Cr ispr-cas fusion proteins with adenine base editor function, compositions comprising the same, and methods of using the same

Abstract

Described herein are fusion proteins, as well as compositions and systems comprising the fusion proteins. Also described herein are methods of using and / or producing the fusion proteins, such as methods of using the fusion proteins to modify or edit a target nucleic acid.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 585,320, filed September 26, 2023, and U.S. Provisional Patent Application Serial No. 63 / 610,011, filed December 14, 2023, the disclosure of each of which is incorporated herein by reference in its entirety.

[0003] Declaration regarding the electronic document of the sequence list

[0004] The sequence list in XML format, titled 1499-137WO_ST26.xml, with a size of 734,720 bytes, generated and submitted on September 25, 2024, is hereby incorporated in its entirety by reference for its public release. Technical Field

[0005] This invention relates to fusion proteins, compositions and systems comprising the same, and methods of using them. Background Technology

[0006] Some base editing systems can use a cytosine base editor (CBE) to convert a single C•G to a T•A base pair and rewrite single or multiple bases using the reverse transcriptase activity of an RNA template REDRAW editor. Additionally, an adenine base editor (ABE) can convert a single A•T to a C•G base pair. New base editors may be advantageous. Summary of the Invention

[0007] A first aspect of the present invention relates to a fusion protein comprising a CRISPR-Cas effector protein and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242.

[0008] Another aspect of the invention relates to a fusion protein comprising: adenosine deaminase; a CRISPR-Cas effector protein; and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242.

[0009] Another aspect of the invention relates to a fusion protein comprising: cytosine deaminase; a CRISPR-Cas effector protein; and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242.

[0010] Another aspect of the invention relates to a fusion protein having an amino acid sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 243-256, 288-292, and / or 308.

[0011] Another aspect of the invention relates to a polynucleotide having a sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 266-279, 294-298, and / or 307.

[0012] Another aspect of the invention relates to a complex comprising the fusion protein of the invention and a guide nucleic acid.

[0013] Another aspect of the invention relates to a nucleic acid encoding the fusion protein of the invention.

[0014] Another aspect of the invention relates to an expression cassette, which is codon-optimized for expression in an organism, the expression cassette comprising: a polynucleotide encoding a promoter sequence, and a polynucleotide encoding a fusion protein of the invention, the polynucleotide being codon-optimized for expression in the organism.

[0015] Another aspect of the invention relates to a method for modifying target nucleic acids in cells, the method comprising introducing the expression cassette and / or vector of the invention into the cells, thereby modifying the target nucleic acids in the cells.

[0016] Another aspect of the invention relates to a method for producing the fusion protein of the invention, the method comprising: culturing cells or cell populations transformed with nucleic acids encoding the fusion protein; and isolating the fusion protein to produce the fusion protein.

[0017] Another aspect of the present invention relates to a method for modifying a target nucleic acid, the method comprising contacting the target nucleic acid with the fusion protein and guide nucleic acid of the present invention, thereby modifying the target nucleic acid.

[0018] It should be noted that aspects of the invention described in relation to one embodiment may be incorporated into different embodiments, even if not explicitly described therewith. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. The applicant reserves the right to amend any initially filed claim and / or accordingly file any new claim, including revising any initially filed claim to make it subordinate to and / or incorporated into any feature of any other claim or claims, even if not initially claimed in this manner. These and other objects and / or aspects of the invention will be set forth in detail in the description below. Further features, advantages, and details of the invention will be understood by those skilled in the art upon reading the accompanying drawings and detailed description of the preferred embodiments below, such descriptions being illustrative only. Attached Figure Description

[0019] Figure 1A This is a schematic diagram illustrating an exemplary fusion protein according to some embodiments of the present invention.

[0020] Figure 1B This is a schematic diagram illustrating another exemplary fusion protein according to some embodiments of the present invention.

[0021] Figure 2 This is a graph showing the percentage of A-to-G edits for an exemplary fusion protein (“fusion protein 1”) containing SEQ ID NO: 244 and control proteins at various sites of different target nucleic acids.

[0022] Figure 3 This is a graph showing the percentage of A-to-G edits of another exemplary fusion protein (“fusion protein 2”) containing SEQ ID NO: 243 and control proteins at various sites of different target nucleic acids.

[0023] Figure 4 It is shown Figure 2 and Figure 3 A graph showing the log2 (fold change) of exemplary fusion proteins 1 and 2 at various sites of different target nucleic acids.

[0024] Figure 5 This is a graph showing the percentage of A-to-G edits for an exemplary fusion protein (“fusion protein 3”) containing SEQ ID NO: 246 and control proteins at various sites of different target nucleic acids.

[0025] Figure 6 This is a graph showing the percentage of A-to-G edits for another exemplary fusion protein (“fusion protein 4”) containing SEQ ID NO: 245 and control proteins at various sites of different target nucleic acids.

[0026] Figure 7 It is shown Figure 5 and Figure 6 A graph showing the log2 (fold change) of exemplary fusion proteins 3 and 4 at various sites of different target nucleic acids.

[0027] Figure 8 This is a graph illustrating the editing efficiency of different proteins in maize, including exemplary fusion proteins, using 1% or 10% edit intensity (ES) cutoff values, according to some embodiments of the present invention. Edit intensity is the highest reported edit event and is provided as a percentage.

[0028] Figure 9 This is a graph showing the percentage of A-to-G edits at various sites of the exemplary fusion protein (“fusion protein 1”) containing SEQ ID NO: 244 and the control protein at different target nucleic acids. (n = 3–4 mean ± sem). Statistically significant increases in edits relative to the control are indicated by an asterisk (unpaired Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001).

[0029] Figure 10 This is a graph showing the percentage of A-to-G edits at various sites of the exemplary fusion protein (“fusion protein 3”) containing SEQ ID NO: 246 and the control protein containing different target nucleic acids. (n = 3–4 mean ± sem). Statistically significant increases in edits relative to the control are indicated by an asterisk (unpaired Student’s t-test, *p < 0.05).

[0030] Figure 11 This is a graph showing the percentage of A-to-G edits at various sites of the fusion protein containing SEQ ID NO: 305 (“fusion protein 8”) and the control protein at different target nucleic acids. (n = 3–4 mean ± sem). Statistically significant increases in edits relative to the control are indicated by an asterisk (unpaired Student's t-test, *p < 0.05). Detailed Implementation

[0031] The invention will now be described below with reference to the accompanying drawings and examples, in which embodiments of the invention are illustrated. This description is not intended to be a detailed list of all different ways in which the invention can be practiced or all features that can be added to the invention. For example, features shown with respect to one embodiment may be incorporated into other embodiments, and features shown with respect to a particular embodiment may be removed from that embodiment. Therefore, the invention contemplates that in some embodiments of the invention, any features or combinations of features set forth herein may be excluded or omitted. Furthermore, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art based on this disclosure, and such variations and additions do not depart from the invention. Therefore, the following description is intended to illustrate some specific embodiments of the invention and is not an exhaustive list of all permutations, combinations, and variations thereof.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0033] All publications, patent applications, patents and other references cited in this article are incorporated in their entirety by reference for the purpose of teaching in relation to the sentences and / or paragraphs presented in the references.

[0034] Unless the context otherwise indicates, the various features of the invention specifically intended to be described herein can be used in any combination. Furthermore, the invention contemplates that, in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification states that a composition comprises components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, may be omitted and abandoned, individually or in any combination.

[0035] As used in the description of the invention and the appended claims, unless the context clearly indicates otherwise, the singular forms “a” and “the” are also intended to include the plural forms.

[0036] As used herein, “and / or” means and covers any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative form (“or”).

[0037] As used herein, when the term “about” refers to a measurable value such as amount or concentration, it is intended to cover variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the specified value itself. For example, “about X,” where X is a measurable value, is intended to include X as well as variations of X of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. The ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​thereof.

[0038] As used herein, phrases such as “between X and Y” and “between approximately X and Y” should be interpreted as including both X and Y. As used herein, phrases such as “between approximately X and Y” mean “between approximately X and approximately Y”, and phrases such as “from approximately X to Y” mean “from approximately X to approximately Y”.

[0039] Unless otherwise indicated herein, the enumeration of value ranges herein is intended only as a shorthand method for individually referring to each individual value falling within the range, and each individual value is incorporated into the specification as if it were individually enumerated herein. For example, if ranges 10 to 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0040] As used herein, the terms “comrpise,” “comprises,” and “comprising” specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0041] As used herein, the transitional phrase “consistently of…” means that the scope of the claim should be interpreted to cover the specified materials or steps listed in the claim as well as materials or steps that do not substantially affect the essential and novel characteristics of the claimed invention. Therefore, when used in the claims of this invention, the term “consistently of…” is not intended to be interpreted as equivalent to “comprising”.

[0042] As used herein, the terms “increase,” “increasing,” “enhance,” “enhancing,” “improve,” and “improving” (and their grammatical variations) describe an improvement of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500%, or higher compared to another measurable property or quantity (e.g., a control).

[0043] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “reduction,” “decrease,” and “lower” (and their grammatical variations) describe a reduction of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% compared to another measurable property or quantity (e.g., a control). In some embodiments, reduction may result in no or substantially no (i.e., negligible amounts, such as less than about 10% or even 5%) detectable activity or quantity.

[0044] "Heteronucleotide sequences" or "recombinant nucleotide sequences" are nucleotide sequences that are not naturally associated with the host cell to which they are introduced, including multiple non-natural copies of naturally occurring nucleotide sequences.

[0045] "Natural" or "wild-type" nucleic acids, nucleotide sequences, polypeptide or amino acid sequences refer to naturally occurring or endogenous nucleic acid, nucleotide, polypeptide or amino acid sequences. Therefore, for example, "natural nucleic acid" is a nucleic acid that is naturally present in or endogenous to the reference organism. "Homologous" nucleic acid sequences are nucleotide sequences that are naturally associated with the introduced host cell.

[0046] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” and “polynucleotide” refer to linear or branched, single-stranded or double-stranded RNA or DNA or hybrids thereof. The term also encompasses RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc., may be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications may also be made, such as modifications to the 2'-hydroxyl group in the phosphodiester backbone or the RNA ribosome.

[0047] As used herein, the term "nucleotide sequence" refers to a hybrid of nucleotides or the sequence of these nucleotides from the 5' to 3' ends of a nucleic acid molecule, and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and antisense RNA, any of which may be single-stranded or double-stranded. The terms "nucleotide sequence," "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "recombinant nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably herein and refer to a hybrid of nucleotides. The nucleic acid molecules and / or nucleotide sequences provided herein are presented from left to right in a 5' to 3' orientation and use the standard code representation for nucleotide characters set forth in U.S. Sequence Rules 37 CFR §§1.831-1.835 and World Intellectual Property Organization (WIPO) Standard ST.26. As used herein, "5' region" may refer to the polynucleotide region closest to the 5' end of a polynucleotide. Therefore, for example, elements in the 5' region of a polynucleotide can be located at any position from the first nucleotide at the 5' end of the polynucleotide to the nucleotide in the middle of the polynucleotide. As used herein, "3' region" can refer to the polynucleotide region closest to the 3' end of the polynucleotide. Therefore, for example, elements in the 3' region of a polynucleotide can be located at any position from the first nucleotide at the 3' end of the polynucleotide to the nucleotide in the middle of the polynucleotide.

[0048] As used herein, the term "gene" refers to a nucleic acid molecule capable of producing mRNA, antisense RNA, miRNA, antimicroRNA antisense oligodeoxyribonucleotides (AMOs), etc. A gene may or may not be capable of producing a functional protein or gene product. A gene may include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions).

[0049] Polynucleotides, genes, or polypeptides can be “isolated,” meaning that the nucleic acid or polypeptide is largely or substantially free of components that are normally present with the nucleic acid or polypeptide in its natural state. In some embodiments, such components include other cellular material, culture media from recombinant production, and / or various chemicals used for the chemical synthesis of nucleic acids or polypeptides.

[0050] The term "mutation" refers to point mutations (e.g., missense or nonsense, or insertions or deletions of single base pairs that cause frameshifts), insertions, deletions, and / or truncations. When a mutation is the substitution of one residue within an amino acid sequence by another residue, or the deletion or insertion of one or more residues within a sequence, it is typically described by identifying the original residue, followed by identifying the position of that residue within the sequence and the identity of the newly substituted residue.

[0051] As used in this article, “non-natural mutation” refers to a mutation that is produced through human intervention and is different from naturally occurring mutations present in the same gene or polypeptide (e.g., naturally occurring and not the result of human modification).

[0052] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides through base pairing under permissible salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). Complementarity between two single-stranded molecules can be "partial," where only some nucleotides bind, or it can be complete when there is perfect complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0053] As used herein, “complementary” can mean 100% complementarity with the compared nucleotide sequence, or it can mean less than 100% complementarity (e.g., “substantially complementary”, such as approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.).

[0054] A “part” or “fragment” of a nucleotide sequence or polypeptide (including domains) should be understood to mean a nucleotide sequence or polypeptide of reduced length (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues (e.g., nucleotides or peptides) relative to a reference nucleotide sequence or polypeptide), and respectively comprising, substantially composed of and / or Composed of a nucleotide sequence or polypeptide of consecutive residues that is identical or nearly identical to a reference nucleotide sequence or polypeptide (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical). In some embodiments, a portion of the reference nucleotide sequence or polypeptide is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or higher percentage of the full-length reference nucleotide sequence or polypeptide. Such nucleic acid fragments or portions according to the invention may, where appropriate, be included in larger polynucleotides in which said nucleic acid fragments or portions are components. As an example, the repeat sequence of the guide nucleic acid of the present invention may include a portion of a wild-type CRISPR-Cas repeat sequence (e.g., a wild-type V CRISPR-Cas repeat sequence, such as repeat sequences from CRISPR-Cas systems (including, but not limited to, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c, etc.)). Similarly, a portion of a polypeptide may be included in a larger polypeptide in which a portion of the polypeptide is a component.

[0055] Different nucleic acids or proteins that are homologous are referred to herein as “homologs.” The term homolog includes homologous sequences from the same and other species, as well as orthologous sequences from the same and other species. “Homology” refers to the level of similarity between two or more nucleic acid and / or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention further comprise homologs of the nucleotide sequences and polypeptides of the present invention. As used herein, “orthologous” and “orthologous” refer to homologous nucleotide and / or amino acid sequences in different species that originated from a common ancestral gene during speciation. The homologs or orthologs of the nucleotide sequence of the present invention have basic sequence identity with the nucleotide sequence of the present invention (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%).

[0056] As used herein, “sequence identity” refers to the degree to which two best-aligned polynucleotide or polypeptide sequences remain unchanged throughout the component (e.g., nucleotide or amino acid) alignment window. "Identity" can be readily calculated using known methods, including but not limited to those described in the following literature: *Computational Molecular Biology* (edited by Lesk, AM), Oxford University Press, New York (1988); *Biocomputing: Informatics and Genome Projects* (edited by Smith, DW), Academic Press, New York (1993); *Computer Analysis of Sequence Data, Part I* (edited by Griffin, AM and Griffin, HG), Humana Press, New Jersey (1994); *Sequence Analysis in Molecular Biology* (edited by von Heinje, G.), Academic Press (1987); and *Sequence Analysis Primer* (Gribskov, M. and Devereux, J. (eds.) Stockton Press, New York, (1991).

[0057] As used herein, the term "sequence identity percentage" or "identity percentage" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to the test ("subject") polynucleotide molecule (or its complementary strand) when two sequences are best aligned. In some embodiments, "identity percentage" may refer to the percentage of identical amino acids in the amino acid sequence compared to a reference polypeptide.

[0058] As used herein, in the context of comparisons and alignments of two nucleic acid molecules, nucleotide sequences, or protein sequences for maximum correspondence, the phrase “substantially identical” or “substantially identical” means that two or more sequences or subsequences have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, using one of the following sequence comparison algorithms or as determined by visual inspection. In some embodiments of the invention, substantial identity exists within regions of consecutive nucleotides in the nucleotide sequence of the invention, said regions being of length from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 30 nucleotides to about 40 nucleotides, from about 50 nucleotides to about 60 nucleotides, from about 70 nucleotides to about 80 nucleotides, from about 90 nucleotides to about 100 nucleotides or more nucleotides, and any range therewith, up to the full length of the sequence. In some embodiments, the nucleotide sequence may be substantially identical in at least about 20 nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In some embodiments, substantially identical nucleotide or protein sequences perform substantially the same function as substantially identical nucleotide (or encoded protein sequences).

[0059] For sequence comparisons, a sequence typically serves as a reference sequence for comparison with one or more test sequences. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, the coordinates of the subsequences are specified, along with the sequence algorithm program parameters. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the specified program parameters.

[0060] The optimal alignment of sequences for the comparison window is well known to those skilled in the art and can be performed using tools such as Smith and Waterman’s local homology algorithm, Needleman and Wunsch’s homology alignment algorithm, and Pearson and Lipman’s similarity search method, and optionally through computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA, and TFASTA, which are available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA)) and web-based alignment programs (such as Clustal Omega, EMBOSS Needle, EMBOSS Stretcher, EMBOSS Water, LALIGN, GGSEARCH2SEQ, EMBOS Cons, Kalign, MAFFT, MUSCLE, and T-Coffee). In some embodiments, the “best alignment” of two sequences (e.g., two peptide sequences or two polynucleotide sequences) is the alignment with the highest score, optionally derived from alignments performed using tools such as Smith and Waterman’s local homology algorithm, Needleman and Wunsch’s homology alignment algorithm, Pearson and Lipman’s similarity search method, GAP, BESTFIT, FASTA and TFASTA, Clustal Omega, EMBOSS Needle, EMBOSS Streetcher, EMBOSS Water, LALIGN, GGSEARCH2SEQ, EMBOS Cons, Kalign, MAFFT, MUSCLE and / or T-Coffee. In some embodiments, the “best alignment” of two sequences (e.g., two peptide sequences) is the alignment that provides the highest percentage of sequence identity, optionally allowing the introduction of one or more vacancies into one or both sequences. The "identity score" for alignment fragments used to test and reference sequences is the number of identical components shared by the two alignment sequences divided by the total number of components in the reference sequence fragment (e.g., the entire reference sequence or a smaller defined portion of the reference sequence). Sequence identity percentage is expressed as the identity score multiplied by 100. Comparisons of one or more sequences can be with the full-length sequence or a portion thereof, or with a longer sequence.For the purposes of this invention, the “percentage of identity” and / or the best alignment can be determined using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information, such as BLASTX for nucleotide sequences for translation, BLASTN for polynucleotide sequences, and BLASTP for peptide sequences.

[0061] When two nucleotide sequences hybridize with each other under stringent conditions, they can also be considered substantially complementary. In some representative examples, two nucleotide sequences considered substantially complementary hybridize with each other under highly stringent conditions.

[0062] In nucleic acid hybridization experiments, such as Southern and Northern hybridization, "strict hybridization conditions" and "strict hybridization washing conditions" are sequence-dependent and vary under different environmental parameters. Extensive guidelines on nucleic acid hybridization can be found in Tijssen's *Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes*, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, New York (1993). Typically, highly stringent hybridization and washing conditions are chosen at defined ionic strengths and pH values ​​that are higher than the melting point (T0) of the specific sequence. m It is about 5°C lower.

[0063] T m This is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at defined ionic strength and pH). Very stringent conditions are chosen to be equal to the Ta of a specific probe. mIn Southern or Northern blotting, an example of stringent hybridization conditions for complementary nucleotide sequences with more than 100 complementary residues hybridizing on a filter membrane is overnight hybridization with 50% formamide and 1 mg heparin at 42°C. An example of highly stringent washing conditions is washing with 0.1 5M NaCl for approximately 15 minutes at 72°C. An example of stringent washing conditions is washing with 0.2x SSC for 15 minutes at 65°C (see Sambrook below for a description of the SSC buffer). Typically, a low-stringency wash is performed before a high-stringency wash to remove background probe signal. For example, an example of a moderately stringent wash for duplexes exceeding 100 nucleotides is washing with 1x SSC for 15 minutes at 45°C. An example of a low-stringency wash for duplexes exceeding 100 nucleotides is washing with 4-6x SSC for 15 minutes at 40°C. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ions, typically about 0.01 M to 1.0 M Na ion concentrations (or other salts) at pH 7.0 to 8.3, and temperatures typically at least about 30°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Typically, a signal-to-noise ratio of 2-fold (or higher) observed against unrelated probes in a specific hybridization assay indicates that specific hybridization has been detected. If the nucleotide sequences encode substantially identical proteins, the nucleotide sequences that do not hybridize to each other under stringent conditions will still be substantially identical. This occurs, for example, when copies of nucleotide sequences are generated using the maximum codon degeneracy allowed by the genetic code.

[0064] The polynucleotides and / or recombinant nucleic acid constructs of the present invention can be codon-optimized for expression. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors of the present invention (e.g., containing / encoding polypeptides of the present invention, such as fusion proteins), nucleic acid-binding polypeptides (e.g., DNA-binding polypeptides, such as sequence-specific DNA-binding domains from polynucleotide-guided endonucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), Argonaute proteins, and / or CRISPR-Cas effector proteins), guide nucleic acids, deaminases, and / or reverse transcriptases) can be codon-optimized for expression in organisms (e.g., animals such as humans, plants, fungi, archaea, or bacteria). In some embodiments, the codon-optimized nucleic acid constructs, polynucleotides, expression cassettes, and / or vectors of the present invention have about 70% to about 99.9% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) identity or higher with reference nucleic acid constructs, polynucleotides, expression cassettes, and / or vectors that have not been codon-optimized.

