, C, or G) known to be required for Cas9 activity. Many Cas9 orthologs from a variety of species have been identified, and these proteins share only a few identical amino acids. All identified Cas9 orthologs have the same domain architecture as the central HNH endonuclease domain and the split-type RuvC / RNase H domain. Cas9 proteins share 4 key motifs with conserved structures; motifs 1, 2, and 4 are RuvC-like motifs, while motif 3 is an HNH motif. In contrast, Cas12a proteins from different species may have different PAM sequence requirements compared to the LbCas12a canonical PAM of TTTV.

[0064] Any suitable CRISPR / Cas system can be used. A variety of naming systems can be used to refer to the CRISPR / Cas system. Exemplary naming systems are provided in Makarova, KS et al., "An updated evolutionary classification of CRISPR-Cas systems [updated evolutionary classification of CRISPR-Cas systems]," Nat Rev Microbiol [Natural Review: Microbiology] (2015) 13: 722-736 and Shmakov, S. et al., "Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems [discovery and functional characterization of different 2 types of CRISPR-Cas systems]," Mol Cell [Molecular Cell] (2015) 60: 1-13. The CRISPR / Cas system can be a type I, type II, type III, type IV, type V, type VI system or any other suitable CRISPR / Cas system. As used herein, the CRISPR / Cas system can be a class 1, class 2 or any other suitable classification CRISPR / Cas system. Class 1 or class 2 determinations can be based on genes encoding effector modules. Class 1 systems typically have multi-subunit crRNA-effector complexes, while Class 2 systems typically have single proteins, such as Cas9, Cpf1, C2c1, C2c2, C2c3 or crRNA-effector complexes. Class 1 CRISPR / Cas systems can use a complex of multiple Cas proteins to achieve regulation. Class 1 CRISPR / Cas systems may include, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g., III, IIIA, IIIB, IIIC, IIID) and type IV (e.g., IV, IVA, IVB) CRISPR / Cas types. Class 2 CRISPR / Cas systems may use a single large Cas protein to achieve regulation. Class 2 CRISPR / Cas systems may include, for example, type II (e.g., II, IIA, IIB) and type V CRISPR / Cas types. CRISPR systems may complement each other, and / or may promote CRISPR locus targeting with the aid of trans functional units.

[0065] The Cas protein can be from any suitable organism. Non-limiting examples include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum), Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas nap hthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus varrooxidans watsoni), Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngby asp., Microcoleus chthonoplastes, Oscillatoria sp.), Petrotoga mobilis, Thermosiphoafricanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some aspects, the organism is Streptococcus pyogenes (S. pyogenes). In some aspects, the organism is Staphylococcus aureus (S. aureus). In some aspects, the organism is Streptococcus thermophilus (S. thermophilus). .

[0066] Cas proteins can be derived from a variety of bacterial species, including but not limited to Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum), Lactobacillus rhamnosus, Lactobacillus gasseri, Fingoldella grandis, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp.Torquens), Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris palustris), Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobactereiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinella succinogenes, Campylobacter jejuni subsp.Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes and Francisella novicida.

[0067] Non-limiting examples of Cas proteins include c2c1, C2c2, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Cpf1, Csyl, Csy2, Csy3, Csel (CasA) , Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4 and Cul966, and homologs or modified forms thereof. In some embodiments, the site-directed nuclease of the fusion protein provided herein includes a CRISPR-associated nuclease, wherein the CRISPR-associated nuclease is Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b or Cas14. In some embodiments, the CRISPR-associated nuclease is a Cas9 enzyme. In some embodiments, the CRISPR-associated nuclease is a Cas12a enzyme. In some embodiments, the CRISPR-associated nuclease is a nickase or an inactivated form of a CRISPR-associated nuclease.

[0068] Lachnospiraceae bacterium Cpf1 (LbCpf1) is one of a large group of many Cpf1 proteins. The terms "Cpf1" and "Cas12a" are used interchangeably throughout this disclosure. Cpf1 is a Cas protein. In some embodiments, the site-directed nuclease is a catalytically inactive Cas12a ("dLbCas12a") from Lachnospiraceae bacteria. In other embodiments, the site-directed nuclease is a catalytically active Cas12a from Lachnospiraceae bacteria ("LbCas12a") or Moraxella bovoculi AAX08_00205 ("Mb2Cas12a"). In some embodiments, the site-directed nuclease domain of the fusion protein is a Cas12a protein from any of the following: Lachnospiraceae, Acidaminococcus sp., Moraxella tauroides, Thiomicrospira sp., Moraxella lacunata, Methanomethylophilus alvus, Btyrivibrio sp., or Bacteroidetesoral sp.

[0069] Cas protein can comprise one or more domains.Non-limiting examples of domains include guiding nucleic acid recognition and / or binding domains, nuclease domains (e.g., DNA enzyme or RNA enzyme domains, RuvC, HNH), DNA binding domains, RNA binding domains, helicase domains, protein-protein interaction domains and dimerization domains.Guiding nucleic acid recognition and / or binding domains can interact with guiding nucleic acids. Nuclease domains can include catalytic activity for nucleic acid cleavage. Nuclease domains can lack catalytic activity to prevent nucleic acid cleavage. Cas protein can be a chimeric Cas protein fused with other proteins or polypeptides. Cas protein can be a chimera of various Cas proteins, for example, comprising domains from different Cas proteins.

[0070] The Cas protein used herein can be an active variant, an inactive variant or a fragment of a wild-type or modified Cas protein. Relative to the wild-type form of the Cas protein, the Cas protein can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras or any combination thereof. The Cas protein can be a polypeptide having at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or sequence similarity with a wild-type exemplary Cas protein. The Cas protein can be a polypeptide having at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and / or sequence similarity with a wild-type exemplary Cas protein. Variants or fragments may comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity or sequence similarity to a wild-type or modified Cas protein or portion thereof. Variants or fragments may be targeted to a nucleic acid locus complexed with a guide nucleic acid while lacking nucleic acid cleavage activity.

[0071] In some embodiments, the modified Cas protein has a reduced function relative to the unmodified form. In some embodiments, the modified Cas protein is a functional defective unmodified form. For example, the nuclease defective Cas protein retains the ability to bind DNA, but lacks or has reduced nucleic acid cleavage activity. Cas nucleases (e.g., retaining wild-type nuclease activity, having reduced nuclease activity and / or lacking nuclease activity) can act in the CRISPR / Cas system to regulate the level and / or activity of the target gene or protein (e.g., reduce, increase or eliminate). The Cas protein can bind to the target polynucleotide and prevent transcription by physical barriers or editing nucleic acid sequences to produce non-functional gene products. In some embodiments, the modified Cas protein has no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5% or no more than 1% of the wild-type Cas protein (e.g., Cas9 from Streptococcus pyogenes) function (e.g., nuclease activity). In some embodiments, the modified Cas protein does not have the substantial function of the wild-type Cas protein. When the Cas protein is a modified form that does not have substantial nucleic acid cleavage activity, it can be referred to as enzymatically inactive and / or "dead" (abbreviated as "d"). Dead Cas proteins (e.g., dCas, dCas9) can bind to target polynucleotides, but cannot cleave target polynucleotides. In some aspects, the dead Cas protein is a dead Cas9 protein or a dead Cas12a protein.

[0072] In some embodiments, the modified Cas protein can be a modified Cas "base editor". Base editing enables a target DNA base to be directly and irreversibly converted to another base in a programmable manner without the need for DNA cleavage or donor DNA molecules. For example, Komor et al. (2016, Nature [Nature], 533: 420-424) teach Cas9-cytidine deaminase fusions, in which Cas9 has also been engineered to be inactive and does not induce double-stranded DNA breaks. In addition, Gaudelli et al. (2017, Nature [Nature], doi: 10.1038 / nature24644) teach Cas9 fused to a tRNA adenosine deaminase with impaired catalytic activity, which can mediate the conversion of A / T to G / C in a target DNA sequence. Another class of engineered Cas9 nucleases that can be used as site-directed nucleases in the fusion proteins of the present disclosure are variants that can recognize a wide range of PAM sequences, including NG, GAA, and GAT (Hu et al., 2018, Nature, doi: 10.1038 / nature26155).

[0073] The Cas protein can be modified to optimize the regulation of gene expression. The Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity and / or enzyme activity. The Cas protein can also be modified to change any other activity or property of the protein, such as stability. For example, one or more nuclease domains of the Cas protein can be modified, deleted or inactivated, or the Cas protein can be truncated to remove domains that are unnecessary for protein function or to optimize (e.g., enhance or reduce) the activity of the Cas protein in order to regulate gene expression.

[0074] One or more nuclease domains (e.g., RuvC, HNH) of Cas protein can be deleted or mutated so that they are no longer functional or contain reduced nuclease activity. For example, in a Cas protein (e.g., Cas9) comprising at least two nuclease domains, if one nuclease domain is deleted or mutated, the Cas protein produced, which is called nickase, can produce single-strand breaks at the CRISPR RNA (crRNA) recognition sequence in double-stranded DNA, but does not produce double-strand breaks. This nickase can cleave complementary strands or non-complementary strands, but cannot cleave both at the same time. In certain embodiments, the specificity of targeting double-strand breaks is improved by targeting the nickase to the opposite strands at two locations near the locus. If the nickase cleaves the single strand at two loci, double-strand breaks are formed and can be repaired via HR as described herein. If all nuclease domains of Cas protein (e.g., RuvC and HNH nuclease domains in Cas9 protein; RuvC nuclease domain in Cpf1 egg) are deleted or mutated, the Cas protein produced can have the ability of reducing the two chains of double-stranded DNA cleavage or do not have this ability. Zinc finger nuclease

[0075] In some embodiments, site-directed nucleases suitable for use in the fusion proteins or methods described herein are "zinc finger nucleases" or "ZFNs". ZFN refers to a fusion between a cleavage domain (such as the cleavage domain of Fok1) and at least one zinc finger motif (e.g., at least 2, 3, 4, or 5 zinc finger motifs), which can bind to polynucleotides (such as DNA and RNA). Heterodimerization at certain positions in the polynucleotides of two separate ZFNs in a certain orientation and spacing can lead to the cleavage of the polynucleotides. For example, a ZFN that binds to DNA can induce double-strand breaks in DNA. In order for the two cleavage domains to dimerize and cleave DNA, two separate ZFNs can be bound to opposite strands of DNA through their C-termini in a manner that is separated by a certain distance. In some cases, the linker sequence between the zinc finger domain and the cleavage domain may require that the 5' edge of each binding site be separated by about 5-7 base pairs. In some cases, the cleavage domain is fused to the C-terminus of each zinc finger domain. Exemplary ZFNs include, but are not limited to, those described in Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Gaj et al., Nat Methods, 2012, 9(8):805-7; U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241,573; 7,241,574; 7,585,849; 7,595,376; 6,903,185; 6,479,626; and U.S. Publication Nos. 2003 / 0232410 and 2009 / 0203140.

[0076] In some embodiments, a nuclease comprising a ZFN can produce a double-strand break in a target polynucleotide, such as a DNA. Double-strand breaks in DNA can lead to DNA break repair, thereby allowing the introduction of one or more genetic modifications (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide containing a homology arm of a site flanking the target DNA can be provided. In some embodiments, the ZFN is a zinc finger nickase that induces site-specific single-strand DNA breaks or nicks, thereby leading to HR. A description of zinc finger nickases is found in, for example, Ramirez et al., Nucl Acids Res [Nucleic Acids Research], 2012, 40 (12): 5560-8; Kim et al., Genome Res [Genome Research], 2012, 22 (7): 1327-33. In some embodiments, the ZFN binds to a polynucleotide (e.g., DNA and / or RNA), but cannot cleave the polynucleotide.

[0077] In some embodiments, the cleavage domain of the nuclease comprising the ZFN comprises a modified form of the wild-type cleavage domain. The modified form of the cleavage domain may comprise an amino acid change (e.g., a deletion, insertion, or substitution) that reduces the nucleic acid cleavage activity of the cleavage domain. For example, the modified form of the cleavage domain may have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid cleavage activity of the wild-type cleavage domain. The modified form of the cleavage domain may not have substantial nucleic acid cleavage activity. In some embodiments, the cleavage domain is enzymatically inactive. TAL effector nuclease

[0078] In some embodiments, the site-directed nuclease suitable for use in the fusion proteins, systems or methods described herein is a "TALEN" or "TAL effector nuclease". TALEN refers to an engineered transcription activator-like effector nuclease, which generally contains a central domain and a cleavage domain of a DNA-binding tandem repeat sequence. TALEN can be produced by fusing the TAL effector DNA binding domain with the DNA cleavage domain. In some cases, the DNA-binding tandem repeat sequence comprises a length of 33-35 amino acids and contains two hypervariable amino acid residues at positions 12 and 13 that can recognize at least one specific DNA base pair. The transcription activator-like effector (TALE) protein can be fused to a nuclease such as a wild-type or mutated Fok1 endonuclease or the catalytic domain of Fok1. For use in TALEN, Fok1 has been subjected to several mutations that, for example, improve cleavage specificity or activity. Such TALEN can be engineered to bind to any desired DNA sequence. TALEN can be used to produce genetic modifications (e.g., nucleic acid sequence editing) by generating double-strand breaks in the target DNA sequence, thereby undergoing NHEJ or HR. Double-strand breaks in DNA can lead to DNA break repair, thereby allowing the introduction of one or more genetic modifications (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide containing a homology arm of a site flanking the target DNA can be provided. In some cases, a single-stranded donor DNA repair template is provided to promote HR. Detailed descriptions of TALENs and their use for gene editing are found, for example, in U.S. Pat. Nos. 8,440,431; 8,440,432; 8,450,471; 8,586,363; and 8,697,853; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Beurdeley et al., Nat Commun, 2013, 4:1762; and Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(1):49-55.

[0079] In some embodiments, TALEN is engineered to reduce nuclease activity. In some embodiments, the nuclease domain of TALEN includes a modified form of a wild-type nuclease domain. The modified form of the nuclease domain may include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain may have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5% or no more than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain may not have substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactive.

[0080] In some embodiments, transcription activator-like effector (TALE) protein is fused with a domain that can regulate transcription and does not contain a nuclease. In some embodiments, transcription activator-like effector (TALE) protein is designed to work as a transcription activator. In some embodiments, transcription activator-like effector (TALE) protein is designed to work as a transcription repressor. For example, the DNA binding domain of a transcription activator-like effector (TALE) protein can be fused (for example, connected) with one or more transcription activation domains or with one or more transcription repression domains. Non-limiting examples of transcription activation domains include tetramer repeats (for example, VP64 activation domains) of herpes simplex VP16 activation domains and VP16 activation domains. Non-limiting examples of transcription repression domains include Kruppel associated box domains. Meganuclease

[0081] In some embodiments, the site-directed nuclease suitable for use in the fusion protein, system or method described herein is a meganuclease. A meganuclease generally refers to a rare cutting endonuclease or a homing endonuclease that can be highly specific. A meganuclease can identify a DNA target site ranging in length from at least 12 base pairs, for example, a length from 12 to 40 base pairs, 12 to 50 base pairs or 12 to 60 base pairs. A meganuclease can be a modular DNA binding nuclease, such as any fusion protein or a protein specifying a nucleic acid target sequence comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain. The DNA binding domain can contain at least one motif for identifying single-stranded or double-stranded DNA. A meganuclease can produce double-strand breaks. Double-strand breaks in DNA can result in DNA break repair, thereby allowing the introduction of one or more genetic modifications (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide containing homology arms flanking the site of the target DNA can be provided. A meganuclease can be monomeric or dimeric. In some embodiments, the meganuclease is naturally occurring (found in nature) or wild-type, and in other cases, the meganuclease is non-natural, artificial, engineered, synthetic, rationally designed or artificial. In some embodiments, the meganuclease of the present disclosure includes I-CreI meganuclease, I-CeuI meganuclease, I-Msol meganuclease, I-SceI meganuclease, variants thereof, derivatives thereof and fragments thereof. Detailed descriptions of useful meganucleases and their use in gene editing are found, for example, in Silva et al., Curr Gene Ther, 2011, 11(1):11-27; Zaslavoskiy et al., BMC Bioinformatics, 2014, 15:191; Takeuchi et al., Proc Natl Acad Sci USA, 2014, 111(11):4061-4066 and U.S. Patent Nos. 7,842,489; 7,897,372; 8,021,867; 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,36; and 8,129,134.

[0082] In some embodiments, the nuclease domain of a meganuclease includes a modified form of a wild-type nuclease domain. The modified form of the nuclease domain may include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid cleavage activity of the nuclease domain. For example, the modified form of the nuclease domain may have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid cleavage activity of the wild-type nuclease domain. The modified form of the nuclease domain may not have substantial nucleic acid cleavage activity. In some embodiments, the nuclease domain is enzymatically inactive. In some embodiments, a meganuclease may bind to DNA, but may not cleave DNA. B. Nonspecific terminal processing enzymes

[0083] The fusion proteins provided herein include non-specific end processing enzymes. Non-specific end processing enzymes are polypeptides that modify the ends of polynucleotides in a non-sequence-specific manner. In some embodiments, the non-specific end processing enzyme is a non-specific exonuclease. As used herein, the term "exonuclease" refers to an enzyme that cleaves polynucleotides from the 5' or 3' end. 5' to 3' exonucleases only cleave polynucleotides in the 5' to 3' direction. 3' to 5' exonucleases only cleave polynucleotides in the 3' to 5' direction. Bidirectional exonucleases can cleave polynucleotides in either direction. Suitable exonucleases are described in, for example, Lovett, 2011, ASM Journals [ASM Magazine] EcoSal Plus 4 (2): 10.1128 / ecosalplus.4.4.7 and Shevelev and Hübscher, 2002, Nature Reviews Molecular Cell Biology [Natural Review: Molecular Cell Biology] 3: 364-376.

[0084] In some embodiments, the non-specific exonuclease is T5Exo, Trex2 (a non-processive 3' to 5' exonuclease that functions as a homodimer), Escherichia coli exonuclease I, exonuclease III, exonuclease T, exonuclease IX, exonuclease X, RecJ, Pol II, Pol IIIε, WRN, MRE11, APE1, VDJP, RAD1, RAD9, p53, or Trex1.

[0085] In some embodiments, the non-specific end processing enzyme comprises an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 4, 5, 18, 19, 20, 22, or 58-74. C. Dimerization

[0086] Dimerization refers to the situation that two non-covalently linked protein domains are combined to act as a single unit (i.e., "dimer"). A non-limiting list of the types of dimers includes homodimers, heterodimers, oligomerization / polymerization, autonomous dimerization, and inducible dimerization. See, for example, US2016 / 0024485; US2020 / 0199254; WO 1999 / 010510; WO 2022 / 040909; and US2018 / 163195. In some embodiments, the fusion protein provided herein forms a dimer (e.g., a homodimer). In some embodiments, this is performed by the protein-protein interaction of a non-specific terminal processing enzyme. In some embodiments, the non-specific terminal processing enzyme is capable of dimerization. In some cases, the non-specific terminal processing enzyme is a monomer of a dimerized protein. In some cases, the non-specific terminal processing enzyme comprises a dimerization domain. In some embodiments, a fusion protein comprising a non-specific terminal processing enzyme capable of dimerization will form a fusion protein dimer (or a complex having more than two monomers) via dimerization of the non-specific terminal processing enzyme.

[0087] In some embodiments, the non-specific end processing enzyme is capable of dimerization in its endogenous form. For example, Trex2 is capable of dimerization, and is known to function as a homodimer. In some embodiments, the non-specific end processing enzyme comprises a domain that contributes to dimerization. In some embodiments, the non-specific end processing enzyme is capable of autonomous dimerization.

[0088] In some embodiments, the non-specific terminal processing enzyme is an engineered polypeptide that has acquired dimerization functionality, for example, by adding a dimerization domain. There are a variety of protein dimerization domains known in the art, including, for example, antibody Fc domains and commercially available dimerization systems (e.g., System, Takara Bio USA). In some embodiments, dimerization is achieved by using any of the polypeptide interaction strategies described below in Section III.D. In some cases, the dimerization domain can be located at the N' terminus or C' terminus of the non-specific terminal processing enzyme. D. Additional Fusion Protein Domains

[0089] In some embodiments, fusion proteins provided herein include one or more joints. As used herein, joints (also referred to as spacers) are flexible molecules or flexible molecular segments that connect (join) or connect (connect) two parts (e.g., domains) of fusion proteins or modified proteins as provided herein. In some embodiments, joints are polypeptides. Proteins with domains connected by polypeptide joints are referred to as fusion proteins. In some embodiments, joints are non-peptide joints. Proteins with domains connected by polypeptide joints are referred to as modified proteins. It should be understood that in the case of discussing fusion proteins throughout this disclosure, modified proteins (where feasible) are also generally envisioned.

[0090] The linker can increase the range of orientations that can be adopted by the domain of the fusion protein or modified protein. The linker can be optimized to produce the desired effect in the fusion protein or modified protein. Aspects of linker design and consideration are described in, for example, Chen, X. et al., Adv Drug Deliv Rev. [Advanced Drug Delivery Review] 2013 Oct 15; 65 (10): 1357-1369; and Klein, JS et al. 2014 Protein Eng. Des. Sel. [Protein Engineering Design and Selection] 27 (10): 325-330. In some embodiments, the protein provided herein comprises a peptide linker. In some embodiments, the protein provided herein comprises a non-peptide linker. In some embodiments, the protein provided herein comprises a peptide linker and a non-peptide linker. The protein provided herein may also comprise a plurality of linkers, including at least one peptide linker, at least one non-peptide linker, or at least one peptide linker and at least one non-peptide linker.

[0091] The linker can be short or long, flexible or rigid. See, for example, PCT / US2020 / 051383 (incorporated herein in its entirety by reference) and WO 2020 / 168102 (incorporated herein in its entirety by reference) and US2021 / 0017506 (incorporated herein in its entirety by reference).

[0092] In certain embodiments, the length of the joint can affect one or more functions of the fusion protein. Select the joint to achieve the desired length within the ability of those skilled in the art. In certain embodiments, the length of the peptide joint can be, for example, 5 to 100 or more amino acids (for example, 5aa, 10aa, 15aa, 20aa, 25aa, 30aa, 35aa, 40aa, 45aa, 50aa, 55aa, 60aa, 65aa, 70aa, 75aa, 80aa, 85aa, 90aa, 95aa or 100aa).

[0093] In some cases, the joint sequence can have a conformation of extension and work as an independent domain that does not interact with adjacent protein domains. The joint sequence can be flexible or rigid. Flexible joints provide a certain degree of movement or interaction between polypeptide domains and are generally rich in small or polar amino acids such as Gly and Ser (for example, at least 90%, at least 95%, at least 98%, at least 99% or all of them in the amino acid residues of joints). Rigid joints can be used to maintain a fixed distance between domains and help maintain their independent functions. Joint attachment can be carried out by amide linkage (for example, peptide bond) or other functionalities as discussed further below.

[0094] In some embodiments, a peptide linker described herein comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7. In some embodiments, the linker comprises one or more repeats (e.g., 2 repeats, 3 repeats, 4 repeats, 5 repeats, 6 repeats, or more) of GGGGS (SEQ ID NO: 125) and / or one or more repeats of GSSGSS (SEQ ID NO: 126). Additional exemplary peptide linkers include, but are not limited to, peptide linkers comprising: SGSETPGTSESATPE (SEQ ID NO: 127), SGSETPGTSESATPES (SEQ ID NO: 128), (GGGGS)3 (SEQ ID NO: 129), (GGGGS)5 (SEQ ID NO: 130), (GGGGS)10 (SEQ ID NO: 131), GGGGGGGG (SEQ ID NO: 132), GSAGSAAGSGEF (SEQ ID NO: 133), A(EAAAK)3A (SEQ ID NO: 134), or A(EAAAK)10A (SEQ ID NO: 135). Additional non-limiting exemplary linkers that may be used include those disclosed in PCT / US2020 / 051383; Chen et al., Adv. Drug. Deliv. Rev. 65(10): 1357-1369 (2014) and Rosemalen et al., Biochemistry 2017, 56, 50, 6565-6574, the entire contents of both documents being incorporated herein by reference.

[0095] In some embodiments, the non-peptide linker may include any of a plurality of known chemical linkers. Exemplary chemical linkers may include one or more units of β-alanine, 4-aminobutyric acid (GABA), (2-aminoethoxy) acetic acid (AEA), 5-aminohexanoic acid (Ahx), PEG polymers, and trioxatridecane-succinamic acid (Ttds). In some embodiments, the non-peptide linker comprises one or more units of polyethylene glycol (PEG), which is commonly used as a linker for conjugating polypeptide domains due to its water solubility, lack of toxicity, low immunogenicity, and well-defined chain length. See, for example, Ramirez-Paz, J. et al., PLoS One [Public Library of Science Comprehensive] 13 (7): e0197643 (2018). The number of PEG linkage units can be selected based on the desired linker length.

[0096] The modified protein comprising a non-peptide linker can be produced in a variety of ways. For example, a site-directed nuclease and a non-specific end processing enzyme can be produced separately (e.g., in vitro or by expression in a host cell and purified therefrom) and chemically linked in vitro. In certain embodiments, a site-directed nuclease, a non-specific end processing enzyme and a linker can each be produced separately and chemically linked in vitro. Various chemical linkers can be used to cross-link two amino acid residues.

[0097] Such embodiments are also contemplated herein, wherein the site-directed nuclease and non-specific terminal processing enzyme as described above are used alone (e.g., introduced into cells alone or applied to target nucleic acids alone) and are brought into proximity to form a complex without using a linker as described above. Various methods of forming a complex between two or more polypeptides are known in the art and include, but are not limited to, using protein-protein interaction strategies (e.g., SunTag, coiled coils, etc.), using RNA-aptamers and related binding proteins (e.g., MS2, N22, etc.) and tag: capture strategies. For example, the site-directed nuclease of the present disclosure can include an MS2 RNA aptamer, which will promote interaction with a non-specific terminal processing enzyme comprising an MS2 coat protein.

[0098] In some embodiments, the fusion protein provided herein comprises a targeting sequence that mediates protein localization (or retention) to a subcellular location, such as plasma membrane or a given organelle membrane, nucleus, cytosol, mitochondria, endoplasmic reticulum (ER), Golgi, chloroplast, apoplast, peroxisome or other organelles. For example, the targeting sequence can guide a protein (e.g., a nuclease) to the nucleus using a nuclear localization signal (NLS); guide the protein to the outside of the nucleus of the cell using a nuclear export signal (NES), such as guiding it to the cytoplasm; guide the protein to mitochondria using a mitochondrial targeting signal; guide the protein to the endoplasmic reticulum (ER) using an ER retention signal; guide the protein to peroxisomes using a peroxisome targeting signal; guide the protein to plasma membrane using a membrane localization signal; or a combination thereof. In some embodiments, the fusion protein comprises a nuclear localization signal.Non-limiting examples of NLSs include NLS sequences derived from: the NLS of the SV40 virus large T antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 8); an NLS from a nucleoplasmin (e.g., a nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 136)); a c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 137) or RQRRNELKRSP (SEQ ID NO: 138); an hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQ YFAKPRNQGGY (SEQ ID NO: 139); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKD EQILKRRNV (SEQ ID NO: 140) from the IBB domain of importin-α; the sequence VSRKRPRP (SEQ ID NO: 141) from the myoma T protein. NO:141) and PPKKARED (SEQ ID NO:142); the sequence of human p53 PQPKKKPL (SEQ ID NO:143); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO:144); the sequences of influenza virus NS1 DRLRR (SEQ ID NO:145) and PKQKKRK (SEQ ID NO:146); the sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO:147); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO:148); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:149); the sequence of steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO:150); NO: 150); and the sequence of the Agrobacterium VirD2 protein KRPRDRHDGELGGRKRAR (SEQ ID NO: 151).

[0099] In some embodiments, the fusion proteins provided herein comprise an amino acid sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 50-57.

[0100] Any polypeptide and fusion protein described herein may further include a detectable portion, such as a fluorescent protein or a fragment thereof. Examples of fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP, such as Venus), green fluorescent protein (GFP), and red fluorescent protein (RFP) and derivatives of these proteins, such as mutant derivatives. See, for example, Chudakov et al. "Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues," Physiological Reviews [Physiological Reviews] 90 (3): 1103-1163 (2010); and Specht et al., "A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging [Critical Comparative Review of Fluorescent Tools for Live-Cell Imaging]," Annual Review of Physiology [Physiological Year Review] 79: 93-117 (2017)).

[0101] For example, any of the polypeptides described herein can further include an affinity tag (e.g., a polyhistidine tag (e.g., (His)6 (SEQ ID NO: 152)), an HA tag (e.g., YPYDVPDYA (SEQ ID NO: 153)), an albumin binding protein, alkaline phosphatase, an AU1 epitope, an AU5 epitope, a biotin-carboxyl carrier protein (BCCP), a FLAG epitope (e.g., DYKDDDDK (SEQ ID NO: 154) or a MYC epitope (e.g., EQKLISEEDL (SEQ ID NO: 155)). See Kimple et al., "Overview of Affinity Tags for Protein Purification," Curr. Protoc. Protein Sci. 73: Unit 9.9 (2013). E. Variants

[0102] Variants of the polypeptides disclosed herein are also provided herein. Unless otherwise expressly specified, the polypeptide variants retain their corresponding biological activity. For example, variants of site-directed nuclease polypeptides retain the biological function of the full-length native sequence site-directed nuclease. In another example, variants of non-specific terminal processing enzymes retain the biological function of the full-length native sequence non-specific terminal processing enzymes.

[0103] The modification of any polypeptide or protein provided herein is carried out by known methods. By way of example, modification is carried out in the following manner: the nucleotides in the nucleic acid encoding the polypeptide are subjected to site-specific mutagenesis, thereby producing a DNA encoding the modification, and then the DNA is expressed in recombinant cell culture to produce the encoded polypeptide. The technology for carrying out substitution mutations at predetermined sites in DNA with known sequences is well known. For example, M13 primer mutagenesis and PCR-based mutagenesis methods can be used to produce one or more substitution mutations. Any nucleotide sequence provided herein can be codon optimized to change, for example, to maximize expression in a host cell or an organism.

[0104] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of naturally occurring amino acids, non-natural amino acids, and chemically modified amino acids. Non-natural amino acids (i.e., those not naturally found in proteins) are also known in the art, as described, for example, in Zhang et al. "Protein engineering with unnatural amino acids," Curr. Opin. Struct. Biol. [Structural Biology New View] 23(4): 581-587 (2013); Xie et al. "Adding amino acids to the genetic repertoire [Adding amino acids to the gene library]," 9(6): 548-54 (2005)); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as non-natural amino acids.

[0105] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, the side chain can be modified to include a signaling moiety, such as a fluorophore or a radioactive label. The side chain can also be modified to include a new functional group, such as a thiol, a carboxylic acid, or an amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0106] Conservative amino acid substitutions are also contemplated. By way of example, conservative amino acid substitutions can be made in one or more amino acid residues, for example, in one or more lysine residues of any of the polypeptides provided herein. Those skilled in the art will appreciate that a conservative substitution is the replacement of one amino acid residue with another amino acid residue that is biologically and / or chemically similar. The following eight groups each contain amino acids that are conservative substitutions of one another: 1) Alanine (A), glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); 6) Phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), methionine (M).