[0065] In any of the embodiments described herein, the polynucleotide or nucleic acid construct of the present invention can be operatively associated with a variety of promoters and / or other regulatory elements for expression in an organism or its cells (e.g., mammalian and / or mammalian cells, plant and / or plant cells, etc.). Therefore, in some embodiments, the polynucleotide or nucleic acid construct of the present invention may further comprise one or more promoters, introns, enhancers, and / or terminators operatively linked to one or more nucleotide sequences. In some embodiments, the promoter may be operatively associated with an intron (e.g., the Ubi1 promoter and an intron). In some embodiments, the promoter associated with an intron may be referred to as a “promoter region” (e.g., the Ubi1 promoter and an intron).

[0066] As used herein, “operably linked” or “operably associated” in relation to polynucleotides means that the indicated elements are functionally related to each other and are generally also physically related. Therefore, as used herein, the term “operably linked” or “operably associated” refers to functionally related nucleotide sequences on a single nucleic acid molecule. Thus, a first nucleotide sequence operably linked to a second nucleotide sequence refers to a situation where the first nucleotide sequence and the second nucleotide sequence are in a functional relationship. For example, if a promoter influences the transcription or expression of a nucleotide sequence, then the promoter is operably associated with said nucleotide sequence. Those skilled in the art will understand that a control sequence (e.g., a promoter) need not be adjacent to the nucleotide sequence with which it is operably associated, as long as the function of the control sequence is to direct its expression. Therefore, for example, an untranslated but transcribed nucleic acid sequence may exist between the promoter and the nucleotide sequence, and the promoter can still be considered “operably linked” to the nucleotide sequence.

[0067] As used herein, the terms “link” or “fusion” relating to peptides refer to the covalent linking of one peptide to another. A peptide can be linked or fused to another peptide directly (e.g., via a peptide bond) or via a linker (e.g., a peptide linker) (e.g., at the N-terminus or C-terminus). Direct fusion (e.g., direct linking) of two peptides means that an amino acid residue of the first peptide is covalently linked to an amino acid residue of the second peptide, without any intermediate element between the two amino acid residues. For example, the first and second peptides can be directly linked via a peptide bond between the first and second peptides, without any intermediate element (e.g., a linker) between them. Indirect fusion (e.g., indirect linking) of two peptides means that an intermediate element (e.g., a linker, such as a peptide linker) exists between the two peptides, and said intermediate element is covalently linked to each peptide, optionally said intermediate element connecting one end of the first peptide to one end of the second peptide.

[0068] As used herein, a “fusion protein” refers to two or more polypeptides covalently linked (e.g., directly or indirectly) such that they are transcribed and translated as a single unit to produce a single polypeptide comprising the two or more polypeptides. In some embodiments, the two or more polypeptides may be naturally encoded by separate genes, but are encoded by a single gene as a fusion protein. The term “connector” is recognized in the art and refers to a chemical group or molecule that connects two molecules or portions, such as two polypeptides or domains connecting a fusion protein, such as a CRISPR-Cas effector protein and a peptide tag and / or the polypeptide of interest. A connector may comprise a single linker molecule (e.g., a single amino acid) or may comprise more than one linker molecule. In some embodiments, a connector may be an organic molecule, group, polymer, or chemical moiety, such as a divalent organic moiety. In some embodiments, a connector may be an amino acid or may be a peptide. In some embodiments, a connector is a peptide (e.g., a peptide connector).

[0069] In some embodiments, the length of the peptide linker used in this invention can be from about 2 to about 100 or more amino acids, for example, a length of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56. 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., lengths of about 2 to about 40, about 2 to about 50, about 2 to about 60, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 5). 0, about 5 to about 60, about 9 to about 40, about 9 to about 50, about 9 to about 60, about 10 to about 40, about 10 to about 50, about 10 to about 60, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids to about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 The peptide linker may contain 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., lengths of about 105, 110, 115, 120, 130, 140, 150 or more amino acids). In some embodiments, the peptide linker may be a GS linker. In some embodiments, the peptide linker is a GS linker having 2, 3 or 4 amino acid residues, optionally having 2 or 4 amino acid residues. In some embodiments, the peptide linker has one amino acid sequence from the amino acid sequences of SEQ ID NO: 1-36. In some embodiments, the peptide linker may contain CA, CF, (GGS). n, GS, SG, GSSG (SEQ ID NO: 31), GSSGSS (SEQ ID NO: 32), GSSGSSGS (SEQ ID NO: 33), (GSS) n (SEQ ID NO: 34), (GSS) n GS (SEQ ID NO: 35), S(GGS) n (SEQ ID NO: 25), SGGS (SEQ ID NO: 26), (GSS) n The amino acid sequence is G (SEQ ID NO: 36) or (GGGGS)n (SEQ ID NO: 27), where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the peptide linker may comprise the following amino acid sequence: SGGSGGSGGS (SEQ ID NO: 28). In some embodiments, the peptide linker may comprise the following amino acid sequence: SGSETPGTSES ATPES (SEQ ID NO: 29), also known as the XTEN linker. In some embodiments, the peptide linker may comprise the following amino acid sequence: SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 30), also known as the GS-XTEN-GS linker. In some embodiments, the linker has (GSS). n The sequence G (SEQ ID NO: 36), where n is 3. In some embodiments, the connector has (GSS). n The sequence of G (SEQ ID NO: 36), where n is 5.

[0070] As used herein, the terms “link” or “fusion” relating to polynucleotides refer to the covalent attachment of one polynucleotide to another. In some embodiments, two or more polynucleotide molecules may be linked by a linker, which may be an organic molecule, group, polymer, or chemical motif, such as a divalent organic motif. Polynucleotides may be linked or fused to another polynucleotide via direct covalent linkage or via one or more linker nucleotides (at the 5' or 3' end). In some embodiments, a polynucleotide motif of a certain structure may be inserted into another polynucleotide sequence (e.g., an extension of a hairpin structure in guide RNA). In some embodiments, the linker nucleotide may be a naturally occurring nucleotide. In some embodiments, the linker nucleotide may be a non-naturally occurring nucleotide. Direct fusion of two polynucleotides (e.g., direct linkage) refers to the covalent linkage of a nucleotide of a first polynucleotide to a nucleotide of a second polynucleotide, without any intermediate element between the two polynucleotides. For example, the first and second polynucleotides may be directly linked via a phosphodiester bond between the first and second polynucleotides, without any intermediate element (e.g., a linker) between them. Indirect fusion (e.g., indirect linking) of two polynucleotides refers to the presence of an intermediate element (e.g., a linker, such as a polynucleotide linker) between the two polynucleotides, and the intermediate element is covalently linked to each polynucleotide, optionally the intermediate element linking one end of the first polynucleotide of the two polynucleotides to one end of the second polynucleotide of the two polynucleotides.

[0071] A promoter is a nucleotide sequence that controls or regulates transcription of a nucleotide sequence (e.g., a coding sequence) that is operatively associated with a promoter. The coding sequence controlled or regulated by a promoter can encode polypeptides and / or functional RNA. Typically, a promoter is a nucleotide sequence containing an RNA polymerase II binding site that directs transcription initiation. Typically, a promoter is located 5' or upstream of the starting point of the coding region relative to the corresponding coding sequence. Promoters may contain other elements that act as regulators of gene expression, such as promoter regions. These include TATA box concordant sequences and often include CAAT box concordant sequences (Breathnach and Chambon, (1981) *Annu. Rev. Biochem.* 50:349). In plants, the CAAT box can be replaced by the AGGA box (Messing et al., (1983) *Genetic Engineering of Plants*, T. Kosuge, C. Meredith, and A. Hollaender (eds.), Plenum Press, pp. 211–227). In some embodiments, the promoter region may contain at least one intron (e.g., SEQ ID NO: 37 or SEQ ID NO: 38).

[0072] Promoters that can be used in this invention may include, for example, constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-biased, and / or tissue-specific promoters for the preparation of recombinant nucleic acid molecules, such as “synthetic nucleic acid constructs” or “protein-RNA complexes.” These various types of promoters are known in the art.

[0073] The choice of promoter can vary depending on the temporal and spatial requirements of expression, as well as on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge available in the field, an appropriate promoter can be selected for the specific host organism of interest. Thus, for example, much is known about promoters upstream of genes that are highly constitutively expressed in model organisms, and such knowledge is readily available and can be implemented in other systems where appropriate.

[0074] In some embodiments, promoters that are functional in plants can be used with the constructs of the present invention. Non-limiting examples of promoters that can be used to drive expression in plants include the promoter of the RuBisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr), and the promoter of the repeat carbonic anhydrase gene 1 (Pdca1) (see Walker et al., Plant Cell Reports. 23:727-735 (2005); Li et al., Gene. 403:132-142 (2007); Li et al., Molecular Biology Reports. 37:1143-1154 (2010)). PrbcS1 and Pactin are constitutive promoters, and Pnr and Pdca1 are inducible promoters. Pnr is nitrate-induced and ammonium-inhibited (Li et al., *Genes* 403:132-142 (2007)), and Pdca1 is salt-induced (Li et al., *Reports of Molecular Biology* 37:1143-1154 (2010)). In some embodiments, the promoter used in this invention is the RNA polymerase II (Pol II) promoter. In some embodiments, the U6 promoter or 7SL promoter from maize (Zea mays) can be used in the constructs of this invention. In some embodiments, the U6c promoter and / or 7SL promoter from maize can be used to drive the expression of guide nucleic acids. In some embodiments, the U6c promoter, U6i promoter, and / or 7SL promoter from soybean (Glycine max) can be used in the constructs of this invention. In some embodiments, the U6c promoter, U6i promoter, and / or 7SL promoter from soybean can be used to drive the expression of guide nucleic acids.

[0075] Examples of constitutive promoters that can be used in plants include, but are not limited to, the cestrum virus promoter (CMP) (US Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992), *Molecular Cell Biology* 12:3399-3406; and US Patent No. 5,641,876), the CaMV 35S promoter (Odell et al. (1985), *Nature* 313:810-812), the CaMV 19S promoter (Lawton et al. (1987), *Plant Molecular Biology* 9:315-324), and the nos promoter (Ebert et al. (1987), *Proceedings of the National Academy of Sciences of the United States of America*). (84:5745-5749), Adh promoter (Walker et al. (1987) Proceedings of the National Academy of Sciences 84:6624-6629), sucrose synthase promoter (Yang and Russell (1990) Proceedings of the National Academy of Sciences 87:4144-4148), and ubiquitin promoter. Constitutive promoters derived from ubiquitin accumulate in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, such as sunflower (Binet et al., 1991. Plant Science 79:87-94), maize (Christensen et al., 1989. Plant Molecular Biology 12:619-632), and Arabidopsis (Norris et al., 1993. Plant Molecular Biology 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocotyledonous plant systems, and its sequence and the vector constructed for monocotyledonous plant transformation are disclosed in European Patent Publication EP0342926. The ubiquitin promoter is suitable for expressing the nucleotide sequences of the present invention in transgenic plants, particularly monocotyledonous plants. Furthermore, the promoter expression cassette described by McElroy et al. (Molecular Genetics and Genomics 231:150-160 (1991)) can be readily modified for the expression of the nucleotide sequences of the present invention and is particularly suitable for monocotyledonous plant hosts.

[0076] In some embodiments, tissue-specific / tissue-preferred promoters can be used to express heterologous polynucleotides in plant cells. Tissue-specific or preferred expression patterns include, but are not limited to, green tissue-specific or preferred, root-specific or preferred, stem-specific or preferred, flower-specific or preferred, or pollen-specific or preferred. Promoters suitable for expression in green tissues include a number of promoters that regulate genes involved in photosynthesis, and many of these are cloned from both monocotyledonous and dicotyledonous plants. In one embodiment, the promoter that can be used in this invention is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth and Grula, Plant Molecular Biology 12:579-589 (1989)). Non-limiting examples of tissue-specific promoters include tissue-specific promoters associated with genes encoding seed storage proteins (such as β-conglycinin, cruciferin, napin, and bean globulin), zein or oleosome proteins (such as olein), or proteins involved in fatty acid biosynthesis (including acyl-carrier proteins, stearoyl-ACP desaturases, and fatty acid desaturases (fad 2-1)), as well as other nucleic acids expressed during embryonic development (such as Bce4; see, for example, Kridl et al. (1991), Seed Sci. Res. 1:209-219; and EP Patent No. 255378). Tissue-specific or tissue-preferred promoters that can be used to express the nucleotide sequences of the present invention in plants, particularly maize, include, but are not limited to, promoters that direct expression in roots, pith, leaves, or pollen. For example, such promoters are disclosed in WO 93 / 07278, which is incorporated herein by reference for the content of its promoter disclosure.Other non-limiting examples of tissue-specific or tissue-preferred promoters that can be used in this invention include the cotton RuBisCo promoter disclosed in U.S. Patent 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent 5,604,121; the root-specific promoter described by de Framond (FEBS 290:103-106 (1991); European Patent EP 0452269 granted to Ciba-Geigy); the stem-specific promoter described in U.S. Patent 5,625,136 (granted to Ciba-Geigy), and said promoter drives the expression of the maize trpA gene; the night-blooming jasmine yellow leaf curl virus promoter disclosed in WO 01 / 73087; and pollen-specific or tissue-preferred promoters, including but not limited to ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al., Plant Biotechnol. Reports 9(5):297-306). (2015)), ZmSTK2_USP from maize (Wang et al., Genome 60(6):485-495(2017)), LAT52 and LAT59 from tomato (Twell et al., Development 109(3):705-713(1990)), Zm13 (US Patent No. 10,421,972), PLA2-δ promoter from Arabidopsis thaliana (US Patent No. 7,141,424) and / or ZmC5 promoter from maize (International PCT Publication No. WO 1999 / 042587).

[0077] Other examples of plant tissue-specific / tissue-biased promoters include, but are not limited to, root-hair-specific cis-elements (RHE) (Kim et al., *The Plant Cell*, 18:2958-2970 (2006)), root-specific promoters RCc3 (Jeong et al., *Plant Physiol.*, 153:185-197 (2010)) and RB7 (US Patent No. 5,459,252), lectin promoters (Lindstrom et al., *Der. Genet.*, 11:160-167; and Vodkin, *Prog. Clin. Biol. Res.*, 138:87-98 (1983)), and zeatol dehydrogenase 1 promoter (Dennis et al., *Nucleic Acids Res.*, 1984). 12:3983-4000), S-adenosine-L-methionine synthase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), maize light-harvesting complex promoter (Bansal et al. (1992) Proceedings of the National Academy of Sciences of the United States of America 89:3654-3658), maize heat shock protein promoter (O'Dell et al. (1985) Journal of the European Organization for Molecular Biology (EMBO J)).5:451-458; and Rochester et al. (1986) *European Journal of Molecular Biology* 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase” pp. 29-39 *Genetic Engineering in Plants*, edited by Hollaender, Plummer Press, 1983; and Poulsen et al. (1986) *Molecular Genetics and Genomics* 205:193-200), Ti plasmid mannitol synthase promoter (Langridge et al. (1989) *Proceedings of the National Academy of Sciences* 86:3219-3223), Ti plasmid carmine synthase promoter (Langridge et al. (1989)). Ibid.), Petunia chalcone isomerase promoter (vanTunen et al. (1988) European Organization for Molecular Biology Journal 7:1257-1263), Legume glycine-rich protein 1 promoter (Keller et al. (1989) Genes Dev).3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato glycoprotein promoter (Wenzler et al. (1989) Plant Molecular Biology 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acid Research 18:7449), zein promoter (Kriz et al. (1987) Molecular Genetics and Genomics 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acid Research 18:6425; Reina et al. (1990) Nucleic Acid Research 18:7449; and Wandelt et al. (1989) Nucleic Acid Research 17:2354), globulin-1 promoter (Belanger et al. (1991), Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989), Molecular Genetics and Genomics 215:431-440), PEPCase promoter (Hudspeth and Grula (1989), Plant Molecular Biology 12:579-589), R gene complex-related promoter (Chandler et al. (1989), Plant Cell 1:1175-1183), and chalcone synthase promoter (Franken et al. (1991), European Organization for Molecular Biology Journal 10:2605-2612).

[0078] The pea globulin promoter can be used for seed-specific expression (Czako et al. (1992) Molecular Genetics and Genomics 235:33-40); and seed-specific promoters, disclosed in U.S. Patent No. 5,625,136. Promoters that can be used for expression in mature leaves are promoters that switch at the onset of senescence, such as the SAG promoter from Arabidopsis thaliana (Gan et al. (1995) Science 270:1986-1988).

[0079] Alternatively, promoters that are functional in chloroplasts can be used. Non-limiting examples of such promoters include the phage T3 gene 9 5' UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters that can be used in this invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0080] Other modulating elements that can be used in this invention include, but are not limited to, introns, enhancers, termination sequences, and / or 5' and 3' untranslated regions.

[0081] Introns that can be used in this invention can be introns identified and isolated from plants and then inserted into expression cassettes for plant transformation. As those skilled in the art will understand, introns can contain the sequence required for self-excision and are incorporated into the nucleic acid construct / expression cassette within a frame. Introns can be used as spacers to separate multiple protein-coding sequences within a nucleic acid construct, or introns can be used within a protein-coding sequence, for example, to stabilize mRNA. If used within a protein-coding sequence, it is inserted "within a frame" and includes an excision site. Introns can also associate with promoters to improve or alter expression. As examples, promoter / intron combinations that can be used in this invention include, but are not limited to, promoter / intron combinations of the maize Ubi1 promoter and introns.

[0082] Non-limiting examples of introns that can be used in this invention include introns derived from: ADHI genes (e.g., Adh1-S introns 1, 2, and 6), ubiquitin genes (Ubi1), RuBisCO small subunit (rbcS) genes, RuBisCO large subunit (rbcL) genes, actin genes (e.g., actin-1 introns), pyruvate dehydrogenase kinase genes (pdk), nitrate reductase genes (nr), repeat carbonic anhydrase gene 1 (Tdca1), psbA genes, atpA genes, or any combination thereof.

[0083] As used herein, “editing system” refers to any site-specific (e.g., sequence-specific) nucleic acid editing system now known or developed thereafter that can introduce modifications (e.g., mutations) into nucleic acids in a target-specific manner. For example, editing systems (e.g., site-specific and / or sequence-specific editing systems) may include, but are not limited to, CRISPR-Cas editing systems, broad-spectrum nuclease editing systems, zinc finger nuclease (ZFN) editing systems, transcription activator-like effector nuclease (TALEN) editing systems, base editing systems, and / or leader editing systems, each of which may contain one or more polypeptides and / or one or more polynucleotides that can modify (e.g., mutate) target nucleic acids in a sequence-specific manner when present and / or expressed together in a composition and / or cell (e.g., as a system). In some embodiments, editing systems (e.g., site-specific and / or sequence-specific editing systems) may contain one or more polynucleotides and / or one or more polypeptides, including but not limited to nucleic acid-binding polypeptides (e.g., DNA-binding domains), nucleases, another polypeptide and / or polynucleotide. In some embodiments, a CRISPR-Cas editing system comprising the fusion protein of the present invention is provided and / or used.

[0084] In some embodiments, the editing system comprises one or more sequence-specific nucleic acid-binding peptides (e.g., DNA-binding domains) that may be derived from, for example, polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins. In some embodiments, the editing system comprises one or more cleaving peptides (e.g., nucleases), including but not limited to endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs).

[0085] As used herein, a “nucleic acid binding polypeptide” refers to a polypeptide or domain that binds to and / or is capable of binding to nucleic acids (e.g., target nucleic acids). DNA-binding domains are exemplary nucleic acid binding polypeptides and can be site-specific and / or sequence-specific nucleic acid binding domains. In some embodiments, the nucleic acid binding polypeptide can be a sequence-specific nucleic acid binding polypeptide, such as, but not limited to, sequence-specific binding domains from, for example, polynucleotide-guided endonucleases, CRISPR-Cas effector proteins (e.g., CRISPR-Cas endonucleases), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins. In some embodiments, the nucleic acid binding polypeptide comprises a cleavage domain (e.g., a nuclease domain), such as, but not limited to, endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases, zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs). In some embodiments, a nucleic acid-binding polypeptide associates with and / or is capable of associating with one or more nucleic acid molecules (e.g., forming a complex) (e.g., forming a complex with a guide nucleic acid as described herein), which directs and / or guides the nucleic acid-binding polypeptide to a specific target nucleotide sequence (e.g., a genomic locus) complementary to the one or more nucleic acid molecules (or portions or regions thereof), thereby causing the nucleic acid-binding polypeptide to bind to the nucleotide sequence at a specific target site. In some embodiments, the nucleic acid-binding polypeptide is a CRISPR-Cas effector protein as described herein.