[0107] By way of example, when referring to arginine to serine, conservative substitutions of serine are also contemplated (eg, threonine). Non-conservative substitutions are also contemplated, such as replacing lysine with asparagine. IV. Recombinant Nucleic Acids, Constructs, Vectors and Host Cells

[0108] Also provided herein are recombinant nucleic acids encoding any of the polypeptides described herein. For example, also provided are recombinant nucleic acids encoding polypeptides having at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) identity to any one of SEQ ID NOs: 4-6, 18-20, 22 or 50-74. Also provided are recombinant nucleic acids having at least 70% identity to any one of SEQ ID NOs: 21, 32 or 33.

[0109] Also provided is a DNA construct comprising a promoter operably linked to a recombinant nucleic acid encoding a fusion protein as described herein or its domain. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. A variety of promoters can be used in constructs as described herein. A promoter is a region or sequence that is located upstream and / or downstream of the start of transcription and involves recognition and binding of RNA polymerase and other proteins to initiate transcription.

[0110] As used herein, the term "promoter" refers to a nucleotide sequence, usually upstream (5') of its coding sequence, which controls the expression of the coding sequence by providing recognition of RNA polymerase and other factors required for appropriate transcription."Promoter regulatory sequence" consists of proximal and more distal upstream elements. The promoter regulatory sequence affects the transcription, RNA processing or stability or translation of the relevant coding sequence. Regulatory sequences include enhancers, promoters, non-translated leader sequences, introns and polyadenylation signal sequences. They include natural sequences and synthetic sequences and sequences that may be combinations of synthetic sequences and natural sequences."enhancer" is a DNA sequence that can stimulate promoter activity and can be an intrinsic element of a promoter or an inserted heterologous element to enhance the level or tissue specificity of a promoter. It can operate in two orientations (normal or flipped), and can even play a role when moved to the upstream or downstream of the promoter. The meaning of the term "promoter" includes "promoter regulatory sequence".

[0111] The choice of promoter to be included depends on several factors, including but not limited to efficiency, selectability, inducibility, desired expression level, and cell or tissue preferential expression. It is a routine practice for those skilled in the art to regulate the expression of a sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the sequence.

[0112] It has been shown that certain promoters can direct RNA synthesis at a higher rate than others. These are referred to as "strong promoters". It has been shown that certain other promoters direct RNA synthesis at higher levels only in specific types of cells or tissues, and are often referred to as "tissue-specific promoters", or if the promoter preferentially directs RNA synthesis in certain tissues (RNA synthesis may occur at reduced levels in other tissues), it is referred to as a "tissue-preferred promoter". Because promoters are used to control the expression pattern of one (or more) chimeric genes introduced into plants, there is a continuing interest in isolating novel promoters that can control the expression of one (or more) chimeric genes at certain levels in specific tissue types or at specific plant development stages.

[0113] Some promoters can guide RNA synthesis at relatively similar levels in all tissues of a plant. These are called "constitutive promoters" or "non-tissue-dependent" promoters. Constitutive promoters can be divided into strong, medium, and weak categories according to their effectiveness in guiding RNA synthesis. Because it is necessary to express one (or more) chimeric genes in different tissues of a plant simultaneously in many cases to obtain the desired function of one (or more) gene, constitutive promoters are particularly useful in this regard. Although many constitutive promoters have been found and characterized from plants and plant viruses, there is still a continued interest in isolating more novel constitutive promoters (synthetic or natural), which can control the expression of one (or more) chimeric genes at different levels and control the expression of multiple genes in the same transgenic plant to perform gene stacking.

[0114] The most commonly used promoters include the nopaline synthase (NOS) promoter (Ebert et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 84:5745-5749 (1987)); the octopine synthase (OCS) promoter; cauliflower mosaic virus group promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. [Plant Molecular Biology] 9:315-324 (1987)); the light-inducible promoter from the small subunit of ribulose bisphosphate carboxylase / oxygenase (Pellegrineschi et al., Biochem. Soc. Trans. [Biological Chemical Society Bulletin] 23(2):247-250 (1995)); Adh promoter (Walker et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 84:6624-66280 (1987)); sucrose synthase promoter (Yang et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 87:414-44148 (1990)); R gene complex promoter (Chandler et al., Plant Cell [Plant Cell] 1:1175-1183 (1989)); chlorophyll a / b binding protein gene promoter; etc.

[0115] In addition, it is contemplated that promoters that combine elements from more than one promoter may be useful. For example, U.S. Pat. No. 5,491,288 discloses combining a cauliflower mosaic virus promoter with a histone promoter. Thus, elements from the promoters disclosed herein can be combined with elements from other promoters. Promoters useful for plant transgenic expression include those that are inducible, viral, synthetic, constitutive (Odell Nature 313:810-812 (1985)), temporally regulated, spatially regulated, tissue-specific, and spatiotemporally regulated. Using the regulatory elements described herein, many agronomic genes can be expressed in transformed plants. More specifically, plants can be genetically engineered to express various phenotypes of agronomic interest. "

[0116] In some embodiments of the DNA construct provided herein, the promoter can be a eukaryotic or prokaryotic promoter. In certain embodiments, the promoter is an inducible promoter, a natural inducible promoter (e.g., drought inducible Rab17), a synthetic inducible promoter (e.g., auxin inducible DR5, estradiol inducible XVE / pLex, dexamethasone inducible GVG / Gal4), a constitutive promoter (e.g., ZmUbq1, OsAct1, OsTub3, EF), an egg cell specific promoter (e.g., EC1, EC2, EC3, EC4, EC5), a pollen specific promoter, a apical meristem specific promoter, or a promoter with enriched expression in a conjugant. In certain embodiments, the promoter is a flower mosaic promoter (e.g., ZmBde1, OsAP1). In certain embodiments, the promoter is an ubiquitin 4 promoter, an actin promoter, a tubulin promoter, a MADS box promoter, or a plant virus promoter. Suitable promoters are disclosed, for example, in U.S. Pat. No. 10,519,456 (the entire contents of which are incorporated herein by reference) and PCT / US2022 / 020690 (incorporated herein by reference).

[0117] The recombinant nucleic acid provided herein can be included in an expression cassette for expressing in a target host cell or organism. The cassette will include 5' and 3' regulatory sequences operably connected to the recombinant nucleic acid provided herein that allows the expression of the fusion protein. The cassette can additionally contain at least one other gene or genetic element to be co-transformed into a cell or organism. In the case of including other genes or elements, these components are operably connected. Alternatively, one or more other genes or elements can be provided on a plurality of expression cassettes. This expression cassette is provided with a plurality of restriction sites and / or recombination sites so that the insertion of the polynucleotide is under the transcriptional regulation of the regulatory region. The expression cassette can additionally contain a selective marker gene. The expression cassette will include in the reverse direction of 5' to 3' transcription: transcription and translation initiation region (i.e., promoter), polynucleotides of the present invention, and transcription and translation termination region (i.e., termination region) that work in the target cell or organism. The promoter of the present invention is capable of directing or driving the expression of a coding sequence (i.e., a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, ncRNA, lncRNA, sense RNA or antisense RNA, whether or not the RNA is then translated to produce a protein) in a host cell. The regulatory regions (i.e., the promoter, transcriptional regulatory regions, and translational termination regions) may be endogenous or heterologous to the host cell or to each other. As used herein, "heterologous" with respect to a sequence is a sequence that is derived from a foreign species or, if derived from the same species, has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0118] Additional regulatory signals include, but are not limited to, transcription initiation start sites, operators, activators, enhancers, other regulatory elements, ribosome binding sites, initiation codons, termination signals, etc. See Sambrook et al. (1992) Molecular Cloning: A Laboratory Manual, Maniatis et al., eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Davis et al., eds. (1980) Advanced Bacterial Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), and references cited therein.

[0119] The expression cassette can also include a selective marker gene for selecting transformed cells. For example, marker genes include genes that confer antibiotic resistance, such as those that confer hygromycin resistance, ampicillin resistance, gentamicin resistance, neomycin resistance. Other selective markers are known, and any can be used.

[0120] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the proper orientation and, if appropriate, in the proper reading frame. To this end, adapters or linkers may be employed to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., conversion and transversion) may be involved.

[0121] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the proper orientation and, if appropriate, in the proper reading frame. To this end, adapters or linkers may be employed to join the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., conversion and transversion) may be used.

[0122] A vector is further provided, the vector comprising the recombinant nucleic acid or DNA construct described herein. It is envisioned that the vector has the necessary functional elements for guiding and regulating the transcription of the inserted nucleic acid. These functional elements include, but are not limited to, a promoter, a region upstream or downstream of the promoter (such as an enhancer that can regulate the transcriptional activity of the promoter), a replication origin, an appropriate restriction site for cloning the inserted sequence adjacent to the promoter, an antibiotic resistance gene or other marker that can be used to select cells containing the vector or a vector containing the inserted sequence, an RNA splicing junction, a transcription termination region, or any other region that can be used to promote the expression of the inserted gene or hybrid gene. Generally, see Sambrook et al. Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2012. The vector can be, for example, a plasmid.

[0123] There are many E. coli expression vectors known to those of ordinary skill in the art, which can be used for the expression of nucleic acids. Other microbial hosts suitable for use include bacillus (such as Bacillus subtilis) and other Enterobacteriaceae (such as Salmonella, Senatia) and various Pseudomonas species. In these prokaryotic hosts, expression vectors can also be prepared, which typically contain expression control sequences (for example, replication origins) compatible with host cells. In addition, there will be any number of well-known promoters, such as lactose promoter systems, tryptophan (Trp) promoter systems, beta-lactamase promoter systems or promoter systems from bacteriophage lambda. In addition, yeast expression can be used. Nucleic acids encoding polypeptides of the present invention are provided herein, wherein the nucleic acids can be expressed by yeast. More specifically, the nucleic acids can be expressed by Pichia pastoris or S. cerevisiae.

[0124] Mammalian cells also allow protein to be expressed in an environment conducive to important post-translational modifications, such as folding and cysteine ​​pairing, the addition of complex carbohydrate structures and the secretion of active proteins. Carriers that can be used for expressing active proteins in mammalian cells are known in the art, and can contain genes that confer hygromycin resistance, geneticin or G418 resistance, or other genes or phenotypes suitable for use as selective markers, or methotrexate resistance for gene amplification. A variety of suitable host cell lines capable of secreting complete human proteins have been developed in the art, and include CHO cells, HeLa cells, HEK-293 cells, HEK-293T cells, U2OS cells or any other primary or converted cell lines. Other suitable host cell lines include COS-7 cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as replication origins, promoters, enhancers and necessary information processing sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites and transcription terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, and the like.

[0125] The expression vectors described herein may also include nucleic acids as described herein under the control of an inducible promoter, such as a tetracycline inducible promoter or a glucocorticoid inducible promoter. The nucleic acids of the present invention may also be under the control of a tissue-specific promoter to promote expression of the nucleic acids in specific cells, tissues or organs. Any regulatable promoter is also contemplated, such as a metallothionein promoter, a heat shock promoter, and other regulatable promoters, many examples of which are well known in the art. In addition, the Cre-loxP inducible system and the Flp recombinase inducible promoter system may also be used, both of which are known in the art.

[0126] Insect cells also allow for the expression of polypeptides. Recombinant proteins produced in insect cells using baculovirus vectors undergo post-translational modifications similar to those of wild-type mammalian proteins.

[0127] Host cells are also provided herein, comprising recombinant nucleic acids, DNA constructs and / or vectors as described herein and methods for preparing such cells. In some embodiments, the cell is a plant cell. In some embodiments, the plant cell is a corn plant cell, a soybean plant cell, a rice plant cell, a wheat plant cell or a sunflower plant cell.

[0128] Provided are host cells comprising nucleic acids or vectors as described herein. The host cell can be an in vitro, ex vivo or in vivo host cell. The host cell as provided herein can express fusion proteins. Also provided are cell colonies of any host cell as described herein. In some embodiments, the cell colony comprises a plurality of cells, wherein the plurality of cells comprise a recombinant nucleic acid encoding a fusion protein as described herein. In some embodiments, the cell colony comprises a plurality of cells, wherein the plurality of cells comprise a DNA construct encoding a fusion protein as described herein. In some embodiments, the cell colony comprises a plurality of cells, wherein the plurality of cells comprise a vector, and the vector comprises a recombinant nucleic acid or DNA construct encoding a fusion protein as described herein. In some embodiments, the cell colony comprises a plurality of cells, wherein the plurality of cells comprise a plurality of any host cells as described herein. In some embodiments, a plurality of cells of any cell colony as described herein express a fusion protein as described herein.

[0129] In certain embodiments, the cell provided is stable or transiently expressed fusion protein. The stable expression of fusion protein in the cell refers to any one of nucleic acid, DNA construct or vector as described herein is integrated into the cell genome, thereby allowing the cell to express the fusion protein. Transient expression refers to any one of the nucleic acid, DNA construct and / or vector of the fusion protein directly from being introduced into the cell (that is, the gene encoding the fusion protein is not integrated into the genome of the cell).

[0130] In some embodiments, the cells provided express the fusion protein constitutively or inducibly. Constitutive expression refers to the sustained, continuous expression of a gene (i.e., a protein), while inducible expression refers to the expression of a gene (protein) in response to a stimulus. Inducible expression is usually regulated via an inducible promoter (described above).

[0131] Also provided are cell cultures comprising one or more host cells described herein.Methods for culturing and producing a variety of cells are available in the art, including cells of bacterial (e.g., E. coli and other bacterial strains), animal (particularly mammalian) and archaeal origin. See, e.g., Sambrook, supra; Ausubel, ed. (1995) Current Protocols in Molecular Biology, John Wiley & Sons; and Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, 3rd ed., Wiley-Liss, New York, and references cited therein; Doyle and Griffiths (1997) Mammalian Cell Culture: Essential Techniques, John Wiley and Sons, NY; Humason (1979) Animal Tissue Techniques, 4th ed., WH Freeman and Company; and Ricciardelli et al., (1989) Invitro Cell Culture: Essential Techniques. Dev. Biol. [In vitro Cell and Developmental Biology] 25:1016–1024.

[0132] The host cell can be a prokaryotic cell, including, for example, a bacterial cell. Alternatively, the cell can be a eukaryotic cell, such as a mammalian cell. In certain embodiments, the cell can be a HEK-293T cell, a HEK-293 cell, a Chinese hamster ovary (CHO) cell, a U2OS cell, or any other primary or transformed cell. In certain embodiments, the cell can be a COS-7 cell, a HELA cell, an avian cell, a myeloma cell, a Pichia cell, an insect cell, or a plant cell. A variety of other suitable host cell lines have been developed, and include myeloma cell lines, fibroblast cell lines, and a variety of tumor cell lines such as melanoma cell lines. The vector containing the target nucleic acid segment can be transferred or introduced into the host cell by a well-known method, and these methods vary according to the type of the cell host.

[0133] As used herein, the phrase "introduction" in the context of introducing nucleic acid into a cell (e.g., a prokaryotic cell, a bacterial cell, a eukaryotic cell, a plant cell) refers to translocation of a nucleic acid sequence from outside the cell into the cell. In some cases, the introduction is to translocate the nucleic acid from outside the cell into the nucleus of the cell. In the case of introducing more than one nucleic acid molecule, these nucleic acid molecules can be assembled into a part of a single polynucleotide or nucleic acid construct, or assembled into a separate polynucleotide or nucleic acid construct, and can be located on the same or different nucleic acid constructs. Therefore, such polynucleotides can be introduced into a cell (e.g., a plant cell) in a single transformation event, in a separate transformation event, or, for example, as part of a breeding scheme. It is contemplated that various methods for introducing nucleic acid into a cell include, but are not limited to, electroporation, nanoparticle delivery, gene gun transformation, viral delivery, contact with nanowires or nanotubes, receptor-mediated internalization, translocation via cell-penetrating peptides, translocation mediated by liposomes, DEAE dextran, lipofectamine, calcium phosphate, or any method known or identified in the future for introducing nucleic acid into a prokaryotic or eukaryotic cell host. Targeted nuclease systems (e.g., RNA-guided nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or large-scale TALs (MTs) can also be used to introduce nucleic acids (e.g., nucleic acids encoding fusion proteins described herein) into host cells. See Li et al. Signal Transduction and Targeted Therapy 5, Article No. 1 (2020).

[0134] The transformation of cells can be stable or transient.Therefore, transgenic cells of the present invention, plant cells, plants and / or plant parts can be stably transformed or transiently transformed." transformation " can refer to nucleic acid molecules being transferred into the genome of host cells, causing genetically stable inheritance.In certain embodiments, being introduced into plants, plant parts and / or plant cells is via bacteria-mediated transformation, particle bombardment transformation, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, liposome-mediated transformation, nanoparticle-mediated transformation, polymer-mediated transformation, virus-mediated nucleic acid delivery, whisker-mediated nucleic acid delivery, microinjection, ultrasonic treatment, infiltration, polyethylene glycol-mediated transformation, protoplast transformation or making nucleic acid be introduced into any other electrical, chemical, physical and / or biological mechanism or any combination thereof in plants, plant parts and / or their cells to carry out.

[0135] Procedures for transforming plants are well known and routine in the art and are generally described in the literature. Non-limiting examples of methods for plant transformation include transformation via bacteria-mediated nucleic acid delivery (e.g., via bacteria from the genus Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical) and / or biological mechanism that allows nucleic acid to be introduced into plant cells, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick, BR and Thompson, JE, eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell Mol Biol Lett 7:849-858 (2002)).

[0136] Agrobacterium-mediated transformation is a common method for transforming plants due to its high transformation efficiency and due to its wide applicability with many different species. Agrobacterium-mediated transformation typically involves transferring a binary vector carrying the foreign DNA of interest to an appropriate Agrobacterium strain, which may depend on the complement of the vir genes carried by the host Agrobacterium strain on a co-existing Ti plasmid or chromosomally (Uknes et al., 1993, Plant Cell [Plant Cell] 5: 159-169). Escherichia coli carrying a recombinant binary vector can be used, and an auxiliary Escherichia coli strain (the auxiliary Escherichia coli strain carries a plasmid capable of moving the recombinant binary vector to the target Agrobacterium strain) is passed through a three-parent mating procedure to achieve the transfer of the recombinant binary vector to Agrobacterium. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by nucleic acid transformation ( and Willmitzer 1988, Nucleic Acids Res 16:9877).

[0137] Plant transformation by recombinant Agrobacterium typically involves co-cultivation of Agrobacterium with explants from plants and follows methods well known in the art. Transformed tissue is typically regenerated on selective media carrying antibiotic or herbicide resistance markers between the binary plasmid T-DNA borders.

[0138] Another method for transforming plants, plant parts and plant cells involves advancing inert or biologically active particles on plant tissues and cells. See, for example, U.S. Patent Nos. 4,945,050; 5,036,006 and 5,100,792. Generally speaking, this method involves advancing inert or biologically active particles at plant cells under conditions effective to penetrate the outer surface of the cell and provide incorporation into its interior. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with a vector containing the target nucleic acid. Alternatively, one or more cells can be surrounded by the vector so that the vector is carried into the cell by the excitation of the particle. Biologically active particles (e.g., dried yeast cells, dried bacteria or bacteriophages, each containing one or more nucleic acids intended to be introduced) can also be advanced into plant tissues. As used herein, the phrase "biolistic transformation" refers to a method of introducing RNA or DNA directly into cells (e.g., plant cells) in which the RNA or DNA is mixed with heavy metal particles (e.g., tungsten or gold) and released into the cells (e.g., plant cells) using high-speed pressure to allow the RNA or DNA to penetrate the cells (e.g., penetrate the plant cell wall).

[0139] The CRISPR / Cas system can also be used to edit the genome of a host cell or organism. As described in detail above, the "CRISPR / Cas" system refers to a class of bacterial systems that are widely used to defend against foreign nucleic acids. Any of the CRISPR / Cas system components described herein can be used to introduce fusion proteins, recombinant nucleic acids or systems into the genome of a host cell or organism. Methods for genome editing mediated by the CRISPR / Cas system are known in the art. It should be understood that the use of the CRISPR / Cas system for introducing fusion proteins, recombinant nucleic acids or systems described herein into the genome of a host cell or organism is different from the specific methods and systems provided herein.

[0140] Any fusion protein described herein can be purified or separated from a host cell or a host cell colony. For example, a recombinant nucleic acid encoding any fusion protein described herein can be introduced into a host cell under conditions that allow the expression of the fusion protein. In certain embodiments, the recombinant nucleic acid is optimized for expression codons. After expression in a host cell, a purification method known in the art can be used to separate or purify the fusion protein. V. System

[0141] On the other hand, systems that can be used to edit one or more nucleic acids are provided herein. These systems include one or more of the fusion proteins (or recombinant nucleic acids, constructs, vectors or host cells) described above. In certain embodiments, these systems further include one or more additional elements that can be used to edit one or more nucleic acids. For example, the system provided herein may further include a donor polynucleotide. As another example, a system including a fusion protein containing a Cas nuclease may further include one or more guide nucleic acids and / or one or more donor polynucleotide sequences. Donor polynucleotides and guide nucleic acids are described in detail below. The system provided herein may be used to carry out the method described in Chapter VI of this disclosure. A. Donor polynucleotide

[0142] The systems and methods of the present disclosure may include donor polynucleotides. "Donor polynucleotides", "donor molecules" or "donor templates" are nucleotide polymers or oligomers intended for insertion at target polynucleotides (typically target genomic sites). The donor sequence may be one or more transgenics, expression cassettes or nucleotide sequences of interest. The donor molecule may be a single-stranded, partially double-stranded or double-stranded donor DNA molecule. The donor polynucleotide may be a natural or modified polynucleotide, an RNA-DNA chimera, or a DNA fragment, a single-stranded, or at least partially double-stranded, or completely double-stranded DNA molecule, or a PGR-amplified ssDNA, or at least a portion of a dsDNA fragment. In some embodiments, the donor DNA molecule is a portion of a circularized DNA molecule. In some cases, a completely double-stranded donor DNA may provide increased stability because dsDNA fragments are generally more resistant to nuclease degradation than ssDNA.

[0143] The donor molecule can comprise at least 10 consecutive nucleotides (often referred to as homology arms) wherein the nucleic acid molecule is at least 70% identical to a genomic nucleotide sequence, such that these consecutive nucleotides are sufficient to homologously recombine the donor DNA molecule into the genome of the cell at the targeted genomic DNA sequence following cleavage, for example, by a site-directed nuclease. In some embodiments, the donor DNA molecule can comprise at least about 10, 20, 30, 50, 70, 80, 100, 150, 200, 250, 300, 250, 400, 450, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 7500, 10000, 15,000, or 20,000 nucleotides, including any value within this range not expressly recited herein, wherein the donor DNA molecule is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the genomic nucleic acid sequence. In some embodiments, the donor DNA molecule can be substantially complementary to the genomic nucleic acid sequence. In some embodiments, the donor DNA molecule comprises a heterologous nucleic acid sequence. In some embodiments, the donor DNA molecule comprises at least one expression cassette. In some embodiments, the donor DNA molecule can comprise a transgenic comprising at least one expression cassette. In some embodiments, the donor DNA molecule comprises an allele modification of a gene that is natural to the target genome. The allele modification can include at least one nucleotide insertion, at least one nucleotide deletion and / or at least one nucleotide substitution. In some embodiments, the allele modification can include a small insertion or deletion. In some embodiments, the donor DNA molecule comprises an arm homologous to the target genome site. In some embodiments, the donor DNA molecule comprises at least 100 consecutive nucleotides that are at least 90% identical to the genomic nucleic acid sequence, and optionally can further comprise a heterologous nucleic acid sequence, such as a transgenic.

[0144] The donor polynucleotide can be any suitable nucleic acid. In some embodiments, the donor nucleic acid is a part of a donor template. In some embodiments, the donor template is a part of a plasmid or linear nucleic acid. In some embodiments, the donor nucleic acid is a part of a chromosome.

[0145] In some embodiments, the donor polynucleotide comprises a nucleotide sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:75 or SEQ ID NO:76. B. Guide nucleic acid

[0146] In some cases, the systems and methods described herein include at least one guide nucleic acid polynucleotide. In some cases, the systems and methods described herein include multiple guide nucleic acids. In some embodiments, the polynucleotide can be deoxyribonucleic acid (DNA). In some cases, the DNA sequence can be single-stranded or double-stranded. In some embodiments, the at least one guide nucleic acid polynucleotide can be ribonucleic acid (guide RNA).

[0147] In some embodiments, a nuclease can be compounded with at least one guide RNA polynucleotide. The at least one guide RNA polynucleotide can include a nucleic acid targeting region, which includes a sequence complementary to a nucleic acid sequence on a targeting polynucleotide (such as a targeting genomic locus or gene) to confer sequence specificity targeted by the nuclease. In some embodiments, the at least one guide RNA polynucleotide can include two separate nucleic acid molecules (which can be referred to as dual-guide nucleic acids) or a single nucleic acid molecule (which can be referred to as a single guide nucleic acid (e.g., a single guide RNA or sgRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid including a fused CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). In some embodiments, the guide nucleic acid is a single guide nucleic acid including crRNA. In some embodiments, the guide nucleic acid is a single guide nucleic acid including crRNA but lacking tracrRNA. In some embodiments, the guide nucleic acid is a dual guide nucleic acid including non-fused crRNA and tracrRNA. Exemplary dual guide nucleic acids can include crRNA-like molecules and tracrRNA-like molecules. Exemplary single guide nucleic acids can include crRNA-like molecules. Exemplary single guide nucleic acids can include fused crRNA-like molecules and tracrRNA-like molecules.

[0148] The crRNA can include a nucleic acid targeting segment (e.g., a spacer region) of a guide nucleic acid and a nucleotide stretch that can form one half of a double-stranded duplex of a Cas protein binding segment of a guide nucleic acid.

[0149] The tracrRNA may include a nucleotide segment that forms the other half of the double-stranded duplex of the Cas protein binding segment of the gRNA. The nucleotide segment of the crRNA may be complementary to the nucleotide segment of the tracrRNA and hybridize therewith to form a double-stranded duplex of the Cas protein binding domain of the guide nucleic acid.

[0150] CrRNA and tracrRNA can hybridize to form a guide nucleic acid. CrRNA can also provide a single-stranded nucleic acid targeting segment (e.g., a spacer region) hybridized with a target nucleic acid recognition sequence (e.g., a protospacer). The sequence of crRNA or tracrRNA molecules including the spacer region can be designed to be specific to the species of the guide nucleic acid to be used.

[0151] Whether the nuclease requires only a crRNA molecule or it requires both a crRNA molecule and a tracrRNA molecule (whether covalently linked or not) depends on the CRISPR-associated nuclease used.

[0152] In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid can be between 18 and 72 nucleotides. The length of the nucleic acid targeting region (e.g., spacer region) of the guide nucleic acid can be from about 12 nucleotides to about 100 nucleotides. For example, the length of the nucleic acid targeting region (e.g., spacer region) of the guide nucleic acid can be from about 12 nucleotides (nt) to about 80nt, from about 12nt to about 50nt, from about 12nt to about 40nt, from about 12nt to about 30nt, from about 12nt to about 25nt, from about 12nt to about 20nt, from about 12nt to about 19nt, from about 12nt to about 18nt, from about 12nt to about 17nt, from about 12nt to about 16nt or from about 12nt to about 15nt. Alternatively, the length of the DNA targeting segment can be from about 18 nt to about 20 nt, from about 18 nt to about 25 nt, from about 18 nt to about 30 nt, from about 18 nt to about 35 nt, from about 18 nt to about 40 nt, from about 18 nt to about 45 nt, from about 18 nt to about 50 nt, from about 18 nt to about 60 nt, from about 18 nt to about 70 nt, from about 18 nt to about 80 nt, from about 18 nt to about 90 nt, from about 18 nt to about 100 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, from about 20 nt to about 60 nt, from about 20 nt to about 70 nt, from about 20 nt to about 80 nt, from about 20 nt to about 90 nt, or from about 20 nt to about 100 nt. The nucleic acid targeting region can be at least 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. The nucleic acid targeting region (e.g., a spacer sequence) can be at most 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length.

[0153] In some embodiments, the length of the nucleic acid targeting region (e.g., spacer) of the guide nucleic acid is 20 nucleotides. In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid is 19 nucleotides. In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid is 18 nucleotides. In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid is 17 nucleotides. In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid is 16 nucleotides. In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid is 21 nucleotides. In some embodiments, the length of the nucleic acid targeting region of the guide nucleic acid is 22 nucleotides.

[0154] The length of the nucleotide sequence of the guide nucleic acid complementary to the nucleotide sequence of the target nucleic acid (target sequence) can be, for example, at least about 12 nt, at least about 15 nt, at least about 18 nt, at least about 19 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 35 nt, or at least about 40 nt. The length of the nucleotide sequence of the guide nucleic acid complementary to the nucleotide sequence of the target nucleic acid (target sequence) can be from about 12 nucleotides (nt) to about 80 nt, from about 12 nt to about 50 nt, from about 12 nt to about 45 nt, from about 12 nt to about 40 nt, from about 12 nt to about 35 nt, from about 12 nt to about 30 nt, from about 12 nt to about 25 nt, from about 12 nt to about 20 nt, from about 12 nt to about 19 nt, from about 19 nt to about 20 ... From about 19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 nt to about 35 nt, from about 19 nt to about 40 nt, from about 19 nt to about 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about 60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt, from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, from about 20 nt to about 45 nt, from about 20 nt to about 50 nt, or from about 20 nt to about 60 nt.

[0155] The protospacer sequence of the targeting polynucleotide can be identified by identifying the protospacer adjacent motif (PAM) within the region of interest and selecting a region of desired size upstream or downstream of the PAM as the protospacer. The corresponding spacer sequence can be designed by determining the complementary sequence of the protospacer region.

[0156] Spacer sequences can be identified using a computer program (e.g., machine readable code). The computer program can use variables such as predicted melting temperature, secondary structure formation and predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, GC%, genomic frequency, methylation status, SNP presence, etc.

[0157] The percent complementarity between a nucleic acid targeting sequence (e.g., a spacer sequence of at least one guide polynucleotide as disclosed herein) and a target nucleic acid (e.g., a protospacer sequence of one or more target loci as disclosed herein) can be at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%. The percent complementarity between a nucleic acid targeting sequence and a target nucleic acid can be at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% over about 20 consecutive nucleotides.

[0158] The Cas protein binding segment of the guide nucleic acid can include two nucleotide segments (e.g., crRNA and tracrRNA) that are complementary to each other. Two nucleotide segments (e.g., crRNA and tracrRNA) that are complementary to each other can be covalently linked by intervening nucleotides (e.g., joints in the case of a single guide nucleic acid). Two nucleotide segments (e.g., crRNA and tracrRNA) that are complementary to each other can hybridize to form a double-stranded RNA duplex or hairpin of the Cas protein binding segment, thus producing a stem-loop structure. CrRNA and tracrRNA can be covalently linked via the 3' end of crRNA and the 5' end of tracrRNA. Alternatively, tracrRNA and crRNA can be covalently linked via the 5' end of tracrRNA and the 3' end of crRNA.