[0086] In some embodiments, the editing system comprises or is a ribonucleoprotein, such as an assembled ribonucleoprotein complex (e.g., a ribonucleoprotein comprising a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase and / or reverse transcriptase). In some embodiments, the ribonucleoproteins of the editing system may be assembled together (e.g., a pre-assembled ribonucleoprotein comprising a CRISPR-Cas effector protein, a guide nucleic acid, and optionally a deaminase and / or reverse transcriptase), such as upon contact with a target nucleic acid or upon introduction into a cell (e.g., a mammalian cell or a plant cell) (e.g., upon contacting a component of the ribonucleoprotein with the target nucleic acid and / or upon introducing a component of the ribonucleoprotein into the cell). In some embodiments, the ribonucleoproteins of the editing system may assemble into a complex (e.g., a non-covalently bound complex) upon contact of a portion of the ribonucleoprotein with the target nucleic acid and / or may assemble after and / or during introduction into a plant cell. In some embodiments, the editing system may assemble upon introduction into a plant cell (e.g., assembling into a non-covalently bound complex). In some embodiments, the ribonucleoproteins of the editing system may contact the target nucleic acid and / or may be introduced into a plant cell. In some embodiments, the editing system can be assembled upon introduction into plant cells (e.g., assembled into a non-covalently bound complex). In some embodiments, the ribonucleoprotein may comprise the fusion protein of the present invention (e.g., a polypeptide comprising a CRISPR-Cas effector protein and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242), guide nucleic acid, and optionally a deaminase and / or reverse transcriptase. In some embodiments, the fusion protein of the present invention is used to replace (e.g., substitute) the CRISPR-Cas effector protein (e.g., in the compositions, complexes, kits, methods, and / or systems (such as editing systems) described herein) and / or optionally as a CRISPR-Cas effector protein in the compositions, complexes, ribonucleoproteins, kits, methods, systems, and / or editing systems of the present invention.

[0087] In some embodiments, the editing system of the present invention comprises a reverse transcriptase, an extended guide nucleic acid, and the fusion protein of the present invention. In some embodiments, the fusion protein, the reverse transcriptase, and the extended guide nucleic acid of the present invention may form a complex or may be contained within a complex, the complex being capable of interacting with the target nucleic acid.

[0088] In some embodiments, the guide nucleic acid further comprises a reverse transcriptase template and may be referred to as an extended guide nucleic acid. As used herein, an "extended guide nucleic acid" is a guide nucleic acid as described herein that further comprises a reverse transcriptase template (RTT) and / or a primer binding site (PBS). In some embodiments, the extended guide nucleic acid is an engineered lead editing guide RNA (pegRNA). The extended guide nucleic acid may be a target allele guide RNA (tagRNA) or a stable target allele guide RNA (stagRNA). As used herein, "tagRNA" refers to an extended guide nucleic acid that comprises PBS and an RTT and has target strand complementarity. As used herein, "stagRNA" refers to a tagRNA that comprises a stabilization motif. The stabilization motif may be present at the 3' and / or 5' ends of the tagRNA. In some embodiments, the stabilization motif is present at the 3' end of the tagRNA. Exemplary stabilization motifs include, but are not limited to, recruitment motifs, RNA hairpins, pseudoknot sequences, and / or PP7 motifs (e.g., PP7 RNA hairpin sequences). In some embodiments, the stagRNA is a tagRNA that comprises a PP7 RNA hairpin sequence. In some embodiments, CRISPR-Cas effector proteins (e.g., type II or type V CRISPR-Cas effector proteins), reverse transcriptase, and extended guide nucleic acids may form a complex or be contained within the complex.

[0089] In some embodiments, the extended guide nucleic acid includes an extension portion comprising a primer binding site and a reverse transcriptase template, wherein the reverse transcriptase template contains a modification (e.g., editing) to be incorporated into the target nucleic acid. In some embodiments, the extended guide nucleic acid includes a primer binding site and a modification (e.g., editing) to be incorporated into the target nucleic acid (e.g., reverse transcriptase template) at its 3' end. In some embodiments, the extended guide nucleic acid comprises: (1) a sequence that interacts (e.g., recruits and / or binds) to a CRISPR-Cas effector protein (e.g., a CRISPR-Cas nuclease), (2) a spacer substantially complementary to the first site on the target nucleic acid (e.g., CRISPR RNA (crRNA) (first crRNA) and / or tracrRNA+crRNA (sgRNA)), and (3) a nucleic acid-encoded repair template (e.g., an RNA-encoded repair template) comprising a primer binding site and an RNA template (e.g., encoding a modification to be incorporated into the target nucleic acid). In some embodiments, the extended guide nucleic acid (e.g., extended guide RNA) may include a spacer sequence, a repeat sequence, and an extension portion from 5' to 3', wherein the extension portion from 5' to 3' includes a reverse transcriptase template and a primer binding site. In some embodiments, the extended guide nucleic acid may include a spacer sequence, a repeat sequence, and an extension portion from 5' to 3', wherein the extension portion from 5' to 3' includes a reverse transcriptase template and a primer binding site. In some embodiments, the extended guide nucleic acid may include an extension portion, a spacer sequence, and a repeat sequence from 5' to 3', wherein the extension portion from 5' to 3' includes a primer binding site and a reverse transcriptase template. In some embodiments, the extended guide nucleic acid may include an extension portion, a spacer sequence, and a repeat sequence from 5' to 3', wherein the extension portion from 5' to 3' includes a primer binding site and a reverse transcriptase template.

[0090] According to some embodiments, the extended guide nucleic acid (e.g., pegRNA) may have a structure and / or be designed as described in Anzalone et al., Nature, December 2019; 576(7785): 149-157. In some embodiments, the extended guide nucleic acid comprises a primer binding site (PBS) having a sequence of 1, 2, 3, 4, or 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides and a reverse transcriptase template (RT template) sequence having a sequence of 65 or more nucleotides. In some embodiments, the PBS of the extended guide nucleic acid has a sequence of less than 15 nucleotides and a sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 nucleotides (e.g., a sequence of 5 or 6 nucleotides in length). The RT template sequence may be following the PBS sequence in the 5' to 3' direction. In some embodiments, the RT template sequence of the extended guide nucleic acid has a length greater than 65 nucleotides and may contain about 50 or more heterologous nucleotides relative to the target site (e.g., the target nucleic acid), followed by about 15 or more homologous nucleotides relative to the target site. In some embodiments, the RT template sequence of the extended guide nucleic acid follows the PBS sequence and has a length greater than 65 nucleotides, wherein the sequence comprises more than 50 heterologous nucleotides relative to the target site, followed by more than 15 homologous nucleotides relative to the target site. Thus, in some embodiments, when the extended guide nucleic acid is reverse transcribed, the resulting newly transcribed sequence may hybridize with the uncut strand of the target site and / or be configured to hybridize with the uncut strand of the target site, which may thereby produce heterodouble-helical DNA with large insertions in the newly synthesized strand. After repairing such mismatched DNA, the resulting repaired DNA may contain large insertions (e.g., greater than 50 nucleotides) of the DNA sequence. In some embodiments, the method may provide large deletions (e.g., greater than 50 nucleotides) of the DNA sequence. In some embodiments, PBS and 15 or more nucleotides homologous to the target site may include homology arms, which can be used optionally for homology-directed repair to insert heterologous DNA into the target site. The inserted DNA may correspond to any functional DNA sequence, such as, but not limited to: functional transgenes; DNA fragments inserted into a gene in a manner sufficient to produce hairpin RNA sufficient to silence the homologous gene via RNAi when the gene is transcribed; and / or one or more functional site-specific recombination sites, such as lox, frt, which can then be used for subsequent Cre or Flp-mediated site-specific recombination processes. In some embodiments, the extended guide nucleic acid may be too large to be generated in vivo using the PolIII promoter. In some embodiments, the extended guide nucleic acid may be operatively associated with and / or generated using the PolII promoter.In some embodiments, the DNA-binding polypeptide (e.g., a DNA-binding domain) and / or DNA endonuclease may have and / or be designed to have the structures described in Anzalone et al., Nature, December 2019; 576(7785): 149-157. In some embodiments, the DNA-binding domain and / or DNA endonuclease are CRISPR-Cas polypeptides, such as Cas9 nickase, a nick variant of another CRISPR-Cas polypeptide, or Cas12a.

[0091] In some embodiments, two extended guide nucleic acids (e.g., pegRNAs) may be used (e.g., the editing system may contain two extended guide nucleic acids). One or both of the two extended guide nucleic acids may have the structure and / or be designed as described in Anzalone et al., Nature, December 2019; 576(7785): 149-157. The two extended guide nucleic acids may contain a primer binding site (PBS) having a sequence of 1, 2, 3, 4 or 5 to 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides and a reverse transcriptase template (RT template) sequence having a sequence of 50 or more nucleotides. The RT template sequences of the two extended guide nucleic acids may be complementary to each other, and therefore the polynucleotides reverse transcribed from each RT template will be complementary to each other and will be able to hybridize with each other. This allows intermediates produced by this system and / or method to link two DNA segments together, which would otherwise be separated by more than 50 nucleotides, for example, within a single chromosome, or located on two separate DNA fragments, for example, on two different chromosomes. Following the repair intermediate, the resulting product can produce large deletions, large inversions, or interchromosomal recombinations, depending on the design of the RT template. Since all these products are produced via homology-directed repair, they can be predictably precise and / or reproducible. In some embodiments, the DNA-binding polypeptide (e.g., a DNA-binding domain) and / or DNA endonuclease may have and / or be designed as described in Anzalone et al., Nature, December 2019; 576(7785): 149-157. In some embodiments, the DNA-binding polypeptide and / or DNA endonuclease is a CRISPR-Cas polypeptide, such as the Cas9 nickase, a similar nick variant of another CRISPR-Cas polypeptide, or Cas12a. In some embodiments, the DNA-binding polypeptide and / or DNA endonuclease is a Cas9 nuclease, a similar nuclease derived from another CRISPR Cas polypeptide, or Cas12a. Using a nuclease (instead of a nicking enzyme) can facilitate intrachromosomal or interchromosomal recombination processes through single-stranded annealing of more than 50 nucleotides at the 3' overhang, which will be generated at each of two target sites corresponding to two pegRNA target nucleic acids. In some embodiments, the editing system comprises an extended guide nucleic acid and a guide nucleic acid lacking a reverse transcriptase template and / or primer binding site.

[0092] The extended guide nucleic acid may comprise a CRISPR nucleic acid (e.g., CRISPR RNA, CRISPR DNA, crRNA, crDNA) and / or a CRISPR nucleic acid and a tracr nucleic acid; and (b) an extension comprising a primer binding site and a reverse transcriptase template (RT template), wherein the RT template encodes a modification to be incorporated into the target nucleic acid. The CRISPR nucleic acid may be a type II or type V CRISPR nucleic acid, and / or the tracr nucleic acid may be any tracr corresponding to a suitable type II or type V CRISPR nucleic acid. In some embodiments, the extended guide nucleic acid comprises: (i) a type V CRISPR nucleic acid or a type II CRISPR nucleic acid (e.g., type II or type V CRISPR RNA, type II or type V CRISPR DNA, type II or type V crRNA, or type II or type V crDNA) and / or a CRISPR nucleic acid and a tracr nucleic acid (e.g., type II or type V tracrRNA, type II or type V tracrDNA); and (ii) an extension containing a primer binding site and a reverse transcriptase template (RT template), wherein the type V CRISPR nucleic acid or the type II CRISPR nucleic acid contains a spacer (e.g., the spacer is complementary to a portion of a continuous nucleotide in the first strand of the target nucleic acid) that binds to the first strand (e.g., the target strand) and the primer binding site binds to the first strand (e.g., the target strand). In some embodiments, the extension portion may be fused to the 5' or 3' end of the CRISPR nucleic acid (e.g., from 5' to 3': repeat sequence-spacer-extension or extension-repeat sequence-spacer) and / or fused to the 5' or 3' end of the tracr nucleic acid. In some embodiments, the extension portion of the extended guide nucleic acid from 5' to 3' includes an RT template (RTT) and a primer binding site (PBS) (e.g., 5'-crRNA-spacer-RTT (edit-encoded)-PBS-3'), or from 5' to 3' includes PBS and RTT (e.g., 5'-crRNA-spacer-PBS-RTT (edit-encoded)-3'), depending on the position of the extension portion relative to the CRISPR nucleic acid of the extended guide nucleic acid. For example, in some embodiments, the extension portion of the extended guide nucleic acid from 5' to 3' may include an RT template and a primer binding site (when the extended guide is attached to the 3' end of the CRISPR nucleic acid). In some embodiments, the extended portion of the extended guide from 5' to 3' may include a primer binding site and an RT template (when the extended guide is attached to the 5' end of a CRISPR nucleic acid).

[0093] In some embodiments, the target nucleic acid is double-stranded and comprises a first strand and a second strand, and the primer binding site of the extended guide nucleic acid binds to the second strand of the target nucleic acid (e.g., a non-target, top strand). In some embodiments, the target nucleic acid is double-stranded and comprises a first strand and a second strand, and the primer binding site of the extended guide nucleic acid binds to the first strand of the target nucleic acid (e.g., binds to the target strand, optionally the same strand as the CRISPR-Cas effector protein, the bottom strand). In some embodiments, the target nucleic acid is double-stranded and comprises a first strand and a second strand, and the primer binding site of the extended guide nucleic acid binds to the second strand of the target nucleic acid (e.g., a non-target strand, optionally the strand opposite to the strand recruiting the CRISPR-Cas effector protein). In some embodiments, reverse transcriptase (RT) may be added to the target strand of the target nucleic acid (e.g., the strand complementary to the spacer of the CRISPR nucleic acid of the extended guide nucleic acid and recruiting the CRISPR-Cas effector protein). In some embodiments, reverse transcriptase (RT) is added to the non-target strand of the target nucleic acid (e.g., the strand complementary to the spacer of the same CRISPR nucleic acid and recruiting the CRISPR-Cas effector protein). Example methods and editing systems are described in International Patent Publication No. WO 2021 / 092130, International Patent Publication No. WO 2022 / 098993, and U.S. Patent Application Publication Nos. 2021 / 0147862, 2021 / 0130835, 2021 / 0147862, and 2022 / 0145334, each of which is incorporated herein by reference in its entirety.

[0094] The extended guide nucleic acid RT template can encode one or more modifications (e.g., edits) to be incorporated into the target nucleic acid. These modifications can be located anywhere within the RT template (e.g., the location can be relative to a protospacer adjacent motif (PAM) of the target nucleic acid). In some embodiments, the RT template has modifications at one or more positions from -1 to 23 (e.g., -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) relative to a PAM (e.g., TTTG) in the target nucleic acid. In some embodiments, the RT template may contain modifications located at nucleotide positions -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the RT template may contain modifications at nucleotide positions 4 to 17 (e.g., positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the PAM of the target nucleic acid. In some embodiments, the RT template may contain modifications at nucleotide positions 10 to 17 (e.g., positions 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the PAM of the target nucleic acid. In some embodiments, the RT template may contain modifications at nucleotide positions 12 to 15 (e.g., positions 12, 13, 14, or 15) of the RT template relative to the PAM of the target nucleic acid.

[0095] In some embodiments, the extension portion of the extended guide nucleic acid from 5' to 3' may include an RT template and a primer binding site (e.g., when the extension portion is attached to the 3' end of a CRISPR nucleic acid). In some embodiments, the length of the RT template can be from about 1 nucleotide to about 100 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61). 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides, and any range or value thereof, for example, a length of about 1 nucleotide to about 10 nucleotides, about 1 nucleotide to about 15 nucleotides, about 1 nucleotide to about 20 nucleotides, about 1 nucleotide to about 25 nucleotides, about 1 nucleotide to... About 30 nucleotides, about 1 nucleotide to about 35, 36, 37, 38, 39 or 40 nucleotides, about 1 nucleotide to about 50 nucleotides, about 5 nucleotides to about 15 nucleotides, about 5 nucleotides to about 20 nucleotides, about 5 nucleotides to about 25 nucleotides, about 5 nucleotides to about 30 nucleotides, about 5 nucleotides to about 35, 36, 37, 38, 39 or 40 nucleotides, about 5 nucleotides to about 50 nucleotides, about 8 nucleotides to about 15 nucleotides, about 8 nucleotides to about 20 nucleotides, about 8 nucleotides to about 25 nucleotides, about 8 nucleotides to about 30 nucleotides, about 8 nucleotides to about 35, 36, 37, 38, 39 or 40 nucleotides, about 8 nucleotides to about 50 nucleotides, lengths of about 8 nucleotides to about 100 nucleotides, about 10 nucleotides to about 15 nucleotides, about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 10 nucleotides to about 36 nucleotides, about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 100 nucleotides, and any range or value thereof.In some embodiments, the RT template may be at least 8 nucleotides long, optionally about 8 nucleotides to about 100 nucleotides. In some embodiments, the RT template is 36, 37, 38, 39, or 40 nucleotides long or less (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long), or any value or range thereof (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides long to about 16, 17, 18, 19, ...). (20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In some embodiments, the length of the RT template may be at least 30 nucleotides, optionally from about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides to about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 7 1, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleotides, or any range or value thereof. In some embodiments, the length of the RT template may be about 36, 40, 44, 47, 50, 52, 55, 63, 72, or 74 nucleotides. One or more modifications may be present within the length of the RTT. The one or more modifications may be located at any position within the RTT, wherein the position of the modification may be described relative to the position of the protospacer adjacent motif (PAM) of the target nucleic acid. In some embodiments, the RT template may contain nucleotides located at positions -1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. Modifications at positions 15, 16, 17, 18, 19, 20, 21, 22, or 23. In some embodiments, the RT template may contain modifications at nucleotide positions 4 to 17 (e.g., positions 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the protospacer adjacent motif (PAM) of the target nucleic acid. In some embodiments, the RT template may contain modifications at nucleotide positions 10 to 17 (e.g., positions 10, 11, 12, 13, 14, 15, 16, or 17) of the RT template relative to the protospacer adjacent motif (PAM) of the target nucleic acid.In some embodiments, the RT template may include modifications at nucleotide positions 12 to 15 (e.g., positions 12, 13, 14, or 15) of the RT template relative to the position of the adjacent motif (PAM) of the protospacer relative to the target nucleic acid.

[0096] As used herein, the “primer binding site” (PBS) of the extended portion of an extended guide nucleic acid (e.g., tagRNA) refers to a continuous nucleotide sequence that can bind to a region or “primer” on the target nucleic acid, such as a primer complementary to the target nucleic acid primer. As an example, CRISPR Cas effector proteins (e.g., type II or V, such as Cas 9 or Casl2a) can cleave / cut DNA, and the 3' end of the cleaved DNA acts as a primer for the PBS portion of the extended guide nucleic acid. The PBS can be complementary to the 3' end of the target nucleic acid strand and can bind to or be configured to bind to either the target or non-target strand. The primer binding site can be completely complementary to the primer, or it can be substantially complementary to the primer of the target nucleic acid (e.g., at least 70% complementary (e.g., 70% or about 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or higher)). In some embodiments, the length of the primer binding site in the extended portion can be from about 1 nucleotide to about 100 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, ...). 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides, or any value or range thereof, or about 4 nucleotides to about 85 nucleotides, about 10 nucleotides to about 80 nucleotides, about 20 nucleotides to about 80 nucleotides, about 25 nucleotides to about 80 nucleotides, about 30 nucleotides to about 80 nucleotides, about 40 nucleotides to about 80 nucleotides, about 45 nucleotides to about 80 nucleotides, about 45 nucleotides to about 75 nucleotides, or about 45 nucleotides to about 60 nucleotides, or any range or value thereof.In some embodiments, the length of the PBS may be at least 30 nucleotides, optionally about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides to about 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides, or any range or value thereof. In some embodiments, the length of the PBS may be about 8, 16, 24, 32, 40, 48, 56, 64, 72 or 80 nucleotides.

[0097] In some embodiments, the length of the RTT can be from about 35 nucleotides to about 75 nucleotides, and the length of the PBS can be from about 30 nucleotides to about 80 nucleotides. Optionally, the PBS can contain a length of about 8, 16, 24, 32, 40, 48, 56, 64, 72 or 80 nucleotides, and the RTT can contain a length of about 36, 40, 44, 47, 50, 52, 55, 63, 72 or 74 nucleotides, or any combination of the RTT length and / or PBS length.

[0098] In some embodiments, the extension portion of the extended guide nucleic acid may be fused to the 5' or 3' end of a type II or type V CRISPR nucleic acid (e.g., from 5' to 3': repeat sequence-spacer-extension or extension-repeat sequence-spacer) and / or fused to the 5' or 3' end of the tracr nucleic acid. In some embodiments, when the extension portion is located at the 5' of the crRNA, the type V CRISPR-Cas effector protein is modified to reduce (or eliminate) the self-processing RNAse activity.

[0099] In some embodiments, the extended portion of the extended guide nucleic acid can be linked via a linker to type II or type V CRISPR nucleic acid and / or type II or type V tracrRNA. In some embodiments, the linker is about 1 to about 100 or more nucleotides long (e.g., lengths of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6). 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides, and any range thereof (e.g., lengths of about 2 to about 40, about 2 to about 50, about 2 to about 60, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 50, about 5 ... 60, about 9 to about 40, about 9 to about 50, about 9 to about 60, about 10 to about 40, about 10 to about 50, about 10 to about 60, about 40 to about 100, about 50 to about 100, or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides to about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides (e.g., lengths of about 105, 110, 115, 120, 130, 140, 150 or more nucleotides).

[0100] The guide nucleic acid and / or extended guide nucleic acid may contain one or more recruitment motifs as described herein, which may be attached to the 5' end and / or the 3' end of the guide nucleic acid and / or may be inserted into the guide nucleic acid (e.g., within a hairpin loop of the guide nucleic acid). In some embodiments, the extended guide nucleic acid may be linked to an RNA recruitment motif. The extended guide nucleic acid and / or guide nucleic acid may be linked to one or two or more RNA recruitment motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; e.g., at least 10 to about 25 motifs), optionally wherein two or more RNA recruitment motifs may be the same RNA recruitment motif or different RNA recruitment motifs. In some embodiments, the RNA recruitment motif may be located at the 3' end of the extended portion of the extended guide nucleic acid (e.g., from 5' to 3', repeat sequence-spacer-extension (RT template-primer binding site)-RNA recruitment motif). In some embodiments, the RNA recruitment motif may be embedded in the extended portion of the extended guide nucleic acid.