[0159] The length of the Cas protein binding segment of the guide nucleic acid can be from about 10 nucleotides to about 100 nucleotides, for example, from about 10 nucleotides (nt) to about 20nt, from about 20nt to about 30nt, from about 30nt to about 40nt, from about 40nt to about 50nt, from about 50nt to about 60nt, from about 60nt to about 70nt, from about 70nt to about 80nt, from about 80nt to about 90nt, or from about 90nt to about 100nt. For example, the length of the Cas protein binding segment of the guide nucleic acid can be from about 15 nucleotides (nt) to about 80nt, from about 15nt to about 50nt, from about 15nt to about 40nt, from about 15nt to about 30nt, or from about 15nt to about 25nt.

[0160] The length of the dsRNA duplex of the Cas protein binding segment of the guide nucleic acid can be from about 6 base pairs (bp) to about 50bp. For example, the length of the dsRNA duplex of the protein binding segment can be from about 6bp to about 40bp, from about 6bp to about 30bp, from about 6bp to about 25bp, from about 6bp to about 20bp, from about 6bp to about 15bp, from about 8bp to about 40bp, from about 8bp to about 30bp, from about 8bp to about 25bp, from about 8bp to about 20bp or from about 8bp to about 15bp. For example, the length of the dsRNA duplex of the Cas protein binding segment can be from about 8bp to about 10bp, from about 10bp to about 15bp, from about 15bp to about 18bp, from about 18bp to about 20bp, from about 20bp to about 25bp, from about 25bp to about 30bp, from about 30bp to about 35bp, from about 35bp to about 40bp or from about 40bp to about 50bp.

[0161] In some embodiments, the length of the dsRNA duplex of the Cas protein binding segment can be 36 base pairs. The percentage of complementarity between the nucleotide sequences of the dsRNA duplexes that hybridize to form the protein binding segment can be at least about 60%. For example, the percentage of complementarity between the nucleotide sequences of the dsRNA duplexes that hybridize to form the protein binding segment can be at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or at least about 99%. In some cases, the percentage of complementarity between the nucleotide sequences of the dsRNA duplexes that hybridize to form the protein binding segment is 100%.

[0162] The length of the joint (e.g., the sequence connecting crRNA and tracrRNA in a single guide nucleic acid) can be from about 3 nucleotides to about 100 nucleotides. For example, the length of the joint can be from about 3 nucleotides (nt) to about 90nt, from about 3 nucleotides (nt) to about 80nt, from about 3 nucleotides (nt) to about 70nt, from about 3 nucleotides (nt) to about 60nt, from about 3 nucleotides (nt) to about 50nt, from about 3 nucleotides (nt) to about 40nt, from about 3 nucleotides (nt) to about 30nt, from about 3 nucleotides (nt) to about 20nt or from about 3 nucleotides (nt) to about 10nt. For example, the length of the linker can be from about 3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt. In some embodiments, the linker of the DNA targeting RNA is 4 nt.

[0163] The guide nucleic acid of the system of the present disclosure can include modifications or sequences that provide additional desired features (e.g., stability of modification or regulation; subcellular targeting; tracking with a fluorescent marker; binding sites for proteins or protein complexes, etc.). Examples of such modifications include, for example, a 5' cap (7-methylguanylate cap (m7G)); a 3' polyadenylation tail (3' poly (A) tail); a riboswitch sequence (e.g., to allow regulated stability and / or regulated accessibility of proteins and / or protein complexes); a stability control sequence; a sequence that forms a dsRNA duplex (hairpin); a modification or sequence that targets RNA to a subcellular location (e.g., nucleus, mitochondria, chloroplasts, etc.); a modification or sequence that provides for tracking (e.g., direct conjugation to a fluorescent molecule, conjugation to a portion that facilitates fluorescence detection, a sequence that allows fluorescence detection, etc.); a modification or sequence that provides a binding site for a protein (e.g., a protein that acts on DNA, including transcription activators, transcription repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and combinations thereof).

[0164] The guide nucleic acid may include one or more modifications (e.g., base modifications, backbone modifications) to provide a nucleic acid with new or enhanced features (e.g., improved stability). The guide nucleic acid may include a nucleic acid affinity tag. Nucleoside may be a base-sugar combination. The base portion of the nucleotide may be a heterocyclic base. The two most common types of such heterocyclic bases are purine and pyrimidine. The nucleotide may be a nucleoside further comprising a phosphate group covalently attached to the sugar portion of the nucleoside. For those nucleosides comprising pentofuranosyl sugars, the phosphate group may be attached to the 2', 3' or 5' hydroxyl portion of the sugar. When forming a guide nucleic acid, the phosphate group may covalently attach nucleosides adjacent to each other to form a linear polymer compound. Furthermore, the corresponding ends of this linear polymer compound may be further connected to form a cyclic compound; however, a linear compound may be suitable. In addition, the linear compound may have internal nucleotide base complementarity and may therefore be folded in a manner that facilitates the production of a fully or partially double-stranded compound. In addition, within the guide nucleic acid, the phosphate group may generally be involved in forming a nucleoside-internucleoside backbone of the guide nucleic acid. The linkage or backbone of the guide nucleic acid can be a 3' to 5' phosphodiester linkage.

[0165] The guide nucleic acid can comprise a modified backbone and / or a modified internucleoside linkage. The modified backbone can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0166] Suitable modified guide nucleic acid backbones containing phosphorus atoms therein may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (such as 3'-alkylenephosphonates, 5'-alkylenephosphonates, chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), phosphorodiamidates, thionylphosphoramidates, thionylphosphonates, thionylphosphonate triesters, selenophosphoroates, and boranophosphates with normal 3'-5' linkages, 2'-5' connection analogs, and those with reverse polarity in which one or more internucleotide linkages are 3' to 3', 5' to 5' or 2' to 2' linkages. Suitable guide nucleic acids with reverse polarity may include a single 3' to 3' linkage at the most 3' internucleotide linkage (such as a single reverse nucleotide residue in which a nucleobase is missing or has a hydroxyl group in its place). Various salts (eg, potassium chloride or sodium chloride), mixed salts, and free acid forms may also be included.

[0167] The guide nucleic acid can comprise one or more phosphorothioate and / or heteroatom internucleoside linkages, particularly -CH2-NH-O-CH2-, -CH2-N(CH3)-O-CH2- (methylene(methylimino) or MMI backbone), -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2-, and -ON(CH3)-CH2-CH2- (where the native phosphodiester internucleoside linkage is represented as -OP(=O)(OH)-O-CH2-).

[0168] Instruct nucleic acid can comprise morpholino backbone structure.For example, nucleic acid can comprise 6 yuan morpholino ring to replace ribose ring.In some of these embodiments, diamino phosphorothioate or other non-phosphodiester internucleoside linkage replaces phosphodiester linkage.

[0169] The guide nucleic acid can include such polynucleotide backbones, which are formed by short-chain alkyl or cycloalkyl nucleoside inter-linkages, mixed heteroatoms and alkyl or cycloalkyl nucleoside inter-linkages or one or more short-chain heteroatoms or heterocyclic nucleoside inter-linkages. These can include those with the following: morpholino linkages (formed by the sugar moiety of nucleosides); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formyl acetyl and thioformyl acetyl backbones; methylene formyl acetyl and thioformyl acetyl backbones; ribose acetyl backbones; olefin-containing backbones; aminosulfonate backbones; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S and CH2 components.

[0170] Instruct nucleic acid can include nucleic acid mimics.The term "mock" can be intended to include such polynucleotides, wherein only the furanose ring or the furanose ring and the internucleotide linkage are replaced by non-furanose groups, and only the replacement of the furanose ring can also be referred to as a sugar substitute. The heterocyclic base moiety or the modified heterocyclic base moiety can be retained for hybridization with an appropriate target nucleic acid. A kind of such nucleic acid can be a peptide nucleic acid (PNA). In PNA, the sugar backbone of polynucleotides can be replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. Nucleotide can be retained and directly or indirectly bonded to the aza nitrogen atom of the amide portion of the backbone. The backbone in the PNA compound can include two or more connected aminoethylglycine units that give the PNA an amide-containing backbone. The heterocyclic base moiety can be directly or indirectly combined with the aza nitrogen atom of the amide portion of the backbone.

[0171] Instruct nucleic acid can include connected morpholino units (morpholino nucleic acid), and these morpholino units have heterocyclic bases attached to morpholino rings. Linking groups can connect morpholino monomer units in morpholino nucleic acids. Oligomeric compounds based on nonionic morpholino can have less desirable interactions with cell proteins. Polynucleotides based on morpholino can be nonionic mimics of instructing nucleic acid. Various compounds in morpholino species can be connected using different linking groups. Another type of polynucleotide mimics can be referred to as cyclohexenyl nucleic acid (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced by cyclohexenyl rings. Phosphoramidite monomers protected by CeNA DMT can be prepared and used for oligomeric compound synthesis using phosphoramidite chemistry. CeNA monomers are incorporated into nucleic acid chains to increase the stability of DNA / RNA hybrids. CeNA oligoadenylic acid can form complexes with nucleic acid complements, and these complexes have stability similar to natural complexes. Another modification may include locked nucleic acids (LNA), in which the 2'-hydroxyl group is linked to the 4' carbon atom of the sugar ring, thereby forming a 2'-C, 4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage may be a methylene (-CH2-) group bridging the 2' oxygen atom and the 4' carbon atom, where n is 1 or 2. LNA and LNA analogs may exhibit very high duplex thermal stability (Tm = +3 to +10°C) with complementary nucleic acids, stability to 3'-exonucleolytic degradation, and good solubility properties.

[0172] The guide nucleic acid may comprise one or more substituted sugar moieties. Suitable polynucleotides may comprise a sugar substituent group selected from the group consisting of: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Particularly suitable is O((CH2) n O) m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON((CH2) n CH3)2, wherein n and m are from 1 to about 10. The sugar substituent group may be selected from: C1 to C 10Lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of guide nucleic acid, or group for improving the pharmacodynamic properties of guide nucleic acid and other substituents with similar properties. Suitable modifications may include 2'-methoxyethoxy (2'-O-CH2 CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE, i.e., alkoxyalkoxy group). Another suitable modification may include 2'-dimethylaminooxyethoxy (O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE) and 2'-dimethylaminoethoxyethoxy (also known as 2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), 2'-O-CH2-O-CH2-N(CH3)2.

[0173] Other suitable sugar substituent groups may include methoxy (-O-CH3), aminopropoxy (-OCH2CH2NH2), allyl (-CH2-CH=CH2), -O-allyl (-O--CH2—CH=CH2) and fluorine (F). The 2'-sugar substituent group may be in the arabinose (upper) position or the ribose (lower) position. A suitable 2'-arabinose modification is 2'-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3' position of the sugar on the 3' terminal nucleoside or in the 2'-5' linked nucleotide and the 5' position of the 5' terminal nucleotide. The oligomeric compound may also have a sugar mimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

[0174] The guide nucleic acid may also include nucleobase (or "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)), and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases may include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-sulfhydryl, 8-sulfanyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3',2':4,5)pyrrolo(2,3-d)pyrimidin-2-one).

[0175] Heterocyclic base moieties may include those in which purine or pyrimidine bases are replaced by other heterocycles such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases may be used to increase the binding affinity of polynucleotide compounds. These nucleobases may include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions may increase nucleic acid duplex stability by 0.6°C-1.2°C, and may be suitable base substitutions (e.g., when combined with 2'-O-methoxyethyl sugar modifications).

[0176] The modification of the guide nucleic acid can include chemically connecting to the guide nucleic acid one or more parts or conjugates that can enhance the activity, cellular distribution or cellular uptake of the guide nucleic acid. These parts or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups can include but are not limited to intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers and groups that can enhance the pharmacodynamic properties of oligomers. Conjugate groups can include but are not limited to cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dyes. Groups that enhance pharmacodynamic properties include groups that improve uptake, enhance degradation resistance and / or strengthen sequence-specific hybridization with target nucleic acids. Groups that can enhance pharmacokinetic properties include groups that improve the uptake, distribution, metabolism or excretion of nucleic acids. The conjugate moiety may include, but is not limited to, a lipid moiety such as a cholesterol moiety, cholic acid, a thioether (e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g., dodecandiol or undecyl residues), a phospholipid (e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glyceryl-3-H-phosphonate), a polyamine or a polyethylene glycol chain or adamantaneacetic acid, a palmityl moiety or octadecylamine or a hexylamino-carbonyl-hydroxycholesterol moiety.

[0177] In some embodiments, at least one guide RNA polynucleotide of the systems or methods provided herein can bind to at least a portion of a genome (e.g., a plant genome) or a gene (e.g., a plant gene). In some cases, the at least one guide RNA polynucleotide is capable of complexing with a site-directed nuclease to direct the site-directed nuclease to target a portion of a target nucleic acid (e.g., a site in a genome or gene).

[0178] In some embodiments, the systems described herein include at least one guide RNA polynucleotide capable of forming a complex with the site-directed nuclease portion of the fusion protein of the system. In some embodiments, the systems described herein include at least two (e.g., at least three, at least four, at least five, or at least six) different guide RNA polynucleotides capable of forming a complex with the site-directed nuclease portion of the fusion protein of the system.

[0179] In some embodiments, the guide nucleic acid comprises a nucleotide sequence that is at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs: 2, 3, 11, 12, 23-26, or 28-31.

[0180] Also provided herein are kits comprising components of the systems described in the present disclosure. In some embodiments, the kits comprise one or more of the fusion proteins and / or polynucleotides described herein. VI. Methods

[0181] On the other hand, provided herein are methods for editing one or more nucleic acids using the fusion proteins and / or systems described herein. In some embodiments, these methods include contacting a nucleic acid (i.e., a nucleic acid to be edited) comprising a fusion protein binding site with at least one fusion protein as described herein, wherein contacting the nucleic acid with at least one fusion protein results in editing of the nucleic acid. The nucleic acid (i.e., the nucleic acid to be edited) can be any suitable nucleic acid. In some embodiments, the nucleic acid is part of a chromosome. In some embodiments, the nucleic acid is part of a genome (e.g., a plant genome).

[0182] As described herein and shown in the following examples, the methods provided herein can result in an increase in the frequency of one or more desired nucleic acid editing outcomes (e.g., fragment excision, fragment inversion, fragment replacement via HDR, chromosome rearrangement). By using a fusion protein that targets (i.e., by a site-directed nuclease) to a specific nucleic acid sequence (e.g., a genomic site, a donor template site), the method herein can be used to increase (or reduce) the frequency of one or more desired nucleic acid editing outcomes. In some embodiments, the fusion protein targets a specific chain of nucleic acid. In some embodiments, the fusion protein targets a site upstream or downstream of a nuclease cleavage site. In some embodiments, the fusion protein targets a nucleic acid in a specific orientation.

[0183] In some embodiments, the nucleic acid to be edited by the method comprises a target region. As used herein, "target region" refers to a portion of a nucleic acid targeted for editing. For example, the target region can be a portion of a gene to be edited. In some embodiments, the fusion protein provided herein is targeted to a binding site within and / or outside the target region (e.g., two sites within the target region, a site within the target region, and a site outside the target region or two sites outside the target region), as described below. In some embodiments, each fusion protein binding site is proximal to the nuclease cleavage site. In some embodiments, the target region is flanked by a nuclease cleavage site. In some embodiments, the nucleic acid comprises a first binding site adjacent to the 5' end of the target region and a second binding site adjacent to the 3' end of the target region.

[0184] In some embodiments, as described in detail below, the nucleic acid to be edited comprises a first binding site and a second binding site. In some embodiments, the first binding site and the second binding site are different sequences, and the method includes providing two different fusion proteins, one binding to the first binding site and one binding to the second binding site. In some embodiments, the first binding site and the second binding site are the same sequence, and the method includes providing a fusion protein that can bind to both the first binding site and the second binding site.

[0185] In some embodiments, the method herein includes providing a donor nucleic acid, which includes a third binding site and a fourth binding site. In some embodiments, the third binding site and the fourth binding site are different sequences, and the method includes providing two different fusion proteins, one binding the third binding site and one binding the fourth binding site. In some embodiments, the third binding site and the fourth binding site are the same sequence, and the method includes providing a fusion protein that can bind both the third binding site and the fourth binding site. In addition, in some embodiments, the third and / or fourth binding site is the same sequence as the first and / or second binding site. The first, second, third and fourth binding sites can include any combination of sequences, from all four having the same sequence to all four having different sequences. In some embodiments, nucleic acid (i.e., nucleic acid to be edited) is a part of the first chromosome, and the donor nucleic acid is a part of the second chromosome. In some embodiments, the first chromosome and the second chromosome are different chromosomes. In some embodiments, the first chromosome and the second chromosome are homologous chromosomes. In some embodiments, the first chromosome and the second chromosome are non-homologous chromosomes. In some embodiments, the first chromosome and the second chromosome are identical chromosomes.

[0186] In some embodiments of the methods provided herein, for example, in the exemplary embodiments described below, the site-directed nuclease of at least one fusion protein includes a CRISPR-associated nuclease. In such embodiments, the method may further include providing a guide RNA for targeting the fusion protein to the binding site. In some embodiments, the method includes providing at least one first guide RNA and at least one second guide RNA. In some embodiments, the at least one first guide RNA includes a nucleotide sequence having complementarity with the first binding site of the nucleic acid to be edited. In some embodiments, the at least one second guide RNA includes a nucleotide sequence having complementarity with the second binding site of the nucleic acid to be edited. In some embodiments, in the case where these methods include providing a donor nucleic acid, these methods further include providing at least one third guide RNA and at least one fourth guide RNA. In some embodiments, the at least one third guide RNA includes a nucleotide sequence having complementarity with the third binding site (ie, on the donor nucleic acid). In some embodiments, the at least one fourth guide RNA includes a nucleotide sequence having complementarity with the fourth binding site (ie, on the donor nucleic acid).

[0187] In some embodiments, the frequency of the desired nucleic acid editing outcome can be increased or decreased by targeting the fusion protein to bind within and / or outside the target area. In some embodiments of the methods provided herein, the editing of the nucleic acid is excision (ie, removal), inversion (ie, reversal direction) or replacement of at least a portion of the target area. In some embodiments, the editing is a chromosome rearrangement. In some embodiments, the chromosome rearrangement is a reciprocal translocation. In some embodiments, the chromosome rearrangement is a non-reciprocal translocation. Several non-limiting exemplary embodiments of fusion protein targeting and editing outcomes with increased frequency are discussed below with reference to the accompanying drawings, and various aspects of these methods are discussed in addition thereto. Although exemplary embodiments include Cas enzymes as fusion proteins SDN, and thereby include discussions of gRNA-mediated binding and PAM sequences, it should be understood that other site-directed nucleases (e.g., zinc finger nucleases, TAL effector nucleases, large-range nucleases, etc.) can be used to similarly target binding (e.g., targeting within and / or outside the target area), as discussed in Section III.A above.

[0188] In some embodiments, as shown in Example 1 of the present disclosure, the methods provided herein result in an increase in the frequency of fragment inversions between paired cleavage sites on a nucleic acid (e.g., at a genomic locus). An exemplary embodiment is shown in Figure 1In, wherein the method includes two fusion proteins of the first binding site and the second binding site on the same chain of the DNA polynucleotide. The first binding site is adjacent to the 5' end of the target region (that is, the region is located between the nuclease cleavage sites), and the second binding site is adjacent to the 3' end of the target region. For illustrative purposes, the fusion protein in this embodiment includes LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. The DNA polynucleotide is a part of the ZmDMR6 gene, the first binding site includes a PAM sequence located in the promoter of the gene, and the second binding site includes a PAM sequence located in intron 1 of the gene. LbCas12a (that is, the SDN of the fusion protein) cleaves the polynucleotide from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA 1") is upstream of the cleavage site (that is, outside the target region), the fusion protein bound to the first binding site remains bound to the end of the cleavage polynucleotide outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA 2") is upstream of the cleavage site (i.e., within the target region), the fusion protein bound to the second binding site remains bound to the downstream end of the target region. Without being bound by any particular theory, the dimerization of the fusion protein bound to the first and second binding sites (i.e., by the dimerization of Trex2) will bring the downstream end of the target region and the polynucleotide ends upstream of the target region into close proximity, where they will be connected by DNA repair mechanisms (such as NHEJ or MMEJ). Connection to the remaining polynucleotide ends (i.e., those not bound by the fusion protein) by DNA repair mechanisms therefore results in inversion of the target region.

[0189] In some embodiments, as described in Example 3 of the present disclosure, the methods provided herein result in an increase in the frequency of fragment excision (i.e., removal) between paired cleavage sites on a nucleic acid (e.g., at a genomic locus). An exemplary embodiment is shown in Figure 2In, wherein the method includes two fusion proteins of the first binding site and the second binding site on the opposite strand of the DNA polynucleotide. The first binding site is adjacent to the 5' end of the target region, and the second binding site is adjacent to the 3' end of the target region. For illustrative purposes, the fusion protein in this embodiment includes LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. The DNA polynucleotide is a part of the ZmDMR6 gene, the first binding site includes a PAM sequence located in the promoter of the gene, and the second binding site includes a PAM sequence located in intron 1 of the gene. LbCas12a (i.e., the SDN of the fusion protein) cleaves the polynucleotide from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA 1") is upstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the first binding site remains bound to the end of the cleavage polynucleotide outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA 2") is downstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the second binding site remains bound to the cleavage polynucleotide end outside and downstream of the target region. Without being bound by any particular theory, dimerization of the fusion protein bound to the first binding site and the second binding site will bring the cleavage polynucleotide ends upstream and downstream of the target region into close proximity, where they will be connected by DNA repair mechanisms (such as NHEJ or MMEJ), resulting in excision of the target region.

[0190] In certain embodiments, the increase of the frequency of fragment excision as described above can also be achieved by using a method for targeting a fusion protein in a target area. An exemplary embodiment of this method includes two fusion proteins of the first binding site and the second binding site on the opposite strand of a DNA polynucleotide. The first binding site is adjacent to the 5' end of the target area, the second binding site is adjacent to the 3' end of the target area, and both the first binding site and the second binding site are in the target area. Therefore, the fusion protein remains bound to the end of the target area after the cleavage mediated by SDN. Not subject to any particular theory, the dimerization of the fusion protein bound to the first binding site and the second binding site will make the cleavage polynucleotide ends of the target area close to each other, wherein they will be connected (i.e., forming a circular polynucleotide) by a DNA repair mechanism (such as NHEJ or MMEJ). The connection of the remaining polynucleotide ends (i.e., those not bound by the fusion protein) by the DNA repair mechanism therefore leads to the excision of the target area.

[0191] In some embodiments, the method provided herein further includes providing a donor nucleic acid comprising a fusion protein binding site. As described above, donor nucleic acids can be used together with SDN to provide a template for homology-directed repair (HDR). In some embodiments, donor nucleic acids can also be used to provide a replacement fragment to be inserted to replace the target region. In some embodiments, donor nucleic acids can be used to promote translocation (e.g., chromosomal translocation). By using a fusion protein targeting a specific sequence in both the nucleic acid to be edited and the donor nucleic acid, the method herein can be used to increase (or reduce) the frequency of one or more desired nucleic acid editing outcomes. In some embodiments, the donor nucleic acid provided in the method herein includes a third binding site and a fourth binding site (i.e., wherein the nucleic acid to be edited includes a first binding site and a second binding site) and a donor nucleotide region. As used herein, "donor nucleotide region" refers to a portion of a donor nucleic acid flanked by a fusion protein binding site. Several non-limiting exemplary embodiments of methods including donor nucleic acids are discussed below with reference to the accompanying drawings.

[0192] In some embodiments, as described in Example 5 of the present disclosure, the methods provided herein result in an increase in the frequency of fragment replacement or targeted insertion between paired cleavage sites on a nucleic acid (e.g., at a genomic locus). Two exemplary embodiments are shown in Figure 3A and Figure 3BIn, wherein the method includes two fusion proteins of the first binding site and the second binding site on the opposite strands of the DNA polynucleotide (i.e., the nucleotide to be edited, shown here as "genomic DNA") and two fusion proteins of the third binding site and the fourth binding site on the opposite strands of the donor nucleic acid (shown here as "donor DNA"). In two embodiments, the first binding site is adjacent to the 5' end of the target region (i.e., the region is between the nuclease cleavage sites), the second binding site is adjacent to the 3' end of the target region, the third binding site is adjacent to the 5' end of the donor nucleotide region (i.e., the donor nucleotide region is between the nuclease cleavage sites), and the fourth binding site is adjacent to the 3' end of the donor nucleotide region. For illustrative purposes, the fusion proteins in these embodiments include LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. LbCas12a (i.e., the SDN of the fusion protein) cleaves polynucleotides (i.e., both nucleotides to be edited and donor nucleic acids) from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleavage polynucleotide. Because the first binding site (indicated by "gRNA-a") is upstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the first binding site remains bound to the cleavage polynucleotide end outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA-b") is downstream of the cleavage site (i.e., outside the target region), the fusion protein bound to the second binding site remains bound to the cleavage polynucleotide end outside the target region and downstream thereof. Because the third binding site (indicated by "gRNA-a / c") is downstream of the cleavage site (i.e., in the donor nucleotide region), the fusion protein bound to the third binding site remains bound to the cleavage polynucleotide end of the donor nucleotide region (i.e., the upstream end of the donor nucleotide region). Because the fourth binding site (indicated by "gRNA-b / d") is upstream of the cleavage site (i.e., within the donor nucleotide region), the fusion protein bound to the fourth binding site remains bound to the cleavage polynucleotide end of the donor nucleotide region (i.e., to the downstream end of the donor nucleotide region). Without being bound by any particular theory, the dimerization of the fusion protein bound to the first and third binding sites (i.e., by the dimerization of Trex2) will bring the cleavage polynucleotide end upstream of the target region into close proximity with the upstream end of the donor nucleotide region, and the dimerization of the fusion protein bound to the second and fourth binding sites will bring the cleavage polynucleotide end downstream of the target region into close proximity with the downstream end of the donor nucleotide region. Connection of the closely approached polynucleotide ends by DNA repair mechanisms (such as NHEJ or MMEJ) will result in replacement of the target region with the donor nucleotide region.The donor nucleotide region can also be inserted in the reverse orientation (ie, if the fusion protein that binds to the first and fourth binding sites dimerizes and the fusion protein that binds to the second and third binding sites dimerizes).

[0193] exist Figure 3A In the example shown in , the donor nucleotide region does not contain homology arms, so NHEJ or MMEJ repair is more likely than HDR. Figure 3B In the embodiment shown in , the donor nucleotide region comprises a homology arm. Without being bound by any particular theory, in Figure 3B In the embodiments shown in , dimerization of the fusion protein bound to the first and third binding sites and dimerization of the fusion protein bound to the second and fourth binding sites will promote HDR-mediated repair by bringing the donor nucleotide region homology arms into close proximity with the homologous sequences in the nucleotide to be edited (e.g., genomic DNA).

[0194] In some embodiments, as described in Example 7 of the present disclosure, the methods provided herein result in an increase in the frequency of translocation between two polynucleotides (e.g., between two chromosomes). Exemplary embodiments are shown in Figure 4In, wherein the method includes two fusion proteins of the first binding site and the second binding site on the opposite strand of the DNA polynucleotide (i.e., the nucleotide to be edited, shown here as "acceptor chromosome") and two fusion proteins of the third binding site and the fourth binding site on the opposite strand of the donor nucleic acid (shown here as "donor chromosome"). The first binding site is adjacent to the 5' end of the target region (i.e., the region is between the nuclease cleavage sites), the second binding site is adjacent to the 3' end of the target region, the third binding site is adjacent to the 5' end of the donor nucleotide region (i.e., the region of the donor nucleotide is between the nuclease cleavage sites), and the fourth binding site is adjacent to the 3' end of the donor nucleotide region. For illustrative purposes, the fusion protein in these embodiments includes LbCas12a as SDN connected to Trex2 exonuclease as a non-specific end processing enzyme. LbCas12a (i.e., the SDN of the fusion protein) cleaves polynucleotides (i.e., both nucleotides to be edited and donor nucleic acids) from PAM in the 3' direction, and the fusion protein remains bound to the binding site containing PAM on the end of the cleaved polynucleotide. Because the first binding site (indicated by "gRNA-a") is upstream of the cleavage site (i.e., outside the target region), the fusion protein combined with the first binding site remains combined with the cleavage polynucleotide end outside the target region and upstream thereof. Because the second binding site (indicated by "gRNA-b") is downstream of the cleavage site (i.e., outside the target region), the fusion protein combined with the second binding site remains combined with the cleavage polynucleotide end outside the target region and downstream thereof. Because the third binding site (indicated by "gRNA-c") is upstream of the cleavage site (i.e., outside the donor nucleotide region), the fusion protein combined with the third binding site remains combined with the cleavage donor nucleotide end outside the donor nucleotide region and upstream thereof. Because the fourth binding site (indicated by "gRNA-d") is downstream of the cleavage site (i.e., outside the donor nucleotide region), the fusion protein combined with the fourth binding site remains combined with the cleavage donor nucleotide end outside the donor nucleotide region and downstream thereof. Without being bound by any particular theory, dimerization of the fusion protein bound to the first and fourth binding sites (i.e., dimerization by Trex2) will bring the cleavage polynucleotide end upstream of the target region into close proximity with the cleavage polynucleotide end downstream of the donor nucleotide region, and dimerization of the fusion protein bound to the second and third binding sites will bring the cleavage polynucleotide end downstream of the target region into close proximity with the cleavage polynucleotide end upstream of the donor nucleotide region. Ligation of the closely approached polynucleotide ends by DNA repair mechanisms (such as NHEJ or MMEJ) will result in translocation of the polynucleotide (and excision of the target and donor nucleotide regions).

[0195] Table 1 summarizes the expected editing outcomes induced by the methods of the present disclosure using various fusion protein targeting strategies. Table 1. Expected editing outcomes.

[0196] The method herein includes at least providing a fusion protein and a nucleic acid to be edited, and may also include providing a donor nucleic acid and / or at least one guide RNA. Any suitable technique may be used to provide these various components. For example, providing a fusion protein may include introducing the fusion protein into a cell or introducing a recombinant nucleic acid, construct or vector encoding the fusion protein into a cell. Similarly, gRNA may be provided by introducing gRNA itself or a nucleic acid sequence encoding gRNA. In some embodiments, the fusion protein and gRNA may be encoded by the same DNA construct or vector. Examples Example 1. Using LbCas12a-Trex2 fusions to increase the frequency of segment inversions between paired guide RNA targeting sites in the maize genome

[0197] This example is to demonstrate that the LbCas12a-Trex2 fusion can promote segment inversion between two paired gRNA targeting sites on the same chromosome and on the same strand (e.g., Figure 1 The corn downy mildew resistance 6 (DMR6) gene (nucleotide sequence shown in SEQ ID NO: 1) was selected as an example to demonstrate this design. Similar designs can be applied to any other genomic loci in any organism. Table 2. Sequences used in Example 1.