[0101] In some embodiments, the editing system includes an extended guide nucleic acid linked to an RNA recruitment motif and a reverse transcriptase as a reverse transcriptase fusion protein, wherein the reverse transcriptase fusion protein comprises a reverse transcriptase polypeptide fused to an affinity polypeptide that binds to the RNA recruitment motif, wherein the extended guide nucleic acid binds to a target nucleic acid and the RNA recruitment motif binds to the affinity polypeptide, thereby recruiting the reverse transcriptase fusion protein to the extended guide nucleic acid and contacting the target nucleic acid with the reverse transcriptase. In some embodiments, two or more reverse transcriptase fusion proteins may be recruited to the extended guide nucleic acid, thereby contacting the target nucleic acid with two or more reverse transcriptase fusion proteins.

[0102] As used herein, the term “transgenic” or “transgenic” refers to at least one nucleic acid sequence obtained from or synthesized from the genome of an organism, then introduced into a host cell (e.g., a plant cell) or an organism or tissue of interest, and subsequently integrated into the host genome via a “stable” transformation or transfection method. Conversely, the term “transient” transformation, transfection, or introduction refers to the manner in which a molecular tool comprising at least one nucleic acid (DNA, RNA, single-stranded or double-stranded, or a mixture thereof) and / or at least one amino acid sequence, optionally containing a suitable chemical or biological agent, is introduced to achieve transfer to at least one compartment of interest within a cell, including but not limited to the cytoplasm, organelles (including the nucleus, mitochondria, vacuoles, chloroplasts), or membrane, resulting in transcription and / or translation and / or association and / or activity of at least one introduced molecule, without achieving stable integration or incorporation into the genome, and thus the corresponding at least one molecule introduced into the cellular genome is not inherited. The term “transgenic-free” means the absence or absence of a transgene in the genome of the host cell, tissue, or organism of interest.

[0103] In some embodiments, the polynucleotide and / or nucleic acid constructs of the present invention may be “expression cassettes” or may be contained within expression cassettes. As used herein, “expression cassette” means a recombinant nucleic acid molecule that contains, for example, the nucleic acid constructs of the present invention (e.g., polynucleotides encoding fusion proteins of the present invention, polynucleotides encoding nucleases, polynucleotides encoding reverse transcriptases, polynucleotides encoding reverse transcriptase fusion proteins, polynucleotides encoding peptide tags, polynucleotides encoding affinity polypeptides, polynucleotides encoding glycosylation enzymes, and / or polynucleotides containing guide nucleic acids), wherein said nucleic acid constructs are operatively associated with at least a control sequence (e.g., a promoter). Thus, some embodiments of the present invention provide expression cassettes designed to express, for example, nucleic acid constructs of the present invention. When the expression cassette contains more than one polynucleotide, the polynucleotides may be operatively linked to a single promoter driving the expression of all polynucleotides, or the polynucleotides may be operatively linked to one or more separate promoters (e.g., three polynucleotides may be driven by one, two, or three promoters in any combination). Therefore, for example, the polynucleotide encoding the fusion protein of the present invention contained in the expression cassette and the polynucleotide containing the guide nucleic acid can each be operatively associated with a single promoter, or one or more of the polynucleotides can be operatively associated with any combination of individual promoters (e.g., two or three promoters), which may be the same as or different from each other.

[0104] In some embodiments, expression cassettes containing the polynucleotide / nucleic acid constructs of the present invention can be optimized for expression in organisms (e.g., animals, plants, bacteria, etc.).

[0105] Expression cassettes containing nucleic acid constructs of the present invention can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components (e.g., a promoter from a host organism is operatively linked to a polynucleotide of interest to be expressed in the host organism, wherein the polynucleotide of interest originates from an organism different from the host, or is not typically associated with the promoter). Expression cassettes can also be naturally occurring, but already obtained in recombinant forms suitable for heterologous expression.

[0106] The expression cassette may optionally include a transcription and / or translation termination region (i.e., a termination region) and / or an enhancer region that functions in a selected host cell. Various transcription terminators and enhancers are known in the art and can be used in the expression cassette. The transcription terminator is responsible for terminating transcription and correcting polyadenine acidification of mRNA. The termination region and / or enhancer region may be native to the transcription initiation region, native to the gene encoding a CRISPR-Cas effector protein or the gene encoding the fusion protein of the present invention, native to the host cell, or native to another source (e.g., foreign or heterologous to the promoter, the gene encoding the CRISPR-Cas effector protein, the host cell, or any combination thereof).

[0107] The expression cassette of the present invention may also include a polynucleotide encoding a selectable marker that can be used to select transformed host cells. As used herein, “selectable marker” means a polynucleotide sequence that, when expressed, confers a unique phenotype on host cells expressing the marker, thereby allowing such transformed cells to be distinguished from cells without the marker. Such polynucleotide sequences may encode selectable or screenable markers, depending on whether the marker confers a trait that can be selected by chemical means (e.g., by using a selector (e.g., antibiotics, etc.)) or whether the marker is a trait that can be identified simply by observation or testing (e.g., by screening (e.g., fluorescence)). Numerous examples of suitable selectable markers are known in the art and can be used in the expression cassette described herein.

[0108] The expression cassettes, nucleic acid molecules / constructs, and polynucleotide sequences described herein can be used in conjunction with vectors. The term "vector" refers to a composition for transferring, delivering, or introducing nucleic acids (or multiple nucleic acids) into a cell. A vector may contain a nucleic acid construct that contains one or more nucleotide sequences to be transferred, delivered, or introduced into a cell. Vectors for transforming host organisms are well known in the art. Non-limiting examples of vectors in general categories include viral vectors (e.g., adeno-associated virus (AAV) vectors), plasmid vectors, phage vectors, phage particle vectors, granular vectors, fosmid vectors, phages, artificial chromosomes, microcircular or double-stranded or single-stranded linear or circular Agrobacterium binary vectors, which may or may not be self-transferable or mobile. In some embodiments, viral vectors may include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or herpes simplex virus vectors. Vectors as defined herein can transform prokaryotic or eukaryotic hosts by integration into the cellular genome or by their presence outside the chromosome (e.g., autonomously replicating plasmids with origins of replication). Additionally, shuttle vectors are included, meaning DNA vectors capable of naturally or intentionally replicating in two different host organisms, which may be selected from actinomycetes and related species, bacteria, and eukaryotes (e.g., higher plants, mammals, yeast, or fungal cells). In some embodiments, the nucleic acid in the vector is controlled by a suitable promoter or other regulatory element and is operatively linked to a suitable promoter or other regulatory element for transcription in a host cell. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter and / or other regulatory elements, while in the case of cDNA, this may be controlled by a suitable promoter and / or other regulatory elements for expression in a host cell. Therefore, the nucleic acid constructs of the present invention and / or expression cassettes containing them may be contained in vectors described herein and known in the art.

[0109] As used herein, “contact,” “contacting,” “contacted,” and their grammatical variations refer to bringing together components of a desired reaction under conditions suitable for carrying out the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking, and / or cutting). Thus, for example, a target nucleic acid can be contacted with a nucleic acid construct of the present invention, which comprises, for example, a polynucleotide encoding a fusion protein of the present invention and optionally a guide nucleic acid, under conditions of expression of the fusion protein and formation of a complex with the guide nucleic acid, the complex hybridizing with the target nucleic acid, and optionally, the fusion protein of the present invention can recruit one or more additional components (e.g., reverse transcriptases, glycosylation inhibitors, and / or deaminases) to the fusion protein (and therefore to the target nucleic acid), or fuse one or more additional components with the fusion protein to modify the target nucleic acid. In some embodiments, the fusion protein of the present invention is optionally located at the target nucleic acid via non-covalent interactions. Methods for recruiting one or more additional components (e.g., reverse transcriptases and / or deaminases) utilizing other protein-protein interactions, RNA-protein interactions, and / or chemical interactions can be used. In some embodiments, the target nucleic acid may be contacted with a ribonucleoprotein comprising the fusion protein of the present invention and optionally a guide nucleic acid, and the ribonucleoprotein hybridizes with the target nucleic acid. Optionally, the fusion protein of the present invention may recruit one or more additional components (e.g., reverse transcriptase, glycosylation inhibitors, and / or deaminases) to the fusion protein (and thus to the target nucleic acid), or fuse one or more additional components with the fusion protein to modify the target nucleic acid.

[0110] In some embodiments, under conditions of expressing a fusion protein, the target nucleic acid can be contacted with a nucleic acid construct of the present invention, the nucleic acid construct encoding the fusion protein of the present invention and optionally a guide nucleic acid, or the target nucleic acid can be contacted with the fusion protein of the present invention and optionally a guide nucleic acid. The fusion protein of the present invention can form a complex with the guide nucleic acid, and the complex can hybridize with the target nucleic acid, and / or the fusion protein of the present invention can recruit the target nucleic acid, which can cause modification of the target nucleic acid.

[0111] As used herein, “modifying” or “modification” of a target nucleic acid includes editing (e.g., mutation), covalent modification, exchange / substitution of nucleic acid / nucleotide bases, deletion, cleavage, and / or nicking of the target nucleic acid to provide a modified nucleic acid and / or alteration of transcriptional control of the target nucleic acid to provide a modified nucleic acid. In some embodiments, modification may include insertions and / or deletions of any size and / or any type of single-base alteration (SNP). In some embodiments, modification includes an SNP. In some embodiments, modification includes the exchange and / or substitution of one or more (e.g., 1, 2, 3, 4, 5, or more) nucleotides. In some embodiments, the length of the insertion or deletion can be from about 1 base to about 30,000 bases or more (e.g., lengths of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38). 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 8 6, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1 500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500 00, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,500, 26,000, 26,500, 27,000, 27,500, 28,000, 28,500, 29,000, 29,500, 30,000 or more bases or any value or range thereof. Therefore, in some embodiments, the length of the insertion or deletion may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250260, 270, 280, 290, 300 to approximately 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 bases, or any range or value thereof; lengths of approximately 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 1 70, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 bases to approximately 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 4 70, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 78 0, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 bases or longer, or any value or range thereof; lengths of approximately 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710,720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 180 0, 1900, 2000 bases to about 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10,000 bases or longer, or any value or range thereof; or a length of about 400, 410, 420, 430, 440, 450, 460, 470, 480 bases. 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690 or 700 bases to approximately 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 86 0, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 bases or longer, or any value or range thereof. In some embodiments, the length of the insertion or deletion may be about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, or 10,000 bases to about 10,500, 11,000, 11,500, or 12,000. 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500, 25,000, 25,50026,000, 26,500, 27,000, 27,500, 28,000, 28,500, 29,000, 29,500, or 30,000 bases or longer, or any value or range thereof.

[0112] As used herein, “recruit,” “recruiting,” or “recruitment” refers to the attraction of one or more polypeptides or polynucleotides to another polypeptide or polynucleotide (e.g., a specific location in the genome) using protein-protein interactions, nucleic acid-protein interactions (e.g., RNA-protein interactions), and / or chemical interactions. Protein-protein interactions may include, but are not limited to, peptide tags (epitopes, multimerized epitopes) and corresponding affinity peptides, RNA recruitment motifs and corresponding affinity peptides, and / or chemical interactions. Examples of chemical interactions that can be used with peptides and polynucleotides for recruitment purposes include, but are not limited to, rapamycin-induced FRB-FKBP dimerization; biotin-streptavidin interactions; SNAP tagging (Hussain et al., Curr Pharm Des. 19(30):5437-42 (2013)); Halo tagging (Los et al., ACS Chemical Biology. 3(6):373-82 (2008)); CLIP tagging (Gautier et al., Chemistry & Biology. 15:128-136 (2008)); compound-induced DmrA-DmrC heterodimerization (Tak et al., Nat Methods. 14(12):1163-1166 (2017)); and / or bifunctional ligand approaches (e.g., chemically induced dimerization (Voß et al., Current Opinions on Chemical Biology 28:194-201 (2015)), (e.g., dihydrofolate reductase (DHFR) (Kopyteck et al., CellCehm Biol 7(5):313-321 (2000)). In some embodiments, the recruitment methods and / or systems of the present invention use protein-protein interactions and / or nucleic acid-protein interactions (e.g., RNA-protein interactions) to attract peptides or polynucleotides to another peptide or polynucleotide (e.g., to a specific location in the genome).

[0113] In the context of the polynucleotide or editing system of interest, “introducing,” “introduce,” “introduced” (and its grammatical variations) means presenting the nucleotide sequence of interest (e.g., polynucleotide, nucleic acid construct, and / or guide nucleic acid) and / or editing system (e.g., polynucleotide, polypeptide, and / or ribonucleic acid) to a host organism or the organism’s cells (e.g., host cells; for example, plant cells) in a manner that allows the nucleotide sequence and / or editing system to enter the cell. Thus, for example, a nucleic acid construct of the present invention encoding the fusion protein and / or guide nucleic acid of the present invention can be introduced into the cells of an organism, thereby transforming the cells with the polypeptide, CRISPR-Cas effector protein, guide nucleic acid, and reverse transcriptase. In some embodiments, the fusion protein and / or guide nucleic acid of the present invention can be introduced into the cells of an organism, optionally wherein the polypeptide and guide nucleic acid may be contained in a complex (e.g., a ribonucleic acid). In some embodiments, the organism is a eukaryote (e.g., a mammal, such as a human).

[0114] As used herein, the term "conversion" refers to the introduction of nucleic acids, peptides, and / or ribonucleoproteins (e.g., heterologous nucleic acids, peptides, and / or ribonucleoproteins) into a cell. Cellular conversion can be stable or transient. Thus, in some embodiments, host cells or host organisms can be stably converted using the polynucleotide / nucleic acid molecules of the present invention. In some embodiments, host cells or host organisms can be transiently converted using the nucleic acid constructs, peptides, and / or ribonucleoproteins of the present invention.

[0115] In the context of polynucleotides, peptides, and / or ribonucleoproteins, "transient conversion" means that polynucleotides, peptides, and / or ribonucleoproteins are introduced into the cell without integrating into the cell's genome.

[0116] In the context of polynucleotides being introduced into cells, "stable introduction" or "being stably introduced" means that the introduced polynucleotides are stably incorporated into the cell's genome, and thus the cell is stably transformed by the polynucleotides.

[0117] As used herein, "stable transformation" or "being stably transformed" means that a nucleic acid molecule is introduced into a cell and integrated into the cell's genome. Therefore, the integrated nucleic acid molecule can be inherited by its offspring, and more specifically, by offspring across multiple generations. As used herein, "genome" includes both nuclear and plasmid genomes, and therefore includes the integration of nucleic acids into, for example, chloroplast or mitochondrial genomes. As used herein, stable transformation can also refer to transgenes maintained outside of chromosomes, for example, as microchromosomes or plasmids.

[0118] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blotting, which can detect the presence of peptides or polypeptides encoded by one or more transgenes introduced into an organism. Stable transformation of cells can be detected, for example, by Southern blotting of genomic DNA and nucleic acid sequences of cells, wherein the nucleic acid sequences specifically hybridize with the nucleotide sequences of transgenes introduced into an organism (e.g., mammals, plants, etc.). Stable transformation of cells can also be detected, for example, by Southern blotting of RNA and nucleic acid sequences of cells, wherein the nucleic acid sequences specifically hybridize with the nucleotide sequences of transgenes introduced into a host organism. Stable transformation of cells can also be detected, for example, by polymerase chain reaction (PCR) or other amplification reactions well known in the art, which employ specific primer sequences that hybridize with the target sequence of the transgene, resulting in the amplification of the transgene sequence, thereby allowing detection of the transgene sequence according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0119] Therefore, in some embodiments, the nucleotide sequences, polynucleotides, nucleic acid constructs, and / or expression cassettes of the present invention can be transiently expressed and / or can be stably incorporated into the genome of a host organism. Thus, in some embodiments, the nucleic acid constructs of the present invention can be transiently introduced into cells together with guide nucleic acids, and therefore, no DNA is maintained in the cells.

[0120] The nucleic acid constructs, peptides, and / or ribonucleoproteins of the present invention can be introduced into cells by any method known to those skilled in the art. In some embodiments, the transformation methods include, but are not limited to, transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide and / 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 electro, chemical, physical (mechanical), and / or biological mechanisms, including any combination thereof, that introduce nucleic acids into cells (e.g., plant or animal cells). In some embodiments of the present invention, cell transformation comprises nuclear transformation. In some embodiments, cell transformation comprises plastid transformation (e.g., chloroplast transformation). In some embodiments, the recombinant nucleic acid constructs of the present invention can be introduced into cells by conventional breeding techniques.

[0121] Procedures for transforming eukaryotes and prokaryotes are well known and routine in the art and are described throughout the literature (see, for example, Jiang et al., 2013. Nature Biotechnol. 31:233-239; Ran et al., Nature Protocols 8:2281-2308 (2013)). General guidelines for various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants”, 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)).

[0122] Therefore, nucleotide sequences, polypeptides, and / or ribonucleoproteins can be introduced into a host organism or its cells in a variety of ways well known in the art. The methods of the present invention do not rely on specific methods for introducing one or more nucleotide sequences, polypeptides, and / or ribonucleoproteins into an organism, but only on their entry into the interior of at least one cell of the organism. In cases where more than one nucleotide sequence, polypeptide, and / or ribonucleoprotein is to be introduced, they can be assembled as part of a single nucleic acid construct, or assembled into separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Thus, nucleotide sequences, polypeptides, and / or ribonucleoproteins can be introduced into the cell of interest in a single transformation event and / or in separate transformation events, or alternatively, in relevant cases, the nucleotide sequence can be incorporated into the plant, for example, as part of a breeding program. In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell or a mammalian cell such as a human cell).

[0123] In some embodiments, the nucleic acid construct of the present invention (e.g., a polynucleotide and / or guide nucleic acid encoding the fusion protein of the present invention and / or an expression cassette and / or vector containing thereof) can be operatively linked to at least one regulatory sequence, optionally wherein the at least one regulatory sequence can be codon-optimized for expression in plants. In some embodiments, the at least one regulatory sequence can be, for example, a promoter, operon, terminator, or enhancer. In some embodiments, the at least one regulatory sequence can be a promoter. In some embodiments, the regulatory sequence can be an intron. In some embodiments, the at least one regulatory sequence can be, for example, a promoter operatively associated with an intron or a promoter region containing an intron. In some embodiments, the at least one regulatory sequence can be, for example, a ubiquitin promoter and its associated intron (e.g., Medicago truncatula and / or maize and its associated intron). In some embodiments, the at least one regulatory sequence can be a terminator nucleotide sequence and / or an enhancer nucleotide sequence.

[0124] In some embodiments, the nucleic acid construct of the present invention can be operatively associated with a promoter region comprising introns, wherein the promoter region may optionally be a ubiquitin promoter and introns (e.g., alfalfa (Medicago) or maize ubiquitin promoter and introns, such as SEQ ID NO: 37 or SEQ ID NO: 38). In some embodiments, the nucleic acid construct of the present invention operatively associated with a promoter region comprising introns may be codon-optimized for expression in plants.

[0125] In some embodiments, the nucleic acid constructs of the present invention may encode one or more (e.g., 1, 2, 3, 4 or more) polypeptides of interest. The one or more polypeptides of interest may be codon-optimized for expression in eukaryotes (e.g., humans or plants). In some embodiments, the fusion protein of the present invention may comprise one or more (e.g., 1, 2, 3, 4 or more) polypeptides of interest.

[0126] The polypeptides of interest that can be used in this invention may include, but are not limited to, polypeptide or protein domains having the following: deaminase activity, nicking enzyme activity, recombinase activity, transposase activity, methyltransferase activity, glycosylation enzyme (DNA glycosylation enzyme) activity, glycosylation enzyme inhibitor activity (e.g., uracil-DNA glycosylation enzyme inhibitor (UGI)), reverse transcriptase, peptide tag (e.g., GCN4 peptide tag), demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity, double-stranded RNA cleavage activity, restriction endonuclease activity (e.g., Fok1), nucleic acid binding activity, methyltransferase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, polymerase activity, ligase activity, helicase activity, nuclear localization sequence or activity, affinity polypeptide, peptide tag and / or photolyase activity. In some embodiments, the peptide of interest is a Fok1 nuclease or a uracil-DNA glycosylation inhibitor. When encoded in nucleic acids (polynucleotides, expression cassettes, and / or vectors), the encoded peptide or protein domain can be codon-optimized for expression in an organism.

[0127] In some embodiments, the editing system of the present invention comprises the fusion protein of the present invention, said fusion protein comprising a CRISPR-Cas effector protein. As used herein, a “CRISPR-Cas effector protein” is a protein or polypeptide that cleaves, breaks, or nicks nucleic acids; binds nucleic acids (e.g., target nucleic acids and / or guide nucleic acids); and / or identifies, recognizes, or binds to guide nucleic acids as defined herein. In some embodiments, a CRISPR-Cas effector protein may be an enzyme (e.g., a nuclease, endonuclease, nicking enzyme, etc.) and / or may act as an enzyme. In some embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease. In some embodiments, a CRISPR-Cas effector protein comprises nuclease activity and / or nicking enzyme activity, comprises a nuclease domain whose nuclease activity and / or nicking enzyme activity has been reduced or eliminated, comprises single-stranded DNA cleavage activity (ssDNAse activity) or has ssDNAse activity that has been reduced or eliminated, and / or comprises self-processing RNAse activity or has self-processing RNAse activity that has been reduced or eliminated. CRISPR-Cas effector proteins may bind to target nucleic acids. CRISPR-Cas effector proteins can be type I, II, III, IV, V, or VI CRISPR-Cas effector proteins. In some embodiments, the CRISPR-Cas effector protein can originate from a type I, II, III, IV, V, or VI CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein can originate from a type II or V CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein can be a type II CRISPR-Cas effector protein, such as the Cas9 effector protein. In some embodiments, the CRISPR-Cas effector protein can be a type V CRISPR-Cas effector protein, such as the Cas12 effector protein. In some embodiments, the CRISPR-Cas effector protein can be Cas12a, and optionally may have the amino acid sequence of any of SEQ ID NO: 39-65 and / or the nucleotide sequence of any of SEQ ID NO: 66-69. In some embodiments, the CRISPR-Cas effector protein may be an active Cas12a and optionally may have the amino acid sequence of SEQ ID NO: 46, 55, or 56. In some embodiments, the CRISPR-Cas effector protein may be an inactive (i.e., dead) Cas12a and optionally may have the amino acid sequence of SEQ ID NO: 59, 60, or 61.In some embodiments, the CRISPR-Cas effector protein may be Cas12b, and optionally may have the amino acid sequence of SEQ ID NO: 70. In some embodiments, the CRISPR-Cas effector protein may be Cas12f, and optionally may have the amino acid sequence of SEQ ID NO: 71. In some embodiments, the CRISPR-Cas effector protein may be Cas12i, and optionally may have the amino acid sequence of SEQ ID NO: 72.

[0128] Exemplary CRISPR-Cas effector proteins include, but are not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3'', Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, and Cmr. 3. Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5 nucleases, optionally wherein the CRISPR-Cas effector protein can be Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c effector proteins.

[0129] In some embodiments, the CRISPR-Cas effector protein that can be used in this invention may contain mutations in its nuclease active site and / or nuclease domain (e.g., RuvC, HNH, such as the RuvC site of the Cas12a nuclease domain; or the RuvC site and / or HNH site of the Cas9 nuclease domain). CRISPR-Cas effector proteins with mutations in their nuclease active site and / or nuclease domain that result in the protein lacking nuclease activity are generally referred to as “inactive” or “dead,” such as dCas9. In some embodiments, CRISPR-Cas effector proteins with mutations in their nuclease active site and / or nuclease domain may have impaired or reduced activity (e.g., nickase activity) compared to the same CRISPR-Cas effector protein without mutations.

[0130] The CRISPR Cas9 effector protein or Cas9 used in this invention can be any known or later identified Cas9 nuclease. In some embodiments, Cas9 can be a protein derived from: for example, *Streptococcus* species (e.g., *Streptococcus pyogenes*, *Streptococcus thermophilus*), *Lactobacillus* species, *Bifidobacterium* species, *Kandleria* species, *Leuconostoc* species, *Oenococcus* species, *Pediococcus* species, *Weissella* species, and / or *Olsenella* species. In some embodiments, the CRISPR-Cas effector protein may be Cas9, and optionally may have the nucleotide sequence of any of SEQ ID NO: 73-87 and / or the amino acid sequence of any of SEQ ID NO: 88-97.

[0131] In some embodiments, the CRISPR-Cas effector protein may be Cas9 derived from Streptococcus pyogenes and / or may recognize the PAM sequence motifs NGG, NAG, NGA (Mali et al., Science 2013; 339(6121): 823-826). In some embodiments, the CRISPR-Cas effector protein may be Cas9 derived from Streptococcus thermophiles and / or may recognize the PAM sequence motifs NGNG and / or NNAGAAW (W = A or T) (see, for example, Horvath et al., Science, 2010; 327(5962): 167-170, and Deveau et al., Journal of Bacteriology 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from *Streptococcus mutans* and / or capable of recognizing the PAM sequence motif NGG and / or NAAR (R = A or G) (see, for example, Deveau et al., *Journal of Biology* 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from *Streptococcus aureus* and / or capable of recognizing the PAM sequence motif NNGRR (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from *Streptococcus aureus* and / or capable of recognizing the PAM sequence motif NGRRT (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be a Cas9 derived from *Streptococcus aureus* and / or capable of recognizing the PAM sequence motif NGRRV (R = A or G). In some embodiments, the CRISPR-Cas effector protein may be Cas9 derived from Neisseria meningitidis, and / or may recognize the PAM sequence motif NGATT or NGCTT (R = A or G, V = A, G, or C) (see, for example, Hou et al., PNAS 2013, 1-6). In the foregoing embodiments in this paragraph, N in the PAM sequence motif may be any nucleotide residue, such as any one of A, G, C, or T.In some embodiments, the CRISPR-Cas effector protein may be Cas13a derived from Leptotrichia shahii and / or a protospacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif that can recognize a single 3' A, U, or C, which may be located within the target nucleic acid.

[0132] The type V CRISPR-Cas effector protein that can be used in embodiments of the present invention can be any type V CRISPR-Cas nuclease. Exemplary type V CRISPR-Cas effector proteins include, but are not limited to, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c1, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, and / or Cas14c nucleases. In some embodiments, the type V CRISPR-Cas effector protein can be Cas12a. In some embodiments, the type V CRISPR-Cas effector protein can be a nicking enzyme, optionally a Cas12a nicking enzyme.

[0133] In some embodiments, the CRISPR-Cas effector protein can be a type V clustered regularly spaced short palindromic repeat (CRISPR)-Cas nuclease. Cas12a differs from the more well-known type II CRISPR Cas9 nuclease in several respects. For example, Cas9 recognizes a G-rich protospacer adjacent motif (PAM) (3'-NGG) at the 3' of its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, target nucleic acid, target DNA), while Cas12a recognizes a T-rich PAM (5'-TTN, 5'-TTTN) at the 5' of the target nucleic acid. In fact, the orientation of Cas9 and Cas12a binding to their guide RNA is almost opposite with respect to their N and C ends. Furthermore, the Cas12a enzyme uses a single guide RNA (gRNA, CRISPR array, crRNA) instead of the dual guide RNAs (sgRNA (e.g., crRNA and tracrRNA)) found in the native Cas9 system, and Cas12a processes its own gRNA. In addition, Cas12a nuclease activity produces staggered DNA double-strand breaks, rather than blunt ends produced by Cas9 nuclease activity, and Cas12a relies on a single RuvC domain to cut both DNA strands, while Cas9 uses both HNH and RuvC domains for cutting.

[0134] The CRISPR Cas12a effector protein that can be used in this invention can be any known or later identified Cas12a (formerly known as Cpf1) (see, for example, U.S. Patent No. 9,790,490, the disclosure of which regarding the sequence of Cpf1 (Cas12a) is incorporated herein by reference). The term "Cas12a" refers to an RNA-guided protein that may have nuclease activity, the protein comprising a guide nucleic acid binding domain and an active, inactive, or partially active DNA cleaving domain, thereby the RNA-guided nuclease activity of Cas12a can be active, inactive, or partially active, respectively. In some embodiments, the Cas12a that can be used in this invention may contain a mutation in the nuclease active site (e.g., the RuvC site of the Cas12a domain). Cas12a that has a mutation in its nuclease domain and / or nuclease active site and therefore no longer contains nuclease activity is generally referred to as dead Cas12a (e.g., dCas12a). In some embodiments, Cas12a that has a mutation in its nuclease domain and / or nuclease active site may have impaired activity, for example, it may have reduced cleavage enzyme activity. In some embodiments, Cas12a may have an amino acid sequence of either SEQ ID NO: 39-65 or 299-301.

[0135] In some embodiments, the CRISPR-Cas effector protein is an engineered protein having an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with one or more of SEQ ID NO: 98-189 and / or 280-287. In some embodiments, the CRISPR-Cas effector protein is an engineered protein (e.g., an engineered CRISPR-Cas effector protein) comprising (i) a CRISPR-Cas effector peptide (e.g., a Cas12a peptide and / or domain) and (ii) a peptide heterologous to the CRISPR-Cas effector peptide. The CRISPR-Cas effector peptide may be a portion of a CRISPR-Cas effector protein (e.g., wild-type Cas12a or inactivated Cas12a). For example, an engineered CRISPR-Cas effector protein may comprise (i) a portion of any of SEQ ID NO: 39-65 or 299-301 and (ii) a polypeptide heterologous to a portion of any of SEQ ID NO: 39-65 or 299-301. In some embodiments, the engineered CRISPR-Cas effector protein comprises at least one non-natural mutation compared to a CRISPR-Cas effector protein (e.g., wild-type Cas12a or inactivated Cas12a). In some embodiments, the engineered CRISPR-Cas effector protein has a similar structure and / or function compared to a wild-type CRISPR-Cas effector protein, and / or comprises all or a portion of a wild-type CRISPR-Cas effector protein or an inactivated CRISPR-Cas effector protein. In some embodiments, the engineered CRISPR-Cas effector protein does not exist in nature (e.g., comprises a non-natural mutation) and may be modified (e.g., mutated) compared to a wild-type CRISPR-Cas effector protein and / or an inactivated CRISPR-Cas effector protein. In some embodiments, the engineered CRISPR-Cas effector protein comprises the Cas12a polypeptide and lacks a portion of Cas12a. In some embodiments, the engineered CRISPR-Cas effector protein comprises the Cas12a polypeptide and a domain not present in Cas12a. In some embodiments, the engineered CRISPR-Cas effector protein comprises all or some of the following: a wedge domain, a Rec1 domain, a Rec2 domain, a PAM-interacting domain, a RuvC domain, a bridging helix, and / or a Nuc domain, each of which may be derived from Cas12a and / or a protein having a sequence of any of SEQ ID NO: 39-65 or 299-301.In some embodiments, the engineered CRISPR-Cas effector protein comprises all or part of the Cas12a domain having a structure as described in Yamano, Takashi et al., Molecular Cell 67: 633-645 (2017).

[0136] In some embodiments, the engineered CRISPR-Cas effector protein comprises all or a portion of an α-helix recognition (REC) leaf, optionally wherein the domains of all or a portion of the REC leaf in the engineered protein may have a different sequence and / or structure than those in Cas12a. The REC leaf may contain a Rec1 domain and a Rec2 domain. The Rec1 domain may contain 13 α-helices, and / or the Rec2 domain may contain 10 α-helices and two β chains that may form small antiparallel sheets. In some embodiments, the engineered CRISPR-Cas effector protein comprises an HNH domain (e.g., the HNH domain of Cas9, optionally having the sequence of any one of SEQ ID NO: 88-97) located between a first polypeptide containing all or a portion of the Rec1 domain and a second polypeptide containing all or a portion of the Rec2 domain, wherein the first and second polypeptides may each be independently derived from Cas12a and / or proteins having the sequence of any one of SEQ ID NO: 39-65 or 299-301. In some embodiments, the engineered CRISPR-Cas effector protein comprises all or a portion of the RuvC domain. In some embodiments, the engineered CRISPR-Cas effector protein comprises an HNH domain (e.g., the HNH domain of Cas9, optionally having the sequence of any of SEQ ID NO: 88-97) located between a first polypeptide comprising all or a portion of the Rec1 domain and a second polypeptide comprising all or a portion of the Rec2 domain, wherein the first and second polypeptides may each be independently derived from Cas12a and / or proteins having the sequence of any of SEQ ID NO: 39-65 or 299-301, and the engineered protein may comprise all or a portion of the RuvC domain. In some embodiments, the engineered CRISPR-Cas effector protein is as described in U.S. Patent Application Publication No. 2002 / 0112473 and / or PCT / US2023 / 063398. As those skilled in the art will understand, some domains (e.g., the wedge domain and RuvC domain of Cas12a) are not sequentially continuous and can be divided into two or more (e.g., two, three, four or more) discontinuous sequences. In some embodiments, engineered CRISPR-Cas effector proteins contain the Cas12a domain.

[0137] In some embodiments, the engineered CRISPR-Cas effector protein optionally includes all or a portion of one or more of the following in the direction from the N-terminus to the C-terminus: a first portion of a wedge-shaped domain (WED-1), a Rec1 domain, a Rec2 domain, a second portion of a wedge-shaped domain (WED-2), a PAM interaction domain (PI), a third portion of a wedge-shaped domain (WED-3), a first portion of a RuvC domain (RuvC-1), a bridging helix, a second portion of a RuvC domain (RuvC-2), a Nuc domain, and / or a third portion of a RuvC domain (RuvC-3). For example, based on SEQ ID NO: 69, an engineered CRISPR-Cas effector protein may be encoded by all or part of one or more of the following: nucleotides 1-69 constituting WED-1, nucleotides 70-1,560 constituting REC leaf, nucleotides 1,561-1,755 constituting WED-2, nucleotides 1,756-2,031 constituting PI, nucleotides 2,032-2,421 constituting WED-3, nucleotides 2,422-2,613 constituting RuvC-1, nucleotides 2,614-2,667 constituting bridged helix, nucleotides 2,668-2,988 constituting RuvC-2, nucleotides 2,989-3,534 constituting Nuc domain, and / or nucleotides 3,535-3,681 constituting RuvC-3. As another example, based on SEQ ID NO: 56, an engineered CRISPR-Cas effector protein may optionally include all or part of one or more of the following in the N-terminal to C-terminal direction: amino acid residues 1-23 constituting WED-1, amino acid residues 24-520 constituting REC leaf, amino acid residues 521-585 constituting WED-2, amino acid residues 586-677 constituting PI, amino acid residues 678-807 WED-3, amino acid residues 808-871 constituting RuvC-1, amino acid residues 872-889 constituting bridged helix, amino acid residues 890-996 constituting RuvC-2, amino acid residues 997-1,178 constituting Nuc domain, and / or amino acid residues 1,179-1,227 constituting RuvC-3.

[0138] In some embodiments, the engineered CRISPR-Cas effector protein comprises all or a portion of an active RuvC domain. In some embodiments, the engineered CRISPR-Cas effector protein comprises all or a portion of an inactivated RuvC domain, optionally having all or a portion of an inactivated RuvC domain with a D10A mutation. In some embodiments, the engineered CRISPR-Cas effector protein comprises all or a portion of an inactivated RuvC domain and, when the engineered protein is best aligned with SEQ ID NO: 59, has an alanine residue at position 831 corresponding to amino acid residue 831 of SEQ ID NO: 59, optionally wherein the mutation is referred to as the D10A and / or D832A mutation. In some embodiments, the engineered CRISPR-Cas effector protein comprises a polypeptide including all or part of an inactivated RuvC domain and has the following mutations: D832A and / or E925A with reference to position numbers of SEQ ID NO: 44, 45, 55, 56 and / or 59 (LbCas12a), D908A and / or E993A with reference to position number of SEQ ID NO: 39 (AsCas12a), D917A and / or E1006A with reference to position number of SEQ ID NO: 43 and / or 58 (FnCas12a), or the corresponding mutations.

[0139] In some embodiments, the CRISPR-Cas effector protein (e.g., an engineered CRISPR-Cas effector protein) is a target chain cleavage enzyme and / or a non-target chain cleavage enzyme. In some embodiments, the CRISPR-Cas effector protein (e.g., an engineered CRISPR-Cas effector protein) is a non-target chain cleavage enzyme.

[0140] In some embodiments, CRISPR-Cas effector proteins can be optimized for expression in organisms such as animals (e.g., mammals, such as humans), plants, fungi, archaea, or bacteria. In some embodiments, CRISPR-Cas effector proteins (e.g., Cas12a peptide / domain or Cas9 peptide / domain) can be optimized for expression in plants.

[0141] The polypeptides (e.g., fusion proteins) of the present invention can be used in combination with guide nucleic acids (e.g., guide RNA (gRNA), CRISPR arrays, CRISPR RNA, crRNA, or extended guide nucleic acids) designed to function in conjunction with CRISPR-Cas effector proteins (e.g., CRISPR-Cas effector proteins present in the fusion protein) to modify target nucleic acids. Guide nucleic acids usable in the present invention may comprise at least one spacer sequence and at least one repeat sequence. The guide nucleic acid may be capable of forming a complex with a CRISPR-Cas effector protein (e.g., with a nuclease domain of a protein) and / or the fusion protein of the present invention, and the spacer sequence may be capable of hybridizing with the target nucleic acid to guide the complex to the target nucleic acid, wherein the target nucleic acid may be modified (e.g., cleaved or edited) and / or regulated (e.g., regulated transcription) by the fusion protein of the present invention optionally present in and / or recruited to the complex.

[0142] In some embodiments, a CRISPR-Cas effector protein containing a Cas9 domain (or a nucleic acid construct encoding it) may be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, and may be in or form a complex.

[0143] Similarly, CRISPR-Cas effector proteins can contain a Cas12a domain (or other selected CRISPR-Cas nucleases, such as C2c1, C2c3, Cas12b, Cas12c, Cas12d, Cas12f, Cas12i, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3'', Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, ... Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5, which can be used in combination with Cas12a guide nucleic acid (or another alternative guide nucleic acid of a CRISPR-Cas nuclease) to modify target nucleic acid, thereby editing the target nucleic acid.

[0144] As used herein, “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA,” “crRNA,” or “crDNA” refers to a nucleic acid containing at least one spacer sequence complementary to (and hybridizing with) a target nucleic acid (e.g., target DNA and / or the original spacer) and at least one repeat sequence (e.g., a repeat sequence, or a fragment or portion thereof, of a type V Cas12a CRISPR-Cas system; a repeat sequence, or a fragment thereof, of a type II Cas9 CRISPR-Cas system; a type V C2c1 CRISPR-Cas system). Repeated sequences or fragments thereof in the Cas system; for example, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12f, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3'', Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2 The repetitive sequences, or fragments thereof, of the CRISPR-Cas system of Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5, wherein the repetitive sequences may be ligated to the 5' end and / or 3' end of the spacer subsequence. In some embodiments, the guide nucleic acid comprises DNA. In some embodiments, the guide nucleic acid comprises RNA (e.g., guide RNA). The gRNA of the present invention can be designed based on a type I, II, III, IV, V, or VI CRISPR-Cas system.

[0145] In some embodiments, the Cas12a gRNA from 5' to 3' may contain a repeating sequence (full length or a portion thereof (“stalk”); e.g., a pseudoknot-like structure) and a spacer subsequence.

[0146] In some embodiments, the guide nucleic acid may comprise more than one repeat-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeat-spacer sequences) (e.g., repeat-spacer-repeat, such as repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer, etc.). The guide nucleic acid of the present invention is synthetic, artificial, and does not exist in nature. The gRNA can be long and can be used as an aptamer (e.g., in the MS2 recruitment strategy) or other RNA structures suspending spacers.

[0147] As used herein, "repetitive sequence" means, for example, any repetitive sequence of a wild-type CRISPR-Cas locus (e.g., Cas9, Cas12a, C2c1, etc.), or a repetitive sequence of a synthetic crRNA that functions in conjunction with a CRISPR-Cas effector protein encoded by the nucleic acid construct of the present invention. The repetitive sequence that can be used in the present invention can be any known or subsequently identified repetitive sequence of a CRISPR-Cas locus (e.g., type I, II, III, IV, V, or VI), or can be a synthetic repetitive sequence designed to function in type I, II, III, IV, V, or VI CRISPR-Cas systems. The repetitive sequence may contain hairpin structures and / or stem-loop structures. In some embodiments, the repetitive sequence may form a pseudo-knot-like structure (i.e., a "stalk") at its 5' end. Therefore, in some embodiments, the repetitive sequence may be identical or substantially identical to repetitive sequences from wild-type CRISPR-Cas loci I, II, III, IV, V, and / or VI. Repetitive sequences from wild-type CRISPR-Cas loci can be determined using established algorithms, such as CRISPRfinder provided via CRISPRdb (see Grissa et al., Nucleic Acid Research, 35 (Web Server Issues): W52-7). In some embodiments, the repetitive sequence or a portion thereof is ligated at its 3' end to the 5' end of a spacer sequence to form a repetitive-spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0148] In some embodiments, the repetitive sequence comprises, is substantially composed of, or is composed of at least 10 nucleotides, depending on the particular repetitive sequence and whether the guide nucleic acid containing the repetitive sequence is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value thereof). In some embodiments, the repeating sequence comprises or consists substantially of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides.

[0149] The repeat sequence linked to the 5' end of the spacer sequence may contain a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of a wild-type repeat sequence). In some embodiments, the length of a portion of the repeat sequence linked to the 5' end of the spacer sequence may be about five to about ten consecutive nucleotides (e.g., about 5, 6, 7, 8, 9, or 10 nucleotides), and it has at least 90% sequence identity with the same region (e.g., the 5' end) of the wild-type CRISPR Cas repeat nucleotide sequence (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher percentage). In some embodiments, a portion of the repeat sequence may include a pseudoknot-like structure (e.g., a "stalk") at its 5' end.