[0198] DMR6 is a well-characterized plant susceptibility gene first studied in Arabidopsis. It is expected that knocking out this gene in other plant species will confer resistance to a variety of pathogens. However, due to the high GC% of the DMR6 protein coding sequence, it is difficult to find that the canonical TTTN PAM of the gene can be knocked out by a simple insertion and deletion mutation induced by Cas12a. In order to overcome this difficulty, a guide RNA is designed to target a target sequence adjacent to the TTTG PAM in the promoter region (SEQ ID NO: 2), and a second guide RNA is designed to target a sequence adjacent to the TTTA PAM in the first intron (SEQ ID NO: 3). The co-expression of the two guide RNAs together with the LbCas12a nuclease should allow the gene to be knocked out by excising or inverting the genomic fragment between the two targeting sites, which contains the entire first exon. Guide RNA design and expected outcomes are shown in Figure 5 middle.

[0199] Previous internal studies have shown that it is more difficult to use Cas12a to excise or invert the genomic sequence between the paired guide RNA targeting sites compared to Cas9. This can be explained by the following phenomenon: Cas12a produces double-strand breaks with sticky ends, which may be more difficult to separate from each other than the blunt ends produced by Cas9. In order to improve the frequency of fragment excision and inversion, various DNA exonucleases (including bacteriophage T5 exonucleases (T5Exo, SEQ ID NO: 4) (a 5' to 3' exonuclease), and mouse 3' repair exonucleases 2 (Trex2, SEQ ID NO: 5) (a 3' to 5' exonuclease)) are fused with LbCas12a (SEQ ID NO: 6) by flexible GS linkers (SEQ ID NO: 7), with the purpose of trimming and disengaging sticky ends immediately after LbCas12a produces double-strand breaks. This hypothesis (where T5Exo is used as an example) is shown in Figure 6 middle.

[0200] Construct a variety of binary vectors to test the efficacy of Cas12a-exonuclease fusions in corn, as summarized in Table 2. The protein coding sequence of each Cas12a-exonuclease fusion (with nuclear localization signal peptide (SEQ ID NO:8) on both ends) is optimized according to corn codon usage, and is expressed by an expression cassette operably connected to sucrose ubiquitin 4 promoter (SEQ ID NO:9) and Agrobacterium nopaline synthase terminator (SEQ ID NO:10). Two guide RNAs (SEQ ID NO:11 and 12) are expressed in tandem arrays under rice U6 promoter (SEQ ID NO:13), and transcripts are processed into separate mature crRNAs by the inherent RNase activity of LbCas12a. The Escherichia coli phosphomannose isomerase gene (SEQ ID NO:14) operably connected to corn ubiquitin 1 promoter (SEQ ID NO:15) and Agrobacterium nopaline synthase terminator (SEQ ID NO:10) serves as a selection marker in all vectors. Table 3. DNA constructs used in Example 1.

[0201] Each construct was delivered into callus cells of immature maize embryos derived from Syngenta's proprietary inbred line AX5707 by Agrobacterium tumefaciens-mediated transformation or biolistic transformation (if the construct was unstable in Agrobacterium). Transformed callus was subjected to mannose selection and plantlets were regenerated by standard tissue culture procedures. Regenerated plantlets were sampled for DNA extraction, where transgenic plants with the construct were identified by TaqMan assay.

[0202] PCR was designed to amplify the genomic sequence around and between the two targeting sites and used to characterize the editing outcome in transgenic plants. The expected amplicon size using the wild-type template was 1,416 bp. Excision of the fragment between the targeting sites resulted in a short of about 350 bp. Sanger sequencing was performed on the amplicon of each transgenic plant to identify the inverted allele and further characterize the repair junction at the targeting site.

[0203] The editing outcomes are summarized in Table 4. Compared with the control construct without the fused exonuclease domain, the N-terminal and C-terminal fusions of T5 exonuclease did not result in the desired frequency of excision or inversion. In contrast, the N-terminal and C-terminal fusions of Trex2 both resulted in a significantly high inversion frequency of about 20%, while moderately increasing the excision frequency. In transgenic plants with construct 25962 (encoding C-terminal LbCas12a-Trex2 fusions), there were three plants in which the 1kb fragment targeted by inversion was inverted in two alleles, and there were two plants in which the targeted fragment was excised from one allele and inverted in another allele. The fully sequenced alleles and the unedited wild-type reference sequences generated by editing with the C-terminal LbCas12a-Trex2 fusions are shown in Table 5 below. Most repair junctions only lose a few additional base pairs. In Table 5, gRNA1 target and flanking sequences (100 bp on each side) are underlined; gRNA2 target and flanking sequences (100 bp on each side) are in italics. PAM sequences are in bold and protospacers are double underlined. Sequences in lowercase are the reverse complements of the reference sequences. Nucleotides in square brackets are nucleotides that were inserted or mutated during the repair process. Table 4. Summary of editing outcomes. Example 2. Dissecting the molecular mechanism conferring the high inversion rate induced by the LbCas12a-Trex2 fusion.

[0204] This example was to examine the necessity of Trex2 dimerization and / or 3'-5' exonuclease activity in inducing high inversion rates by LbCas12a-Trex2 fusions. CD , SEQ ID NO: 18), dimerization-deficient Trex2 mutant (Trex2 DD , SEQ ID NO: 19) and a single-chain Trex2 homodimer (scTrex2, SEQ ID NO: 20) were fused to the C-terminus of LbCas12a, respectively, and the inversion-inducing efficacy of each fused variant was compared with that of the original LbCas12a-Trex2 fusion. Based on previous studies (Chen et al., Nucleic Acids Res. [Nucleic Acids Research] April 2007; 35(8): 2682-2694.; Delacote et al., PLoS One. [Public Library of Science Comprehensive] 2013; 8(1): e53217.), Trex2 CD Loss of exonuclease activity but retains the ability to form homodimers; Trex2 DD Impaired in forming homodimers and thus also lacking exonuclease activity; scTrex2 is able to post-translationally form intramolecular dimers that exhibit exonuclease activity but are no longer able to mediate dimerization between the two fusion proteins.

[0205] Expression of LbCas12a-Trex2 was generated by mutating the codons encoding amino acid residues H188 and D193 in construct 25962 to alanine encoding codons CD Construct 27431 of LbCas12a-Trex2 was generated by mutating the codons encoding amino acid residues E29, K59, N94, R107, and E191 in 25962 to alanine encoding codons. DD Construct 27432 of LbCas12a-scTrex2 was generated by inserting a DNA sequence encoding a polypeptide (TPPQTGLDVPY) linker and a second recoded Trex2 monomer (SEQ ID NO: 21) just upstream of the C-terminal NLS. All constructs used the same paired gRNA targeting ZmDMR6. Table 6. Sequences used in Example 2. Table 7. DNA constructs used in Example 2.

[0206] Transgenic plants were generated and analyzed as described in Example 1. The efficiency of the targeted inversion of each construct was compared with the efficiency of constructs 25962 and 26297, summarized in Table 8. CD The fusion can still induce the same high frequency of segment inversion as the original LbCas12a-Trex2 fusion (if not higher). DD The inversion rate induced by the fusion body dropped to only one-third of the inversion rate induced by the original LbCas12a-Trex2 fusion body. Part of the editing outcome produced by LbCas12a-scTrex2 was unexpected - the editing efficiency at the gRNA1 target dropped to about one-third of the efficiency produced by other constructs at the same target. However, in 13 plants where two targets were edited, targeted inversions were identified in one plant, and its frequency (7.7%) was comparable to that of 27432 (6.0%). Taken together, these results indicate that the dimerization ability of Trex2, rather than the 3'-5' exonuclease activity, is required for the high inversion rate induced by the LbCas12a-Trex2 fusion body. Table 8. Summary of editing outcomes. *Underestimated due to unexpectedly low editing efficiency at the gRNA1 target. Example 3. Use of LbCas12a-Trex2 fusions to increase the frequency of fragment excision between paired guide RNA targeting sites in the maize genome.

[0207] This example is to demonstrate that the LbCas12a-Trex2 fusion is combined with a pair of gRNAs targeting the same chromosome, with binding sites on opposite strands and both outside the target region (e.g., Figure 2 The combination can significantly increase the frequency of target area resection.

[0208] In Example 1, two gRNAs in the same orientation are selected to excise the first exon of the ZmDMR6 gene. When LbCas12a-Trex2 fusions are used in combination with two gRNAs, compared with non-fusion controls, the frequency of desired excision is moderately increased (see Table 4). Based on the dimerization hypothesis described in Example 2, it is predicted that two gRNAs with binding sites on opposite chains outside the target area can further increase the excision frequency induced by LbCas12a-Trex2. In order to design such gRNA pairs, ZmDMR6-crRNA1 is retained and paired with the target in the downstream of the target of ZmDMR6-crRNA2 and in the two gRNAs on the complementary chain (respectively ZmDMR6-crRNA3 (SEQ ID NO: 23) and ZmDMR6-crRNA4 (SEQ ID NO: 24)) ( Fig. 7A ). Table 9. Sequences used in Example 3.

[0209] The coding sequence of ZmDMR6-crRNA2 in construct 25962 was replaced by the coding sequence of ZmDMR6-crRNA3 and ZmDMR6-crRNA4, respectively, to generate fusion constructs 26710 and 26711. The coding sequence of ZmDMR6-crRNA2 in construct 26297 was replaced by the coding sequence of ZmDMR6-crRNA3 and ZmDMR6-crRNA4, respectively, to generate non-fusion control constructs 26712 and 26713. Table 10. DNA constructs used in Example 3.

[0210] Generate and analyze transgenic plants as described in Example 1, except that the reverse primer for PCR was redesigned. The editing outcomes are summarized in Table 11. Interestingly, all four constructs lead to extremely low to zero inversion frequencies. The excision frequency produced by all four constructs is less than 10%, but the presence of Trex2 almost doubles the excision frequency between ZmDMR6-crRNA1 and ZmDMR6-crRNA4. Other binding sites (in Table 1) that are expected to significantly improve the design of the excision frequency between the two target sites are also tested. Table 11. Summary of editing outcomes.

[0211] The ZmWaxy1 gene was selected to test the prediction that two gRNAs with binding sites on opposite strands within the target region can significantly increase the frequency of excision induced by LbCas12a-Trex2. ZmWaxy1-crRNA1 (SEQ ID NO: 25) was designed to target a sequence adjacent to the TTTG PAM in exon 4 on the coding strand, while ZmWaxy1-crRNA5 (SEQ ID NO: 26) was designed to target a sequence adjacent to the TTTA PAM in the promoter region on the complementary strand ( Figure 7B ).

[0212] The coding sequences of ZmDMR6-crRNA1 and ZmDMR6-crRNA2 in construct 25962 were replaced by the coding sequences of ZmWaxy1-crRNA1 and ZmWaxy1-crRNA5, respectively, to generate the fusion construct 26958. The coding sequences of ZmDMR6-crRNA1 and ZmDMR6-crRNA2 in construct 26297 were replaced by the coding sequences of ZmWaxy1-crRNA1 and ZmWaxy1-crRNA5, respectively, to generate the non-fusion control construct 26961. Transgenic plants were similarly generated and analyzed as described in Example 1, and the editing outcomes are also summarized in Table 9. The gRNA pair resulted in a high (about 20%) excision frequency even in the absence of Trex2; however, the addition of Trex2 almost tripled the excision frequency to a surprising 58.6%. The results of the excision experiment in this example are inconsistent with the dimerization hypothesis, but can be explained by an alternative hypothesis based on repair inhibition. For example, the presence of Trex2 may inhibit immediate repair by canonical NHEJ between the two ends at one DSB site (which may be due to exonuclease activity), but leave two unbound ends at the open two DSB sites for NHEJ repair. NHEJ repair between the two unbound ends can then lead to a preferred repair outcome, depending on the orientation of the gRNA target site. Example 4. Use of LbCas12a-Trex2 fusions to increase the frequency of segment inversion and excision between paired guide RNA targeting sites in the soybean genome.

[0213] This example is to demonstrate the efficacy of LbCas12a-Trex2 fusions in increasing the frequency of segment inversion and excision in the dicot crop soybean. The soybean FATTY ACID DESATURASE 2A (FAD2-1A) gene (SEQ ID NO: 27) was selected for the experiment.

[0214] The first gRNA, GmFAD2A-crRNA1 (SEQ ID NO: 28) is designed to target a sequence adjacent to the TTTG PAM in the FAD2-1A promoter region on the coding strand. The second gRNA, GmFAD2A-crRNA2 (SEQ ID NO: 29) is designed to target a sequence adjacent to the TTTG PAM on the coding strand near the 3' splice site of the first intron. The distance between the crRNA1 and crRNA2 target sites is about 1.1 kb. The third gRNA, GmFAD2A-crRNA3 (SEQ ID NO: 30) is designed to target a sequence adjacent to the TTTG in the second exon on the complementary strand. The fourth gRNA, GmFAD2A-crRNA4 (SEQ ID NO: 31) is designed to target a sequence adjacent to the TTTG in the second exon on the coding strand. The distance between the crRNA3 and crRNA4 target sites is about 1 kb ( Figure 8 ).

[0215] The following outcome predictions are based on the corn results described in Examples 1 and 3. GmFAD2A-crRNA1 and crRNA2 are in the same orientation, so when the two gRNAs are co-expressed with LbCas12a-Trex2 fusions in soybean cells, it is expected that they induce a significantly higher frequency of inversion of the fragment between the two gRNA target sites compared with LbCas12a co-expression. GmFAD2A-crRNA3 and crRNA4 bind to the opposite strands within the target region, so when the two gRNAs are co-expressed with LbCas12a-Trex2 fusions in soybean cells, it is expected that they induce a significantly higher frequency of excision of the fragment between the two gRNA target sites compared with LbCas12a co-expression.

[0216] Binary vectors constructed to test the efficacy of Cas12a-exonuclease fusions in soybean are summarized in Table 12. Arabidopsis codon optimized coding sequences (SEQ ID NO: 32) of LbCas12a-Trex2 fusions or Arabidopsis codon optimized coding sequences (SEQ ID NO: 33) of LbCas12a were operably linked to Figwortmosaic virus (FMV) enhancers (SEQ ID NO: 34), promoters of Arabidopsis EF1A elongation factor genes (SEQ ID NO: 35) and Agrobacterium nopaline synthase terminators (SEQ ID NO: 10) for constitutive expression in soybean cells. Two paired gRNAs (SEQ ID NO: 36 and 37, respectively) in tandem arrays and flanked by hammerhead structures (HH) and hepatitis delta virus (HDV) ribozymes were expressed under the soybean ubiquitin promoter (SEQ ID NO: 38). The transcripts containing the gRNA array are processed into individual mature crRNAs by the self-cleavage of the ribozyme and the inherent RNase activity of LbCas12a. The aminoglycoside 3'-adenyl transferase gene (SEQ ID NO: 39) operably connected to the promoter of the soybean EF1 elongation factor gene (SEQ ID NO: 40) and the terminator of the pea (pea (Pisum sativum)) ribulose-1,5-bisphosphate carboxylase (rbcS2) small subunit E9 gene (SEQ ID NO: 41) confers resistance to the antibiotic spectinomycin in plant cells and serves as a plant selection marker in all vectors. Table 12. Sequences used in Example 4. Table 13. DNA constructs used in Example 4.

[0217] Each construct is delivered into the stem cells in the cotyledons of Syngenta's proprietary soybean stock line 06KG by Agrobacterium tumefaciens-mediated transformation. The transformed cotyledons are subjected to spectinomycin selection and plantlets are regenerated by tissue culture procedures. The regenerated plantlets are sampled for DNA extraction, where transgenic plants with the constructs are identified by TaqMan assays.