[0150] As used herein, a “spacer sequence” is a nucleotide sequence complementary to a target nucleic acid (e.g., target DNA) (e.g., a protospacer). The spacer sequence may be completely or substantially complementary to the target nucleic acid (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percentages)). Therefore, in some embodiments, the spacer sequence may have one, two, three, four, or five mismatches compared to the target nucleic acid, and these mismatches may be sequential or discontinuous. In some embodiments, the spacer sequence may be 70% complementary to the target nucleic acid. In other embodiments, the spacer nucleotide sequence may be 80% complementary to the target nucleic acid. In other embodiments, the spacer nucleotide sequence may have 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% complementarity with the target nucleic acid (protospacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. The length of the spacer sequence may be from about 15 nucleotides to about 30 target nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value thereof). Thus, in some embodiments, the spacer sequence may have complete or substantially complementary complementarity in regions of the target nucleic acid (e.g., the protospacer) of at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the spacer is about 20 nucleotides long. In some embodiments, the spacer is about 21, 22, or 23 nucleotides long.

[0151] In some embodiments, the 5' region of the spacer sequence of the guide nucleic acid may be completely complementary to the target nucleic acid, while the 3' region of the spacer may be substantially complementary to the target nucleic acid (as in the spacer of a type V CRISPR-Cas system), or the 3' region of the spacer sequence of the guide nucleic acid may be completely complementary to the target nucleic acid, while the 5' region of the spacer may be substantially complementary to the target nucleic acid (as in the spacer of a type II CRISPR-Cas system), and therefore, the overall complementarity of the spacer sequence to the target nucleic acid may be less than 100%. Thus, for example, in the guide nucleic acid of a type V CRISPR-Cas system, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 nucleotides in the 5' region (i.e., the seed region) of a 20-nucleotide spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary to the target nucleic acid (e.g., at least about 70% complementary). In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 nucleotides and any range thereof) of the 5' end of the spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary to the target nucleic acid (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percentage)).

[0152] As another example, in the guide nucleic acid of the type II CRISPR-Cas system, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 nucleotides in the 3' region (i.e., the seed region) of a 20-nucleotide spacer sequence can be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target nucleic acid (e.g., at least about 70% complementary). In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any range thereof) of the 3' end of the spacer sequence may be 100% complementary to the target nucleic acid, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target nucleic acid (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher percentage, or any range or value thereof)). The recruitment guide RNA further includes one or more recruitment motifs as described herein, which may be attached to the 5' or 3' end of the guide, or may be inserted into the recruitment guide nucleic acid (e.g., within a hairpin loop).

[0153] In some embodiments, the seed region of the spacer may be about 8 to about 10 nucleotides long, about 5 to about 6 nucleotides long, or about 6 nucleotides long.

[0154] In some embodiments, the guide nucleic acid further comprises a reverse transcriptase template and may be referred to as an extended guide nucleic acid.

[0155] The guide nucleic acid and / or extended guide nucleic acid may contain one or more recruitment motifs as described herein, which may be attached to the 5' end and / or the 3' end of the guide nucleic acid and / or may be inserted into the guide nucleic acid (e.g., within the hairpin loop of the guide nucleic acid).

[0156] The terms “target nucleic acid,” “target DNA,” “target nucleotide sequence,” “target region,” and “target region in the genome” are used interchangeably herein and refer to a region in the genome of an organism (e.g., a plant) that contains a sequence that is completely complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) to the spacer sequence in the guide nucleic acid as defined herein. The target nucleic acid is targeted by an editing system (or a component thereof) as described herein. Target regions that can be used in CRISPR-Cas systems can be located immediately adjacent to the 3' (e.g., in type V CRISPR-Cas systems) or immediately adjacent to the 5' (e.g., in type II CRISPR-Cas systems) of the PAM sequence in an organism's genome (e.g., plant genome or mammalian genome, e.g., human genome). The target region can be selected from any region immediately adjacent to the PAM sequence, consisting of at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, etc.).

[0157] As used herein, a “protospacer sequence” or “protospacer” means a sequence that is completely or substantially complementary to (and can hybridize with) the spacer sequence of a guide nucleic acid. In some embodiments, a protospacer is all or part of a target nucleic acid as defined herein that is completely or substantially complementary to (and hybridizes with) the spacer sequence of a CRISPR repeat sequence-spacer sequence (e.g., guide nucleic acid, CRISPR array, crRNA).

[0158] In the case of type V CRISPR-Cas (e.g., Cas12a) and type II CRISPR-Cas (Cas9) systems, the flanking element of the protospacer sequence is (e.g., immediately adjacent) the protospacer adjacent motif (PAM). For type V CRISPR-Cas systems, the PAM is located at the 5' end of the non-target strand and at the 3' end of the target strand (see below as an example).

[0159]

[0160] In the case of type II CRISPR-Cas systems (e.g., Cas9), the PAM is located immediately adjacent to the 3' of the target strand. In type I CRISPR-Cas systems, the PAM is located at the 5' of the target strand. There is no known PAM for type III CRISPR-Cas systems. Makarova et al. described the nomenclature for all classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722-736 (2015)). R. Barrangou described the guide structure and PAM (Genome Biology 16:247 (2015)).

[0161] Typical Cas12a PAMs are rich in T. In some embodiments, a typical Cas12a PAM sequence may be 5'-TTN, 5'-TTTN, or 5'-TTTV. In some embodiments, a typical Cas9 (e.g., Streptococcus pyogenes) PAM may be 5'-NGG-3'. In some embodiments, atypical PAMs may be used, but may be less efficient.

[0162] Those skilled in the art can determine the additional PAM sequence using established experimental and computational methods. Thus, for example, experimental methods include targeting a sequence with all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as through transformation of the target plasmid DNA (Esvelt et al. 2013 *Nature Methods* 10:1116-1121; Jiang et al. 2013 *Nature Biotechnology* 31:233-239). In some aspects, computational methods may include performing a BLAST search on natural spacers to identify the original target DNA sequence in a phage or plasmid and comparing these sequences to determine conserved sequences adjacent to the target sequence (Briner and Barrangou. 2014. *Applied and Environmental Microbiology* 80:994-1001; Mojica et al. 2009. *Microbiology* 155:733-740).

[0163] In some embodiments, the present invention provides expression cassettes and / or vectors comprising nucleic acid constructs of the present invention (e.g., one or more components of the editing system of the present invention). In some embodiments, expression cassettes and / or vectors comprising nucleic acid constructs of the present invention and / or one or more guide nucleic acids may be provided. In some embodiments, the nucleic acid constructs of the present invention encode the fusion protein of the present invention and one or more guide nucleic acids, said nucleic acid constructs may be contained on the same expression cassette or vector or on a separate expression cassette or vector containing one or more guide nucleic acids. When a nucleic acid construct encoding a fusion protein or component of the editing system of the present invention is contained on a separate expression cassette or vector containing guide nucleic acids, the target nucleic acid and the expression cassette or vector encoding the fusion protein or component of the editing system of the present invention and the guide nucleic acid may contact each other in any order (e.g., provided together), such as before, simultaneously with, or after the expression cassette containing guide nucleic acids (e.g., contacted with the target nucleic acid).

[0164] Methods for recruiting one or more components of an editing system to each other and / or target nucleic acids are known in the art and may include the use of peptide tags or affinity peptides that interact with peptide tags. In some embodiments, the guide nucleic acid may be linked to an RNA recruitment motif, and the fusion protein of the present invention may be linked to an affinity peptide capable of interacting with an RNA recruitment motif, thereby recruiting the fusion protein of the present invention to the target nucleic acid. Alternatively, chemical interactions may be used to recruit peptides (e.g., the fusion protein of the present invention) to the target nucleic acid.

[0165] As used herein, a “recruiting motif” refers to one half of a binding pair that can be used to recruit a compound bound by the recruitment motif to another compound comprising the other half of the binding pair (i.e., a “corresponding motif”). The recruitment motif and the corresponding motif can be non-covalently bound. In some embodiments, the recruitment motif is an RNA recruitment motif (e.g., an RNA recruitment motif capable of binding to and / or configured to bind to an affinity peptide), an affinity peptide (e.g., an affinity peptide capable of binding to and / or configured to bind to an RNA recruitment motif and / or a peptide tag), or a peptide tag (e.g., a peptide tag capable of binding to and / or configured to bind to an affinity peptide). For example, when the recruitment motif is an RNA recruitment motif, the corresponding motif of the RNA recruitment motif can be an affinity peptide that binds to the RNA recruitment motif. Another example is when the recruitment motif is a peptide tag, the corresponding motif of the peptide tag can be an affinity peptide that binds to the peptide tag. Therefore, a compound containing a recruitment motif (e.g., an affinity polypeptide) can be recruited to another compound (e.g., a guide nucleic acid) containing a corresponding motif (e.g., an RNA recruitment motif).

[0166] Peptide tags (e.g., epitopes) that can be used in this invention may include, but are not limited to, GCN4 peptide tags (e.g., Sun-Tag), c-Myc affinity tags, HA affinity tags, His affinity tags, S affinity tags, methionine-His affinity tags, RGD-His affinity tags, FLAG octapeptides, strep tags or strep tag II, V5 tags, and / or VSV-G epitopes. Any epitope that can be linked to a polypeptide and has a corresponding affinity polypeptide that can be linked to another polypeptide can be used as a peptide tag in this invention. In some embodiments, the peptide tag may comprise one or two or more copies of the peptide tag (e.g., repeating units, polymerized epitopes (e.g., tandem repeat sequences)) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more repeating units). In some embodiments, the affinity peptide that interacts with / binds to the peptide tag may be an antibody. In some embodiments, the antibody may be an scFv antibody. In some embodiments, the affinity peptides that bind to the peptide tag may be synthetic (e.g., evolved for affinity interactions) and include, but are not limited to, affibody, anti-carrier protein, monobody, and / or DARPin (see, for example, Sha et al., ProteinSci. 26(5):910-924 (2017); Gilbreth (Current Opinion StrucBiol. 22(4):413-420 (2013)); U.S. Patent No. 9,982,053), each of which is incorporated herein by reference in its entirety for the purposes of its teachings concerning affibody, anti-carrier protein, monobody, and / or DARPin.

[0167] In some embodiments, the guide nucleic acid may be linked to an RNA recruitment motif, and the polypeptide to be recruited (e.g., the fusion protein of the present invention) may be fused with an affinity polypeptide that binds to the RNA recruitment motif, wherein the guide binds to the target nucleic acid, and the RNA recruitment motif binds to the affinity polypeptide, thereby recruiting the polypeptide to the guide and contacting the target nucleic acid with the polypeptide (e.g., the fusion protein of the present invention). In some embodiments, two or more polypeptides may be recruited to the guide nucleic acid, thereby contacting the target nucleic acid with two or more polypeptides (e.g., one or more fusion proteins of the present invention).

[0168] In some embodiments of the invention, the guide RNA may be linked to one or two or more RNA recruitment motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; e.g., at least 10 to about 25 motifs), wherein optionally the two or more RNA recruitment motifs may be the same RNA recruitment motif or different RNA recruitment motifs. In some embodiments, the RNA recruitment motif and corresponding affinity peptide may include, but are not limited to, a telomerase Ku-binding motif (e.g., Ku-binding hairpin) and a corresponding affinity peptide Ku (e.g., Ku heterodimer), a telomerase Sm7-binding motif and a corresponding affinity peptide Sm7, an MS2 phage operon stem loop and a corresponding affinity peptide MS2 capsid protein (MCP), a PP7 phage operon stem loop and a corresponding affinity peptide PP7 capsid protein (PCP), an SfMu phage Com stem loop and a corresponding affinity peptide Com RNA-binding protein, a PUF binding site (PBS) and an affinity peptide Pumilio / fem-3 mRNA-binding factor (PUF), and / or synthetic RNA aptamers and aptamer ligands as the corresponding affinity peptides. In some embodiments, the RNA recruitment motif and corresponding affinity peptide may be an MS2 phage operon stem loop and an affinity peptide MS2 capsid protein (MCP). In some embodiments, the RNA recruitment motif and corresponding affinity peptide can be a PUF binding site (PBS) and an affinity peptide Pumilio / fem-3 mRNA binding factor (PUF). Exemplary RNA recruitment motifs and corresponding affinity peptides that can be used in this invention may include, but are not limited to, SEQ ID NO:190-200.

[0169] In some embodiments, the components used to recruit peptides and nucleic acids may include components that act through chemical interactions, which may include, but are not limited to, rapamycin-induced FRB-FKBP dimerization; biotin-streptavidin; SNAP tag; Halo tag; CLIP tag; compound-induced DmrA-DmrC heterodimer; bifunctional ligands (e.g., chemically induced dimerization).

[0170] As described herein, a “peptide tag” can be used to recruit one or more peptides. A peptide tag can be any peptide capable of being bound by a corresponding motif (such as an affinity peptide). A peptide tag may also be referred to as an “epitope” and, when provided in multiple copies, as a “multiplying epitope.” Example peptide tags may include, but are not limited to, GCN4 peptide tags (e.g., Sun-Tag), c-Myc affinity tags, HA affinity tags, His affinity tags, S affinity tags, methionine-His affinity tags, RGD-His affinity tags, FLAG octapeptides, strep tags or strep tag II, V5 tags, and / or VSV-G epitopes. In some embodiments, a peptide tag may also include a phosphorylated tyrosine residue in a specific sequence environment recognized by an SH2 domain, a characteristic shared sequence containing a phosphoserine residue recognized by a 14-3-3 protein, a proline-rich peptide motif recognized by an SH3 domain, a PDZ protein-protein interaction domain, or a PDZ signal sequence, and an AGO hook motif from plants. Peptide tags are disclosed in WO2018 / 136783 and U.S. Patent Application Publication No. 2017 / 0219596, the contents of which are incorporated herein by reference. Peptide tags that can be used in this invention may include, but are not limited to, SEQ ID NO: 201 and SEQ ID NO: 202. Affinity peptides that can be used for peptide tags include, but are not limited to, SEQ ID NO: 203.

[0171] Peptide tags may be contained in or present in one copy or two or more copies of a peptide tag (e.g., polymerized peptide tags or polymerized epitopes) (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 9, 20, 21, 22, 23, 24, or 25 or more peptide tags). When polymerized, peptide tags may be directly fused to each other, or they may be linked to each other via one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, optionally about 3 to about 10, about 4 to about 10, about 5 to about 10, about 5 to about 15, or about 5 to about 20 amino acids, and any value or range thereof). Therefore, in some embodiments, the CRISPR-Cas effector proteins and / or peptides of the present invention may be fused with one peptide tag or with two or more peptide tags, optionally wherein the two or more peptide tags are fused to each other via one or more amino acid residues. In some embodiments, the peptide tag that can be used in the present invention may be a single copy of a GCN4 peptide tag or epitope, or may be a polymerized GCN4 epitope comprising about 2 to about 25 or more copies of the peptide tag (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more copies of the GCN4 epitope, or any range thereof).

[0172] In some embodiments, the peptide tag may be fused to a CRISPR-Cas peptide or domain. In some embodiments, the peptide tag may be fused to or linked to the C-terminus of a CRISPR-Cas effector protein to form a CRISPR-Cas fusion protein. In some embodiments, the peptide tag may be fused to or linked to the N-terminus of a CRISPR-Cas effector protein to form a CRISPR-Cas fusion protein. In some embodiments, the peptide tag may be fused within a CRISPR-Cas effector protein (e.g., the peptide tag may be within a loop region of the CRISPR-Cas effector protein). In some embodiments, the peptide tag may be fused to the fusion protein of the present invention.

[0173] "Affinity peptide" (e.g., "recruiting peptide") refers to any peptide capable of binding to its corresponding peptide tag, peptide tag, or RNA recruitment motif. Affinity peptides with peptide tags can be, for example, antibodies and / or single-chain antibodies that specifically bind to said peptide tag. In some embodiments, the peptide tag antibody can be, but is not limited to, scFv antibodies. In some embodiments, the affinity peptide can be fused to or linked to the N-terminus of the fusion protein of the present invention. In some embodiments, the affinity peptide is stable under reducing conditions in cells or cell extracts.

[0174] The nucleic acid constructs and / or guide nucleic acids of the present invention may be contained in one or more expression cassettes as described herein. In some embodiments, the nucleic acid constructs of the present invention may be contained in the same or separate expression cassettes or vectors as those containing guide nucleic acids and / or extended guide nucleic acids.

[0175] In some embodiments, the nucleic acid constructs, expression cassettes, or vectors of the present invention optimized for expression in organisms (e.g., humans or plants) may be approximately 70% to 100% identical (e.g., approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) of the present invention.

[0176] When used in combination with guide nucleic acids, the nucleic acid constructs of the present invention (and expression cassettes and / or vectors containing them) can be used to modify target nucleic acids and / or their expression. The target nucleic acid can be contacted with the nucleic acid constructs of the present invention and / or expression cassettes and / or vectors containing them before, simultaneously with, or after contacting the target nucleic acid with the guide nucleic acid / recruiting guide nucleic acid and / or the expression cassettes and vectors containing it.

[0177] According to embodiments of the present invention, a fusion protein is provided herein. The fusion protein of the present invention comprises a CRISPR-Cas effector protein and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242. Therefore, the fusion protein of the present invention comprises a CRISPR-Cas effector protein fused (directly or indirectly) to a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242. A polypeptide having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242 can be heterologous to a CRISPR-Cas effector protein. The fusion protein of the present invention can comprise two or more polypeptides (e.g., 2, 3, 4, 5, or more), said polypeptides being identical or different, and each polypeptide having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242, and optionally, the two or more polypeptides can be fused in tandem (directly or indirectly). In some embodiments, the fusion protein of the present invention comprises two or more polypeptides (e.g., 2, 3, 4, 5 or more), each polypeptide having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 241 or SEQ ID NO: 242, and optionally, the two or more polypeptides may be fused in tandem (directly or indirectly).

[0178] In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241 or SEQ ID NO: 242, and the length of the polypeptide is about 10 to about 40 amino acid residues. For example, the length of the polypeptide can be from 10, 11, 12, 13, 14, or 15 amino acid residues to 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues, or any value or range thereof. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241 or SEQ ID NO: 242, and the length of the polypeptide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241, and the length of the polypeptide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241, and the polypeptide having a length of 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues.In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 242, and the length of the polypeptide is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 242, and the polypeptide having a length of 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues.

[0179] The fusion protein of the present invention may comprise a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241, and an arginine or lysine residue (e.g., R9 or K9) at amino acid residue 9 of the polypeptide. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241, and an arginine residue (R9) at amino acid residue 9 of the polypeptide. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 241, an arginine residue at amino acid residue 9 (R9) of the polypeptide, and the polypeptide having a length of 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0180] The fusion protein of the present invention may comprise a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 242, and arginine or lysine at one or more of amino acid residues 9, 13, and / or 19 (e.g., R9 or K9, R13 or K13, and / or R19 or K19) of the polypeptide. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 242, and arginine at amino acid residue 9 (R9), arginine at amino acid residue 13 (R13), and arginine at amino acid residue 19 (R19) of the polypeptide. In some embodiments, the fusion protein of the present invention comprises a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 242, arginine at amino acid residue 9 (R9), arginine at amino acid residue 13 (R13), and arginine at amino acid residue 19 (R19); and the length of the polypeptide is 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acid residues.

[0181] In some embodiments, the CRISPR-Cas effector protein is directly fused to the peptide (e.g., linked by a peptide bond), without any intermediate element between the CRISPR-Cas effector protein and the peptide. In some embodiments, a linker (e.g., a peptide linker) fuses / conjugates the CRISPR-Cas effector protein and the peptide. The linker can be a peptide linker with a length of 1, 5, 10, or 15 amino acid residues up to 20, 25, 30, 35, 45, or 50 amino acid residues. In some embodiments, the fusion protein of the present invention comprises a peptide linker with a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acid residues. The fusion protein of the present invention may comprise one or more linkers (e.g., 1, 2, 3, 4, or more) (e.g., one or more peptide linkers). In some embodiments, the fusion protein of the present invention comprises two linkers (e.g., two peptide linkers). In some embodiments, the fusion protein of the present invention comprises peptide linkers comprising glycine and / or serine. In some embodiments, the fusion protein of the present invention comprises peptide linkers having an amino acid sequence having one of SEQ ID NO: 1-36 or a sequence selected from the following: CA, CF, (GGS). n , GS, SG, where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

[0182] In some embodiments, the fusion protein of the present invention may comprise a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242, said polypeptide being fused (directly or indirectly) to the N-terminus and / or C-terminus of a CRISPR-Cas effector protein. In some embodiments, the polypeptide is fused to the N-terminus of the CRISPR-Cas effector protein. In some embodiments, the polypeptide is fused to the C-terminus of the CRISPR-Cas effector protein.

[0183] The fusion protein of the present invention may comprise a CRISPR-Cas effector protein having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, or 299-301. In some embodiments, the CRISPR-Cas effector protein has a sequence of one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, or 299-301. In some embodiments, the fusion protein of the present invention comprises one or more (e.g., 1, 2, 3 or more) polypeptides having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% with respect to one or more of SEQ ID NO: 241-242. In some embodiments, the fusion protein of the present invention comprises two or more polypeptides and / or two or more copies of the polypeptides having a sequence identity of at least about 70% with respect to one or more of SEQ ID NO: 241-242, and the two or more copies of the polypeptides having a sequence identity of at least about 70% with respect to one or more of SEQ ID NO: 241-242. In some embodiments, the fusion protein of the present invention comprises a CRISPR-Cas effector protein fused (directly or indirectly) to a polypeptide, the effector protein having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, and 299-301, and the polypeptide having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242. In some embodiments, the fusion protein of the present invention comprises a CRISPR-Cas effector protein fused to a polypeptide having a sequence having one of SEQ ID NO: 241-242.

[0184] The fusion protein of the present invention may comprise a polypeptide of interest, said polypeptide of interest being fused (directly or indirectly) to the N-terminus or C-terminus of a CRISPR-Cas effector protein of the fusion protein. In some embodiments, the fusion protein of the present invention comprises two or more (e.g., 2, 3, 4 or more) polypeptides of interest that are the same as or different from each other. In some embodiments, the fusion protein of the present invention comprises a deaminase (e.g., adenosine deaminase and / or cytidine deaminase). Two or more (e.g., 2, 3, 4 or more) deaminases that are the same as or different from each other may be present in the fusion protein of the present invention. The deaminase may be fused (directly or indirectly) to the N-terminus or C-terminus of a CRISPR-Cas effector protein of the fusion protein of the present invention. In some embodiments, the fusion protein of the present invention comprises adenosine deaminase and cytidine deaminase. In some embodiments, the fusion protein of the present invention is fused with a cytosine deaminase (e.g., having all or a portion of a cytosine deaminase having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 204-213), optionally wherein the cytosine deaminase is fused (directly or indirectly) to the N-terminus or C-terminus of a CRISPR-Cas effector protein of the fusion protein. In some embodiments, the fusion protein of the present invention is fused with a glycosylation inhibitor, optionally wherein the glycosylation inhibitor is a uracil glycosylation inhibitor (UGI), for example, having all or a portion of a UGI having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 214. In some embodiments, the fusion protein of the present invention is fused with the cytosine deaminase and the glycosylation inhibitor in any order (directly or indirectly). In some embodiments, the fusion protein of the present invention is fused with an adenine deaminase (e.g., all or a portion of an adenine deaminase having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 215-225), optionally wherein the adenine deaminase is fused (directly or indirectly) to the N-terminus or C-terminus of the CRISPR-Cas effector protein of the fusion protein. In some embodiments, the fusion protein of the present invention is fused with an adenine deaminase and a cytosine deaminase in any order (directly or indirectly), optionally wherein the adenine deaminase is fused (directly or indirectly) to one of the N-terminus or C-terminus of the CRISPR-Cas effector protein of the fusion protein, and the cytosine deaminase is fused (directly or indirectly) to the other of the N-terminus or C-terminus of the CRISPR-Cas effector protein.In some embodiments, the fusion protein of the present invention comprises a reverse transcriptase, optionally wherein the reverse transcriptase is fused (directly or indirectly) to the N-terminus or C-terminus of a CRISPR-Cas effector protein of the fusion protein. In some embodiments, the fusion protein of the present invention is fused with a nuclear localization signal (NLS). The fusion protein of the present invention may comprise one or more (e.g., 1, 2, 3 or more) nuclear localization signals. In some embodiments, the nuclear localization signal has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 226-228. In some embodiments, the fusion protein of the present invention comprises an NLS fused (directly or indirectly) to the N-terminus or C-terminus of a CRISPR-Cas effector protein of the fusion protein. In some embodiments, the NLS is present at the N-terminus and / or C-terminus of the fusion protein of the present invention.

[0185] Exemplary fusion proteins according to some embodiments of the present invention are shown in Figure 1A and Figure 1B .like Figure 1A As shown, NLS can be present at both the N-terminus and C-terminus of the fusion protein of the present invention, a deaminase (e.g., adenosine deaminase) can be fused to the N-terminus of the CRISPR-Cas effector protein via a linker (e.g., a peptide linker), and a polypeptide having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO:241-242 can be fused to the C-terminus of the CRISPR-Cas effector protein via a linker (e.g., a peptide linker). Figure 1BAs shown, the fusion protein of the present invention may comprise two deaminases. In some embodiments, the fusion protein of the present invention comprises: an adenosine deaminase, a CRISPR-Cas effector protein, and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242, optionally wherein the adenosine deaminase is fused (directly or indirectly) to the N-terminus of the CRISPR-Cas effector protein, and / or wherein the polypeptide is fused (directly or indirectly) to the C-terminus of the CRISPR-Cas effector protein. In some embodiments, the fusion protein of the present invention comprises, from the N-terminus to the C-terminus, the following: a polypeptide of interest (e.g., adenosine deaminase, cytosine deaminase, and / or reverse transcriptase), a CRISPR-Cas effector protein (e.g., a CRISPR-Cas effector protein having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, or 299-301), and a polypeptide having a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one of SEQ ID NO: 241-242.

[0186] In some embodiments, the fusion protein of the present invention has an amino acid sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 243-256, 288-292, or 308. In some embodiments, the fusion protein of the present invention has an amino acid sequence comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 266-279, 294-298, and / or 307. In some embodiments, the polynucleotides of the present invention having a sequence of one of SEQ ID NO: 266-279, 294-298 or 307 encode fusion proteins of SEQ ID NO: 243-256, 288-292 or 308, respectively.

[0187] Compared to CRISPR-Cas effector proteins lacking at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242, the fusion proteins of the present invention may have at least one improvement (e.g., modified editing window, increased nuclease activity, increased stability, increased binding to nucleic acids, etc.). In some embodiments, compared to proteins having the amino acid sequence of any one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, or 299-301, the fusion proteins of the present invention have at least one improvement (e.g., increased activity and / or increased binding). In some embodiments, the fusion protein of the present invention can increase and / or stabilize the fusion protein at or near the target nucleic acid (e.g., compared to the stability of proteins or complexes lacking a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242, and / or compared to proteins having an amino acid sequence having any of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, or 299-301). In some embodiments, the increased stability can be determined by increased editing compared to a control and / or by editing efficiency (EE) and / or editing intensity.

[0188] Any deaminase domain / peptide that can be used for base editing is applicable to this invention. As used herein, “cytosine deaminase” and “cytidine deaminase” refer to a polypeptide or its domain that catalyzes or is capable of catalyzing the deamination of cytosine, because said polypeptide or domain catalyzes or is capable of catalyzing the removal of an amine group from a cytosine base. Thus, cytosine deaminase can lead to the conversion of cytosine to thymidine (via a uracil intermediate), resulting in a C-to-T or G-to-A conversion in the complementary strand of the genome. Therefore, in some embodiments, the cytosine deaminase encoded by the polynucleotide of this invention produces a C→T conversion in the sense (e.g., “+”; template) strand of the target nucleic acid or a G→A conversion in the antisense (e.g., “-”; complementary) strand of the target nucleic acid. In some embodiments, the cytosine deaminase encoded by the polynucleotide of this invention produces a C-to-T, G, or A conversion in the complementary strand of the genome.

[0189] The cytosine deaminases that can be used in this invention can be any known or later identified cytosine deaminase from any organism (see, for example, U.S. Patent No. 10,167,457 and Thuronyi et al., Nature Biotechnology 37:1070-1079 (2019), the disclosure of each of these documents regarding its cytosine deaminase is incorporated herein by reference). Cytosine deaminases can catalyze the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Therefore, in some embodiments, the deaminase or deaminase domain that can be used in this invention can be a cytidine deaminase domain that catalyzes the hydrolysis and deamination of cytosine to uracil. In some embodiments, the cytosine deaminase can be a variant of a naturally occurring cytosine deaminase, including but not limited to primates (e.g., humans, monkeys, chimpanzees, gorillas), dogs, cows, rats, or mice. Therefore, in some embodiments, the cytosine deaminase that can be used in the present invention may be about 70% to about 100% identical to wild-type cytosine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to naturally occurring cytosine deaminase, and any range or value thereof).

[0190] In some embodiments, the cytosine deaminase used in this invention may be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase may be APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, human activation-inducible deaminase (hAID), rAPOBEC1, FERNY and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g19570), and its evolved versions. Evolved deaminases are disclosed, for example, in U.S. Patent No. 10,113,163, Gaudelli et al., Nature 551(7681):464-471 (2017), and Thuronyi et al. (Nature Biotechnology 37: 1070-1079 (2019)), the disclosure of each of these documents regarding its deaminase and evolved deaminases is incorporated herein by reference. In some embodiments, the cytosine deaminase may be the APOBEC1 deaminase having the amino acid sequence of SEQ ID NO: 204. In some embodiments, the cytosine deaminase may be the APOBEC3A deaminase having the amino acid sequence of SEQ ID NO: 205. In some embodiments, the cytosine deaminase may be the CDA1 deaminase, optionally having the nucleotide sequence of SEQ ID NO: 206. In some embodiments, the cytosine deaminase may be the FERNY deaminase, optionally having the amino acid sequence of SEQ ID NO: 209. In some embodiments, the cytosine deaminase may be rAPOBEC1 deaminase, optionally having the amino acid sequence of SEQ ID NO: 210. In some embodiments, the cytosine deaminase may be hAID deaminase, optionally having the amino acid sequence of SEQ ID NO: 208 or SEQ ID NO: 211.In some embodiments, the cytosine deaminase that can be used in the present invention may be about 70% to about 100% identical in amino acid sequence to naturally occurring cytosine deaminases (e.g., "evolved deaminases") (see, for example, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214) (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%). In some embodiments, the cytosine deaminase used in the present invention may be identical in about 70% to about 99.5% of the amino acid sequence of any of SEQ ID NO: 204-213 (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) (e.g., identical in at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%) of the amino acid sequence of any of SEQ ID NO: 204-213. In some embodiments, the polynucleotide encoding cytosine deaminase may be codon-optimized for expression in plants, and the codon-optimized polypeptide may be approximately 70% to 99.5% identical to a reference polynucleotide.

[0191] As used herein, “adenine deaminase” and “adenosine deaminase” refer to a polypeptide or a domain thereof that catalyzes or is capable of catalyzing the hydrolytic deamination (e.g., removal of an amine group from adenine) of adenine or adenosine. In some embodiments, adenine deaminase may catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminase may catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, an adenine deaminase encoded by a nucleic acid construct of the present invention may produce an A→G conversion in the sense (e.g., “+”; template) strand of a target nucleic acid or a T→C conversion in the antisense (e.g., “-”, complementary) strand of a target nucleic acid. The adenine deaminase that can be used in the present invention may be any known or later identified adenine deaminase from any organism (see, for example, U.S. Patent No. 10,113,163, which, by reference, is incorporated herein by reference the disclosure of its adenine deaminase).

[0192] In some embodiments, adenosine deaminase may be a variant of naturally occurring adenine deaminase. Thus, in some embodiments, adenosine deaminase may be approximately 70% to 100% identical to wild-type adenine deaminase (e.g., approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to naturally occurring adenine deaminase, and any range or value thereof). In some embodiments, the deaminase or deaminase is not naturally occurring and may be referred to as an engineered, mutated, or evolved adenosine deaminase. Therefore, for example, engineered, mutated, or evolved adenine deaminase peptides or adenine deaminase domains can be approximately 70% to 99.9% identical to naturally occurring adenine deaminase peptides / domains (e.g., approximately 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%, and any range or value thereof). In some embodiments, adenosine deaminase may be derived from bacteria (e.g., *Escherichia coli*, *Staphylococcus aureus*, *Haemophilus influenzae*, *Caulobacter crescentus*, etc.). In some embodiments, the polynucleotide encoding the adenosine deaminase polypeptide / domain may be codon-optimized for expression in plants.

[0193] In some embodiments, the adenine deaminase domain may be a wild-type tRNA-specific adenine deaminase domain, such as tRNA-specific adenine deaminase (TadA), and / or a mutated / evolved adenine deaminase domain, such as a mutated / evolved tRNA-specific adenine deaminase domain (TadA*). In some embodiments, the TadA domain may be derived from *Escherichia coli*. In some embodiments, TadA may be modified, for example, truncated, thereby deleting one or more N-terminal and / or C-terminal amino acids relative to the full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and / or C-terminal amino acid residues may be deleted relative to the full-length TadA). In some embodiments, the TadA polypeptide or TadA domain does not contain an N-terminal methionine. In some embodiments, wild-type Escherichia coli TadA comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, mutant / evolved Escherichia coli TadA* comprises the amino acid sequence of any of SEQ ID NO: 216-219. In some embodiments, the polynucleotide encoding TadA / TadA* may be codon-optimized for expression in plants. In some embodiments, the adenine deaminase may comprise SEQ ID NO: The amino acid sequence of any one of SEQ ID NO: 215-225 may be all or part of the amino acid sequence. In some embodiments, adenine deaminase may comprise all or part of the amino acid sequence of any one of SEQ ID NO: 215-225.

[0194] In some embodiments, the nucleic acid constructs of the present invention may further encode glycosylation inhibitors (e.g., uracil glycosylation inhibitors (UGIs), such as uracil-DNA glycosylation inhibitors). In some embodiments, the present invention provides a fusion protein comprising an engineered protein and a UGI and / or one or more polynucleotides encoding thereus, wherein optionally said one or more polynucleotides may be codon-optimized for expression in plants.

[0195] The "uracil glycosylation enzyme inhibitor" used in this invention can be any protein or polypeptide capable of inhibiting uracil-DNA glycosylation enzyme base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI domain used in this invention can be about 70% to about 100% identical to the amino acid sequence of a naturally occurring UGI domain (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical, and any range or value thereof). In some embodiments, the UGI domain may comprise the amino acid sequence of SEQ ID NO:214 or a polypeptide having about 70% to about 99.5% identity with the amino acid sequence of SEQ ID NO:214 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the amino acid sequence of SEQ ID NO:214). For example, in some embodiments, the UGI domain may comprise a fragment of the amino acid sequence of SEQ ID NO: 214, said fragment being 100% identical to a portion of a continuous nucleotide sequence of SEQ ID NO: 214 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., about 10, 15, 20, 25, 30, 35, 40, 45 to about 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides). In some embodiments, the UGI domain may be a variant of a known UGI (e.g., SEQ ID NO: 214) having about 70% to about 99.5% identity with a known UGI (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identity, and any range or value therein). In some embodiments, the polynucleotide encoding the UGI may be codon-optimized for expression in a plant (e.g., a plant), and the codon-optimized polypeptide may be about 70% to about 99.5% identical to the reference polynucleotide.

[0196] As used herein, “reverse transcriptase” includes, but is not limited to, naturally occurring reverse transcriptases, engineered reverse transcriptases, or commercially available reverse transcriptases. Exemplary reverse transcriptases include, but are not limited to, Moloney murine leukemia virus reverse transcriptase (MMLV-RT or M-MuLV-RT), mutant MMLV (e.g., 5M-MMLV), avian myeloma virus reverse transcriptase (AMV-RT), and human immunodeficiency virus reverse transcriptase (HIV-RT). In some embodiments, the reverse transcriptase may be MMLV and / or 5M-MMLV. In some embodiments, the reverse transcriptase may have a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with one or more of SEQ ID NO: 229-240.

[0197] Nucleic acid molecules can encode the fusion protein of the present invention, and said nucleic acid molecules can be present in expression cassettes and / or vectors. The polynucleotides and / or recombinant nucleic acid constructs of the present invention can be codon-optimized for expression. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes and / or vectors of the present invention (e.g., those containing / encoding the fusion protein of the present invention) and / or guide nucleic acids of the present invention can be codon-optimized for expression in organisms (e.g., animals, plants, fungi, archaea, or bacteria). In some embodiments, the expression cassettes and / or vectors of the present invention contain polynucleotides encoding polypeptides having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to one or more of SEQ ID NO: 241-242. In some embodiments, the expression cassette and / or vector of the present invention comprises a polynucleotide encoding a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with one or more of SEQ ID NO: 243-256, 288-292 and / or 308. In some embodiments, the expression cassette and / or vector of the present invention comprises a polynucleotide encoding a promoter sequence and a polynucleotide encoding a fusion protein having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with one or more of SEQ ID NO: 241-256 and / or 288-292. In some embodiments, the expression cassette and / or vector of the present invention comprises a polynucleotide having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 266-279, 294-298, and / or 307. In some embodiments, the expression cassette and / or vector of the present invention comprises a first polynucleotide encoding a promoter sequence and a second polynucleotide having a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 266-279, 294-298, and / or 307. The polynucleotide encoding the fusion protein of the present invention may be codon-optimized for expression in a specific organism (e.g., human or plant). In some embodiments, the organism is an animal (e.g., human), plant, fungus, archaea, or bacterium.

[0198] A method for modifying target nucleic acids in cells is provided, and the method may include introducing an expression cassette and / or vector of the present invention into the cells to provide modified target nucleic acids. In some embodiments, the cells are plant cells, and the method further includes regenerating the plant cells containing the modified target nucleic acids to produce a plant containing the modified target nucleic acids. In some embodiments, the introduction of the expression cassette is performed at a temperature of about 20°C to about 42°C. In some embodiments, the cells are mammalian cells (e.g., human cells).

[0199] This document provides a method for generating the fusion protein of the present invention, which may include: culturing cells or multiple cells transformed with nucleic acids encoding the fusion protein of the present invention; and isolating the fusion protein of the present invention to generate the fusion protein. In some embodiments, the isolation step is performed by dialysis, centrifugation, column purification, etc.

[0200] This document provides a method for performing reverse transcription, which may involve contacting a target nucleic acid with a fusion protein of the present invention. In some embodiments, all or a portion of the fusion protein has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with one or more of SEQ ID NO: 241-256 and / or 288-292. Optionally, the fusion protein of the present invention, together with one or more components, can reverse transcribe the target nucleic acid to provide DNA (e.g., cDNA). In some embodiments, the target nucleic acid is in a plant and / or in a plant cell including a cell wall. In some embodiments, the target nucleic acid is in a mammal and / or in a mammalian cell.

[0201] This document provides a method for modifying a target nucleic acid, which may include contacting the target nucleic acid with a fusion protein and a guide nucleic acid of the present invention. In some embodiments, all or a portion of the fusion protein has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with one or more of SEQ ID NO: 241-256 and / or 288-292. In some embodiments, the target nucleic acid is in a plant and / or in a plant cell including a cell wall. In some embodiments, the target nucleic acid is in a mammal and / or in a mammalian cell. In some embodiments, the method for modifying the target nucleic acid is a template-based editing method. In some embodiments, the method further includes introducing a nucleic acid molecule encoding the fusion protein into a cell and generating the fusion protein in the cell.

[0202] The method of the present invention may include contacting the target nucleic acid with an extended guide nucleic acid. In some embodiments, the extended guide nucleic acid includes a primer binding site, and the target nucleic acid is double-stranded and includes a first strand and a second strand. In some embodiments, the primer binding site of the extended guide nucleic acid binds to either the first strand or the second strand of the target nucleic acid. In some embodiments, the second strand is a non-target strand of the target nucleic acid. In some embodiments, the target nucleic acid is double-stranded and includes a first strand and a second strand, and the primer binding site binds to the first strand of the target nucleic acid. The first strand may be the target strand of the target nucleic acid, and / or the fusion protein of the present invention may be recruited to the first strand. In some embodiments, the target nucleic acid is double-stranded and includes a first strand and a second strand, and the primer binding site of the extended guide nucleic acid binds to the second strand of the target nucleic acid. In some embodiments, the second strand is a non-target strand of the target nucleic acid, and / or the fusion protein is recruited to the second strand. In some embodiments, the fusion protein of the present invention is a double-stranded nuclease that cleaves the first and second strands of the target nucleic acid, causing a double-strand break. In some embodiments, the fusion protein and the extended guide nucleic acid form a complex, or are contained within the complex.

[0203] The method of the present invention may include contacting a target nucleic acid with an extended guide nucleic acid and / or introducing the extended guide nucleic acid into a cell, said extended guide nucleic acid comprising: (i) a CRISPR nucleic acid and / or a CRISPR nucleic acid and a tracr nucleic acid; and (ii) an extension portion comprising a primer binding site and a reverse transcriptase template (RT template). In some embodiments, the extension portion of the extended guide nucleic acid is fused to the 5' or 3' end of the CRISPR nucleic acid (e.g., from 5' to 3': repeat sequence-spacer-extension portion or extension portion-repeat sequence-spacer) and / or fused to the 5' or 3' end of the tracr nucleic acid. In some embodiments, the extension portion of the extended guide nucleic acid from 5' to 3' comprises an RT template and a primer binding site. In some embodiments, the extension portion of the extended guide nucleic acid is located at the 5' of the crRNA.

[0204] In some embodiments, the primer binding site is about one nucleotide to about 100 nucleotides in length. In some embodiments, the primer binding site is at least 45 nucleotides in length, or about 45 nucleotides to about 100 nucleotides in length, for example 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length.

[0205] In some embodiments, the length of the RT template is from about one to about 100 nucleotides, or the length of the RT template can be about 40 nucleotides or less, such as 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide.

[0206] In some embodiments, the extended portion of the extended guide nucleic acid is linked to the CRISPR nucleic acid and / or the tracrRNA via a adapter. The adapter can be about 1 to about 100 nucleotides in length, for example, lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides.

[0207] In some embodiments, in the method of the present invention, the fusion protein of the present invention is fused with one or more components that recruit deaminases and / or reverse transcriptases to the fusion protein. In some embodiments, the fusion protein of the present invention is fused (e.g., linked) with a peptide tag (e.g., an epitope or a polymerized epitope), and the reverse transcriptase is fused (e.g., linked) with an affinity polypeptide that binds to the peptide tag. In some embodiments, the fusion protein of the present invention is fused (e.g., linked) with a peptide tag (e.g., an epitope or a polymerized epitope), and the reverse transcriptase is fused (linked) with an affinity polypeptide that binds to the peptide tag. In some embodiments, the target nucleic acid is contacted with two or more reverse transcriptases.