[0218] PCR is used to detect inversion and excision events in samples.For 27188 and 27194, the forward primer in the upstream of GmFAD2A-crRNA1 target site and the reverse primer in the downstream of GmFAD2A-crRNA2 target site are designed to increase the sequence around and between two target sites.The second " forward primer " (initiated to the same chain as the first forward primer) between GmFAD2A-crRNA1 and GmFAD2A-crRNA2 target sites is designed to increase the inversion fragment when paired with the first forward primer.For 27253 and 27254, the forward primer in the upstream of GmFAD2A-crRNA3 target site and the reverse primer in the downstream of GmFAD2A-crRNA4 target site are designed to increase the sequence around and between two target sites, and the fragment excision between two target sites will cause smaller PCR amplicon.The results of agarose gel electrophoresis based on PCR product are summarized in Table 14. These results confirm the findings in maize: when the two paired target sites are on the same strand (one within the target region and one outside the target region), the editing outcome strongly prefers fragment inversion; when the two paired target sites are on opposite strands and both are within the target region, the editing outcome prefers fragment excision. Table 14. Summary of editing outcomes. Due to the high degree of mosaicism often seen in TO plants generated by organogenesis-based transformation, the proportion of the desired mutation will be determined by NGS and the heritability of the desired mutation will be determined in the T1 population. Example 5. Use of Cas12a-Trex2 fusions to achieve efficient targeted insertion via NHEJ / MMEJ repair in maize.

[0219] This example was to demonstrate that the LbCas12a-Trex2 fusion in combination with a gRNA in a carefully designed orientation can efficiently mediate the insertion of a 3.6-kb expression cassette of the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (CP4 EPSPS; cargo sequence, SEQ ID NO: 42) into a targeted gap in the maize genome via the NHEJ / MMEJ repair pathway (i.e., no long homology is required in the donor DNA).

[0220] Select gRNA pairs ZmDMR6-crRNA1 and ZmDMR6-crRNA3 (see Example 3) that have binding sites on opposite strands outside the target region (e.g., Figure 3A)) to create a gap between the two target sites in the ZmDMR6 gene. The target sequences of crRNA1 and crRNA2 (SEQ ID NOs: 43 and 44, respectively) are added to the 5' and 3' ends of the cargo sequence, respectively, in such a way that the crRNA binding sites are on opposite strands, but within the donor nucleotide region (e.g., Figure 3A ), thereby generating a donor DNA containing the cargo. The donor DNA was cloned into a 1.8-kb mini-pUC57 backbone, thereby generating the donor construct 27022. Table 15. Sequences used in Example 5. Table 16. DNA constructs used in Example 5.

[0221] The linearized plasmid DNA of constructs 26710 and 27022 will be delivered to maize immature embryos by biolistic transformation. The bombarded embryos will be subjected to mannose selection and plantlets will be regenerated by tissue culture. The regenerated plantlets will be sampled for DNA extraction, where transgenic plants with the constructs will be identified by TaqMan assays. As a non-fusion control, transgenic plants transformed with constructs 26712 and 27022 will be produced according to the same procedure.

[0222] Multiple junction PCR will be designed to identify the insertion at the target locus, and each of the junction PCR uses a genome-specific primer and a goods-specific primer. The amplicon of the junction PCR will be subjected to Sanger sequencing to characterize the junction sequence. A long PCR using two genome-specific primers flanking the insertion site will be performed to verify the length of the insertion sequence. Because the goods are inserted via NHEJ / MMEJ repair, it is expected that the goods can be inserted in either direction, and small indels can be found at the junction. Example 6. Use of Cas12a-Trex2 fusions to achieve efficient targeted insertion via homology-directed repair (HDR) in maize.

[0223] This example is to demonstrate that LbCas12a-Trex2 fusions in combination with gRNA in a carefully designed orientation can efficiently mediate the insertion of a 3.8-kb CP4 EPSPS expression cassette (cargo sequence, SEQ ID NO: 45) into a targeted gap in the maize genome via an HDR approach (i.e., with long homology sequences flanking the cargo sequence). The intergenic region on maize chromosome 1 (labeled ZmSH1) was selected to demonstrate this design.

[0224] A gRNA, ZmSH1-crRNA1 is designed to target the sequence adjacent to TTTG PAM on the positive strand (SEQ ID NO: 46). Another gRNA, ZmSH1-crRNA2 is designed to target the sequence adjacent to TTTG PAM on the negative strand (SEQ ID NO: 47). The two gRNA target sites are 167bp apart in the AX5707 genome, and they are on opposite strands outside the target region, thereby causing a gap in ZmSH1 when co-expressed with LbCas12a-Trex2 fusion or non-fusion LbCas12a.

[0225] The 458-bp genomic sequence (SEQ ID NO: 48) upstream of the PAM of the ZmSH1-crRNA1 target was selected as the left homology arm (LHA) to mediate HDR, and was added to the 5' end of the cargo sequence. The 509-bp genomic sequence (SEQ ID NO: 49) downstream of the PAM of the ZmSH1-crRNA2 target was selected as the right homology arm (RHA), and was added to the 3' end of the cargo sequence. The target sequences of ZmSH1-crRNA1 and ZmSH1-crRNA2 (both of which have TTTG PAM added to the 5' end) were added to the 5' end of the left homology arm and the 3' end of the right homology arm, respectively, and added in such a way that the crRNA binding site is on the opposite strand, but in the donor nucleotide region, to produce donor DNA ( Fig. 9 ). Table 17. Sequences used in Example 6.

[0226] Donor DNA, Cas12a-Trex2 expression cassette, gRNA expression cassette and PMI selection marker are built into a single T-DNA region in a binary vector. The resulting construct will be used to transform immature corn embryos, and transgenic plants will be produced by tissue culture with mannose selection and will be identified by TaqMan assay.

[0227] Multiple junction PCR will be designed to identify the desired insertion at the target locus, and each of the junction PCR uses a genome-specific primer and a goods-specific primer. The amplicon of the junction PCR will be subjected to Sanger sequencing to characterize the junction sequence. A long PCR using two genome-specific primers flanking the insertion site will be performed to verify the length of the insertion sequence. Because the goods are inserted via HDR repair, it is expected that the goods will be inserted in the design direction as it is in the donor DNA, and the junction will not have other mutations. Example 7. Use of Cas12a-Trex2 fusion to achieve efficient targeted chromosomal translocation.

[0228] This example is to demonstrate that LbCas12a-Trex2 fusions can efficiently mediate the translocation of chromosome arms between two nonhomologous chromosomes in combination with gRNA in a carefully designed orientation. The soybean FAD2-1A gene on chromosome 10 and its paralog FAD2-1B gene on chromosome 20 were selected as targets to demonstrate this design.

[0229] One gRNA will be designed to simultaneously target a sequence adjacent to the TTTV PAM and present in both FAD2-1A and FAD2-1B. The second gRNA will be designed to simultaneously target another sequence adjacent to the TTTV PAM on the opposite strand and present in both FAD2-1A and FAD2-1B. Co-expression of both gRNA and LbCas12a-Trex2 fusion will create gaps in both FAD2-1A and FAD2-1B, and the fusion molecules will remain bound to the genomic ends flanking the gaps. Figure 4 It is shown in Figure 2 that, at a certain frequency, dimerization of Trex2 will direct the exchange of chromosome arms between chromosomes 10 and 20. Chromosomal translocations will be identified by junctional PCR, each of which uses one FAD2-1A-specific primer and one FAD2-1B-specific primer.

[0230] All patents, patent publications, patent applications, journal articles, books, technical references, etc. discussed in this disclosure are incorporated herein by reference in their entirety for all purposes.

[0231] It should be understood that the figures and descriptions of the present disclosure have been simplified to illustrate elements relevant to a clear understanding of the present disclosure. It should be understood that the figures are presented for illustrative purposes rather than as structural diagrams. Omitted details and modified or alternative embodiments are within the knowledge of those of ordinary skill in the art.

[0232] It is understood that in certain aspects of the present disclosure, a single component may be replaced by multiple components, and multiple components may be replaced by a single component to provide an element or structure or to perform a given one or more functions. Unless such a replacement would not be operable to implement certain embodiments of the present disclosure, such a replacement is considered to be within the scope of the present disclosure.

[0233] The examples presented herein are intended to illustrate the potential and specific implementation of the present disclosure. It is to be understood that these examples are primarily intended to illustrate the purpose of the present disclosure to those skilled in the art. Without departing from the spirit of the present disclosure, these figures or the operations described herein may be changed. For example, in some cases, method steps or operations may be performed or executed in different orders, or operations may be added, deleted or modified.

[0234] Where a numerical range is provided, it is understood that each intervening value between the upper and lower limits of the range (to the smallest decimal place of the lower limit, unless the context clearly dictates otherwise) is also specifically disclosed. Any smaller ranges between any stated value or non-stated intermediate value in the stated range and any other stated value or intermediate value in the stated range are contemplated. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, none, or both of the limits are included in the smaller range is also contemplated within the present technology, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding one or both of those included limits are also contemplated.

[0235] In the foregoing description, many specific details are set forth to provide a more thorough understanding of the present invention. However, it will be clear to those skilled in the art that the present invention described in this disclosure can be practiced without one or more of these specific details. In other cases, features and procedures well known to those skilled in the art are not described to avoid obscuring the present invention. For illustrative and not limiting purposes, embodiments of the present disclosure have been described. Although the present invention is primarily described with reference to specific embodiments, other embodiments that will become clear to those skilled in the art after reading this disclosure are also contemplated, and such embodiments are intended to be included in the inventive method. Therefore, the present disclosure is not limited to the embodiments described above or depicted in the accompanying drawings, and various embodiments and modifications may be made without departing from the scope of the following claims.

Claims

1. A fusion protein comprising a site-directed nuclease linked to a non-specific terminal processing enzyme.

2. The fusion protein of claim 1, wherein the site-directed nuclease comprises a CRISPR-associated nuclease.

3. The fusion protein of claim 2, wherein the CRISPR-associated nuclease is selected from the group consisting of Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and nickases or inactivated forms thereof.

4. The fusion protein of claim 3, wherein the CRISPR-associated nuclease is a Cas9 enzyme.

5. The fusion protein of claim 3, wherein the CRISPR-associated nuclease is a Cas12a enzyme. The fusion protein according to claim 1 , wherein the non-specific end processing enzyme is a non-specific exonuclease.

7. The fusion protein of claim 6, wherein the non-specific exonuclease is T5Exo, Trex2, Escherichia coli exonuclease I, exonuclease III, exonuclease T, exonuclease IX, exonuclease X, RecJ, PolII, PolIIIε, WRN, MRE11, APE1, VDJP, RAD1, RAD9, p53 or Trex1.

8. The fusion protein of any one of claims 1 to 7, wherein the non-specific terminal processing enzyme comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 4, 5, 18, 19, 20, 22, or 58-74.

9. The fusion protein of any one of claims 1 to 8, wherein the non-specific terminal processing enzyme is a monomer of a dimerized protein.

10. The fusion protein of any one of claims 1 to 9, wherein the fusion protein comprises a linker between the site-directed nuclease and the non-specific end processing enzyme. The fusion protein of claim 10 , wherein the linker comprises SEQ ID NO:

7.

12. The fusion protein of any one of claims 1 to 11, wherein the fusion protein comprises a nuclear localization signal.

13. The fusion protein of any one of claims 1 to 12, wherein the fusion protein comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 50-57.

14. A recombinant nucleic acid encoding the fusion protein according to any one of claims 1 to 13.

15. A DNA construct comprising a promoter operably linked to the recombinant nucleic acid of claim 14.

16. A DNA construct as claimed in claim 15, wherein the promoter is at least one of an inducible promoter, a constitutive promoter, an egg cell-specific promoter, a pollen-specific promoter or an apical meristem-specific promoter.

17. A DNA construct as claimed in claim 15 or 16, wherein the promoter is a ubiquitin 4 promoter, an actin promoter, a tubulin promoter, a MADS box promoter or a plant virus promoter.

18. A vector comprising the recombinant nucleic acid of claim 14 or the DNA construct of any one of claims 15 to 17.

19. A cell comprising the recombinant nucleic acid of claim 14, the DNA construct of any one of claims 15 to 17, or the vector of claim 18.

20. The cell of claim 19, wherein the cell is a plant cell.

21. The cell of claim 20, wherein the plant cell is a corn plant cell, a soybean plant cell, a rice plant cell, a wheat plant cell, or a sunflower plant cell.

22. A method for editing a nucleic acid, the method comprising: a. providing at least one fusion protein as described in any one of claims 1 to 13; b. providing the nucleic acid, wherein the nucleic acid comprises a first binding site, a second binding site, and a target region comprising a portion of the nucleic acid, wherein the first binding site is adjacent to the 5' end of the target region and the second binding site is adjacent to the 3' end of the target region; as well as c. contacting the nucleic acid with the at least one fusion protein, wherein the at least one fusion protein specifically binds to the first binding site and the second binding site, thereby resulting in editing of the target region of the nucleic acid.

23. The method of claim 22, wherein the site-directed nuclease of the at least one fusion protein comprises a CRISPR-associated nuclease, and the method further comprises providing at least one first guide RNA and at least one second guide RNA, wherein the at least one first guide RNA comprises a nucleotide sequence having complementarity to the first binding site and the at least one second guide RNA comprises a nucleotide sequence having complementarity to the second binding site.

24. The method of claim 22 or 23, wherein the first binding site and the second binding site are on the same strand.

25. The method of claim 22 or 23, wherein the first binding site and the second binding site are on opposite strands.

26. The method of any one of claims 22 to 25, wherein at least one of the first binding site or the second binding site is within the target region.

27. The method of any one of claims 22 to 26, wherein both the first binding site and the second binding site are within the target region.

28. The method of any one of claims 22 to 25, wherein neither the first binding site nor the second binding site is within the first target region.

29. The method of any one of claims 22 to 28, further comprising providing a donor nucleic acid, wherein the donor nucleic acid comprises a third binding site, a fourth binding site, and a donor nucleotide region, wherein the third binding site is adjacent to the 5' end of the donor nucleotide region and the fourth binding site is adjacent to the 3' end of the donor nucleotide region, and wherein the at least one fusion protein specifically binds to the third binding site and the fourth binding site.

30. The method of claim 29, wherein the site-directed nuclease of the at least one fusion protein comprises a CRISPR-associated nuclease, and the method further comprises providing at least one third guide RNA and at least one fourth guide RNA, wherein the at least one third guide RNA comprises a nucleotide sequence having complementarity to the third binding site and the at least one fourth guide RNA comprises a nucleotide sequence having complementarity to the fourth binding site.

31. The method of claim 29 or 30, wherein the third binding site and the fourth binding site are on the same strand.

32. The method of claim 29 or 30, wherein the third binding site and the fourth binding site are on opposite strands.

33. The method of any one of claims 29 to 32, wherein at least one of the third binding site or the fourth binding site is within the donor nucleotide region.

34. The method of any one of claims 29 to 33, wherein both the third binding site and the fourth binding site are within the donor nucleotide region.

35. The method of any one of claims 29 to 32, wherein neither the third binding site nor the fourth binding site is within the donor nucleotide region.

36. The method of any one of claims 22 to 35, wherein the nucleic acid is part of a first chromosome.

37. The method of any one of claims 29 to 36, wherein the donor nucleic acid is part of a donor template.

38. The method of claim 37, wherein the donor template is a portion of a plasmid or a linear nucleic acid.

39. The method of any one of claims 22 to 38, wherein the editing is an excision, inversion or replacement of at least a portion of the target region.

40. The method of claim 36, wherein the donor nucleic acid is a portion of a second chromosome.

41. The method of claim 40, wherein the first chromosome and the second chromosome are homologous chromosomes or non-homologous chromosomes.

42. The method of claim 40 or 41, wherein the editing is a chromosomal rearrangement or replacement of at least a portion of the target region.

43. The method of claim 42, wherein the chromosomal rearrangement is a reciprocal translocation or a non-reciprocal translocation.

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