[0208] In some embodiments, the extended guide nucleic acid is linked to an RNA recruitment motif, and a reverse transcriptase is fused (e.g., linked) to an affinity polypeptide that binds to the RNA recruitment motif. In some embodiments, the target nucleic acid is contacted with two or more reverse transcriptases. In some embodiments, the extended guide nucleic acid (e.g., extended guide RNA) is linked to two or more RNA recruitment motifs, optionally wherein the two or more RNA recruitment motifs are the same RNA recruitment motif or different RNA recruitment motifs. In some embodiments, at least one of the two or more RNA recruitment motifs is located at the 3' end of the extended portion of the extended guide nucleic acid or embedded in the extended portion.

[0209] In some embodiments, the method of the present invention has improved efficiency in modifying target nucleic acids compared to control methods (e.g., methods performed under the same conditions using control CRISPR-Cas effector proteins (e.g., CRISPR-Cas effector proteins lacking at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242, and / or CRISPR-Cas effector proteins having the amino acid sequence of any one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, or 299-301). Compared to control methods, the method of the present invention can produce increased levels of insertions / deletions and / or increased levels of modification (e.g., precise modification).

[0210] The fusion protein of the present invention can be present in a complex with a guide nucleic acid. The guide nucleic acid can be programmed to function in conjunction with a CRISPR-Cas effector protein present in the fusion protein. In some embodiments, the complexes and / or methods of the present invention may include those described in U.S. Patent Application Publication No. 2021 / 0130835 and / or U.S. Patent Application Publication No. 2022 / 0145334 (the contents of each of these U.S. patent applications are incorporated herein by reference in their entirety), wherein the fusion protein of the present invention is used in place of a CRISPR-Cas effector protein.

[0211] In some embodiments, the editing system of the present invention is used for lead editing. As used herein, “lead editing” and its grammatical variations refer to nucleic acid editing techniques that use a Cas9 nickase domain fused with reverse transcriptase and modify the target nucleic acid without double-strand breaks or donor DNA template. In lead editing, the Cas9 nickase domain cleaves the non-complementary strand of DNA upstream of the PAM site, thereby providing a 3' lobe that is extended with an extended portion including the modification. Further details regarding lead editing can be found in Anzalone et al. (2019) Nature 576, 149-157 and / or U.S. Patent Application Publication No. 2021 / 0147862, the contents of each of which are incorporated herein by reference in their entirety.

[0212] In some embodiments, the editing system of the present invention utilizes the Redraw editing system. Further details regarding the Redraw editing system can be found in U.S. Patent Application Publication No. 2021 / 0130835 and / or U.S. Patent Application Publication No. 2022 / 0145334, the contents of which are incorporated herein by reference in their entirety.

[0213] As described herein, the polypeptides (e.g., fusion proteins), nucleic acids, expression cassettes, and / or vectors of the present invention can be codon-optimized for expression in an organism. An organism that can be used in the present invention can be any organism or its cells for which nucleic acid modifications are applicable. An organism can include, but is not limited to, any animal (e.g., a mammal), any plant, any fungus, any archaea, or any bacteria. In some embodiments, the organism can be a plant or its cells. In some embodiments, the organism is an animal, such as a mammal (e.g., a human).

[0214] The target nucleic acid can be a genomic sequence from any organism (e.g., a eukaryote, such as a mammal or a plant). In some embodiments, the target nucleic acid is a genomic sequence from a model organism, such as, but not limited to, *Escherichia coli*, immortalized human cell lines (e.g., HEK293, HeLa, etc.), *Caenorhabditis elegans*, and / or *Drosophila melanogaster*. In some embodiments, the target nucleic acid is a genomic sequence from a non-model organism. Exemplary non-model organisms include, but are not limited to, crop plants (e.g., fruit crops, vegetable crops, and / or field crops) and / or animals, such as humans, primates, and / or mice. In some embodiments, the non-model organism is a crop plant, such as corn, soybean, wheat, or canola. In some embodiments, the non-model organism is an animal used for testing and / or using human therapeutics.

[0215] The nucleic acid constructs of this invention can be used to modify target nucleic acids of any plant or plant part. Any plant (or plant group, such as genus or higher classification) can be modified using the fusion protein of this invention, including angiosperms, gymnosperms, monocots, dicots, C3, C4, CAM plants, bryophytes, ferns and / or pseudoferns, microalgae and / or macroalgae. Plants and / or plant parts that can be used in this invention can be plants and / or plant parts of any plant species / variety / cultivar. As used herein, the term "plant part" includes, but is not limited to, embryo, pollen, ovule, seed, leaf, stem, bud, flower, branch, fruit, grain, spike, rachis, husk, culm, root, root tip, anther, plant cell (including intact plant cells in a plant and / or plant part), plant protoplast, plant tissue, plant cell tissue culture, plant callus, plant clump, etc. As used herein, "bud" refers to the aboveground part, including leaves and stems. Furthermore, as used herein, "plant cell" refers to the structural and physiological unit of a plant, which includes a cell wall and may also refer to a protoplast. A plant cell can be an isolated single-celled form, a cultured cell, or a higher-level tissue unit, such as part of a plant tissue or organ.

[0216] Non-limiting examples of plants that can be used in this invention include turfgrass (e.g., Kentucky bluegrass, creeping bentgrass, ryegrass, fescue), feather reed, tufted grass, miscanthus, reed, switchgrass, vegetable crops including artichoke, kohlrabi, arugula, leek, asparagus, lettuce (e.g., head lettuce, leaf lettuce, romaine lettuce), taro, melons (e.g., cantaloupe, watermelon, crenshaw, cantaloupe, honeydew melon), brassica crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, headless cabbage, Chinese cabbage, bok choy), artichoke, carrot, bok choy, okra, onion, celery, parsley, chickpea, parsnip, chicory, pepper, potato, cucurbitaceae plants (e.g., zucchini, cucumber, Italian cucumber, squash, pumpkin, cantaloupe, watermelon, honeydew melon), radish, and dry bulb onion. Onion, turnip, eggplant, ginseng, broadleaf chicory, scallions, endive, garlic, spinach, green onions, squash, leafy greens, beets (sugar beets and forage beets), sweet potatoes, chard, horseradish, tomatoes, carrots, and spices; fruit crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, figs, nuts (e.g., chestnuts, pecans, pistachios, hazelnuts, peanuts, walnuts, macadamia nuts, almonds, etc.), citrus (e.g., clementine, kumquats, oranges, grapefruits, tangerines, mandarins, lemons, limes, etc.), blueberries, black raspberries, bosomberries, cranberries, currants, currants, raspberries, strawberries, blackberries, grapes (wine grapes and table grapes), avocados, bananas, kiwis Kiwifruit, persimmon, pomegranate, pineapple, tropical fruits, pears, cantaloupe, mango, papaya, and lychee; field crops such as clover, alfalfa, timothy, evening primrose, silvergrass, corn / corn (feed corn, sweet corn, popcorn corn), hops, jojoba, buckwheat, safflower, quinoa, wheat, rice, barley, rye, millet, sorghum, oats, triticale, tobacco, kapok, legumes (legumes (e.g., green beans and dried beans), lentils, peas, soybeans), oilseeds (rapeseed, mustard, poppy, olive, sunflower, coconut, castor oil plants, cocoa beans, peanuts, oil palm), duckweed, Arabidopsis, fiber plants (cotton, flax, hemp, jute), Cannabis (e.g., hemp). Cannabis sativa, Indian hemp (Cannabis indica and Cannabis ruderalis), Lauraceae plants (cinnamon, camphor) or plants such as coffee trees, sugarcane, tea and natural rubber plants; and / or flower bed plants such as flowering plants, cacti, succulents and / or ornamental plants (e.g. roses, tulips, violets), and trees such as forest trees (broadleaf trees and evergreen trees such as conifers);For example, elm, ash, oak, maple, fir, spruce, cedar, pine, birch, cypress, eucalyptus, willow) as well as shrubs and other seedlings. In some embodiments, the nucleic acid constructs and / or expression cassettes and / or vectors encoding them of the present invention can be used to modify corn, soybean, wheat, canola, rice, tomato, pepper, sunflower, raspberry, blackberry, black raspberry and / or cherry.

[0217] In some embodiments, the present invention provides cells (e.g., plant cells, animal cells, bacterial cells, archaea cells, etc.) comprising the polypeptides, polynucleotides, nucleic acid constructs, expression cassettes, or vectors of the present invention.

[0218] This invention further includes one or more kits for carrying out the methods of this invention. The kits of this invention may include reagents, buffers, and equipment for mixing, measuring, sorting, labeling, etc., as well as instructions for modifying target nucleic acids.

[0219] In some embodiments, the present invention provides a kit comprising one or more polypeptides (e.g., fusion proteins) of the present invention as described herein, nucleic acid constructs of the present invention, and / or expression cassettes and / or vectors and / or cells containing them, and optionally, instructions for use thereof. In some embodiments, the kit may further comprise a CRISPR-Cas guide nucleic acid (corresponding to the CRISPR-Cas effector proteins provided herein, which may be encoded by polynucleotides) and / or expression cassettes and / or vectors and / or cells containing them. In some embodiments, the guide nucleic acid may be provided on the same expression cassette and / or vector as one or more nucleic acid constructs of the present invention. In some embodiments, the guide nucleic acid may be provided on an expression cassette or vector separate from an expression cassette or vector containing one or more nucleic acid constructs of the present invention.

[0220] Therefore, in some embodiments, a kit is provided comprising a nucleic acid construct containing (a) a polynucleotide as provided herein, and (b) a promoter driving the expression of said polynucleotide (a). In some embodiments, the kit may further comprise a nucleic acid construct encoding a guide nucleic acid, wherein said construct contains a cloning site for cloning a nucleic acid sequence identical or complementary to the target nucleic acid sequence into the backbone of the guide nucleic acid.

[0221] In some embodiments, the nucleic acid construct of the present invention may be mRNA, which may encode one or more introns within the encoded polynucleotide. In some embodiments, the nucleic acid construct of the present invention and / or the expression cassette and / or vector containing it may further encode one or more selectable markers that can be used to identify transformants (e.g., nucleic acids encoding antibiotic resistance genes, herbicide resistance genes, etc.).

[0222] The peptides, polynucleotides, nucleic acid constructs, expression cassettes, vectors, compositions, kits, systems, and / or cells of the present invention may comprise all or a portion of the sequences of one or more of SEQ ID NO: 1-308. In some embodiments, if not present in the amino acid sequence described herein and / or encoded in the nucleotide sequence described herein, then the amino acid sequence and / or the nucleotide sequence may further comprise one, two, three, four, or five additional amino acids or corresponding nucleotides, such as methionine or its corresponding nucleotide at amino acid residue 1 of the amino acid sequence, and / or one, two, three, or four additional amino acids (such as glycine or alanine) or their corresponding nucleotides, which may facilitate expression or enable the construction or cloning required for the construction of plasmids, constructs, or vectors. In some embodiments, if present in and / or encoded in the amino acid sequence described herein and the nucleotide sequence described herein, the amino acid sequence and / or nucleotide sequence may lack one, two, three, four, or five amino acids or corresponding nucleotides present at amino acid residues 1-5 at the N-terminus of the amino acid sequence, such as methionine or its corresponding nucleotide at amino acid residue 1 of the amino acid sequence, and / or one, two, three, or four additional amino acids (such as glycine or alanine) or their corresponding nucleotides. This may facilitate expression or enable the construction or cloning of plasmids, constructs, or vectors as required. In some embodiments, the polypeptides, polynucleotides, nucleic acid constructs, expression cassettes, vectors, compositions, kits, systems, and / or cells of the present invention may comprise at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the sequences of one or more of SEQ ID NO: 1-308.

[0223] The invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims of the invention, but are intended to be examples of certain embodiments. Any variations of the exemplary methods that may occur to those skilled in the art are intended to fall within the scope of the invention.

[0224] Example

[0225] Example 1:

[0226] In the absence of antibiotics, HEK293T cells were seeded into 48-well collagen-coated plates (Corning) using DMEM medium. At 70–80% confluence, cells were transfected with 500 ng of fusion protein plasmid and 500 ng of guide RNA plasmid in 1.5 μL LTX (Thermo Fisher Scientific), according to the manufacturer's protocol. Three days later, cells were lysed using a crude extraction method with SDS buffer to isolate genomic DNA.

[0227] Genomic DNA was amplified by PCR using genomic primers with Inmena adaptor overhangs to produce a product of approximately 300 bases. The product was amplified by another round of PCR and then pooled to generate an Inmena barcode library. The library was purified using Sera-Mag® Select (Cytiva) and quantified using the SpectraMax® Quant™ dsDNA assay kit (Molecular Devices). The pooled amplicon library was sequenced using an Inmena MiSeq 2x250.

[0228] The original Inmena MiSeq paired-end reads were trimmed and merged, and aligned using CRISPResso2 (Clement et al. (2019) Nature Biotechnology 37, 224–226), and processed as described above (Kim et al. (2022) Biopreprints 2022.12.13.520319).

[0229] Table 1. Spacer sequences used in guide RNA:

[0230]

[0231] The average observed frequency of fusion proteins 1-4 (SEQ ID NO: 243-246) at the edit sites within the tested spacers and the control adenine-to-guanine edits is provided. Figure 2 , 3In 1, 5, and 6. Each of fusion proteins 1-4 is a fusion protein comprising adenosine deaminase, a CRISPR-Cas effector protein, and a polypeptide having the sequence SEQ ID NO: 241 or 242. The N-terminal fusions of TadA7.10 or TadA8e with Cas9 were used as controls (SEQ ID NO: 264 and 265). For experiments performed with fusion proteins 1 and 3 (SEQ ID NO: 244 and 246, respectively), the control protein had the sequence SEQ ID NO: 265, and for experiments performed with fusion proteins 2 and 4 (SEQ ID NO: 243 and 245, respectively), the control protein had the sequence SEQ ID NO: 264. Figure 4 and Figure 7 The log2 (fold change) of the fusion protein at each test interval (Table 1) is shown.

[0232] Example 2:

[0233] As previously described, maize seed embryo explants (SEE) were transformed and cultured (Ye et al. (2022), Frontiers in Plant Science, 13, 1056190). In short, vectors containing tools and guide expression elements were transformed into Agrobacterium tumefaciens AB32, a carmine-containing strain. The binary vectors were electroporated into competent AB32 cells, followed by selection on LB medium containing 30 mg / L gentamicin and 75 mg / L spectinomycin. Overnight cultures were decelerated, vortexed, and resuspended for inoculation. Prior to co-culture, rehydrated SEEs were immersed in Agrobacterium inoculation, sonicated, isolated, and resuspended in medium. Plants were regenerated after selection on glyphosate-containing medium. After approximately 8 weeks, plants were harvested, and genomic DNA was isolated and submitted for next-generation sequencing.

[0234] Genomic DNA was amplified by PCR using genomic primers with Inmena adductor overhangs to produce a product of approximately 300 bases. The product was amplified by another round of PCR and then pooled to generate an Inmena barcode library. The library was purified using Sera-Mag® Select and quantified using a SpectraMax® Quant™ dsDNA assay kit. The pooled amplicon library was sequenced using an Inmena MiSeq 2x250.

[0235] The original Inminal MiSeq paired-end reads were trimmed and merged, and aligned using CRISPResso2 (Clement et al. (2019) Nature Biotechnology 37, 224–226), and processed as described above (Kim et al. (2022) BioPreprint 2022.12.13.520319).

[0236] Fusion proteins 5 and 6 (SEQ ID NO: 288 and 289, respectively), comprising the peptide of SEQ ID NO: 241 or 242, fused to the C-terminus of Cas12a via the linker of SEQ ID NO: 29, were evaluated in stable maize lines. Fusion protein 7 (SEQ ID NO: 303) comprising the peptide of SEQ ID NO: 302, fused to the C-terminus of Cas12a via the linker of SEQ ID NO: 29, was also evaluated in stable maize lines. For these experiments, a control protein with the sequence of SEQ ID NO: 301 was used. Editing efficiency was determined (Table 2), and the results are provided in [Table 2]. Figure 8 In the study, a genomic locus (locus 171) was selected, which exhibited historically low average editing efficiency, such as 12.5 EE. 10% .

[0237] Table 2. Editing Efficiency:

[0238]

[0239] Editing efficiency (EE) x% The value represents the ratio of the number of sampled plants (n) with an edit intensity (ES) higher than the indicated cutoff value (i.e., 1% or 10% insertion / deletion) to the total number of sampled plants (N). Edit intensity (ES) represents the frequency of insertion / deletion observed at the target site in a single plant.

[0240] Within the tested loci, both fusion proteins 5 and 6 showed improved editing efficiency relative to the control protein. The control protein exhibited a modest editing efficiency (EE) comparable to its historical average of 12.5%. 10% = 9%). However, both fusion protein 5 and fusion protein 6 contribute to EE. 10% Increased by two times or more (EE of fusion protein 5) 10% The percentage was 17%, and fusion protein 6 was 20%. When analyzing data with less stringent cutoff values, relative to the control protein, EE 1% Increased by three times or more (EE relative to control protein) 1% The EE of fusion protein 5 is 10%. 1%(It accounts for 29%, and fusion protein 6 accounts for 33%).

[0241] Example 3:

[0242] In the absence of antibiotics, HEK293T cells were seeded into 48-well collagen-coated plates (Corning). At 70–80% confluence, cells were transfected with 500 ng of fusion protein plasmid and 500 ng of guide RNA plasmid in 1.5 μL LTX (Thermo Fisher Scientific), according to the manufacturer's protocol. After 3 days, cells were lysed using a crude extraction method with Triton™-X buffer to isolate genomic DNA, which was then submitted for next-generation sequencing as described in Example 1.

[0243] Table 3. Spacer sequences used in guide RNA:

[0244]

[0245] The average observed frequency of adenine-to-guanine editing at the editing site within the tested spacers for fusion protein 1 (SEQ ID NO: 244), fusion protein 3 (SEQ ID NO: 246), or fusion protein 8 (SEQ ID NO: 305) and controls is provided. Figure 9-11 Fusion protein 8 (SEQ ID NO: 305) is a fusion protein comprising adenosine deaminase, a CRISPR-Cas effector protein, and a polypeptide having the sequence of SEQ ID NO: 302. The control protein has the sequence of SEQ ID NO: 265.

[0246] Fusion proteins 1 and 3 typically showed improved adenine base editing at all seven loci evaluated. Fusion protein 1 provided the strongest and most consistent improvement. Figure 9 The maximum fold improvement in the activity of fusion protein 1 and fusion protein 3 was observed at PWsp92. Figure 9-10This spacer was somewhat difficult to edit via the Cas9 nuclease (PWsp92: 31.9 ± 10.6% insertions / deletions), while the insertion / deletion frequency ranged from 44.3% to 55.2% at all other spacers (Table 3). This suggests that this site may be partially blocked by Cas9, and the inclusion of SEQ ID NO:241 or SEQ ID NO:242 is confirmed to improve base editing. Overall, Examples 1–3 demonstrate that, compared to controls, the fusion of SEQ ID NO:241 or SEQ ID NO:242 with CRISPR-Cas effector proteins provides improvements in modifications performed on organisms in two different kingdoms under two different gene editing mechanisms (base editing and cleavage), and improves pipeline efficiency by significantly increasing the number of edited plants / cells recovered.

[0247] The foregoing is a description of the invention and should not be construed as limiting it. The invention is defined by the following claims, including their equivalents.

Claims

1. A fusion protein comprising a CRISPR-Cas effector protein and a polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242.

2. The fusion protein of claim 1, further comprising a linker (e.g., a peptide linker) between the CRISPR-Cas effector protein and the polypeptide.

3. The fusion protein according to claim 2, wherein the linker is a peptide linker with a length of 1, 5, 10 or 15 amino acid residues to 20, 25, 30, 35 or 40 amino acid residues.

4. The fusion protein according to any one of claims 2 to 3, wherein the linker is a peptide linker comprising glycine and / or serine.

5. The fusion protein according to any one of claims 2 to 4, wherein the linker is a peptide linker having an amino acid sequence having one of SEQ ID NO: 1-36 or a sequence selected from the following: CA, CF, (GGS) n , GS, SG, where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

6. The fusion protein according to claim 1, wherein the CRISPR-Cas effector protein and the polypeptide are directly fused together, and there is no intermediate element between the CRISPR-Cas effector protein and the polypeptide.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein comprises two or more copies of the polypeptide.

8. The fusion protein according to any one of claims 1 to 7, wherein the polypeptide is fused to the N-terminus of the CRISPR-Cas effector protein.

9. The fusion protein according to any one of claims 1 to 8, wherein the polypeptide is fused to the C-terminus of the CRISPR-Cas effector protein.

10. The fusion protein according to any one of claims 1 to 9, further comprising a deaminase, optionally wherein the fusion protein comprises two or more copies of the deaminase.

11. The fusion protein of claim 10, wherein the deaminase is fused (directly or indirectly) to the N-terminus of the CRISPR-Cas effector protein.

12. The fusion protein according to claim 10 or 11, wherein the deaminase is fused (directly or indirectly) to the C-terminus of the CRISPR-Cas effector protein.

13. The fusion protein according to any one of claims 10 to 12, wherein the deaminase is an adenosine deaminase, and optionally the fusion protein further comprises a cytidine deaminase.

14. The fusion protein according to any one of claims 10 to 12, wherein the deaminase is cytidine deaminase, and optionally the fusion protein further comprises adenosine deaminase.

15. The fusion protein according to any one of claims 1 to 14, further comprising a glycosylation inhibitor, optionally wherein the glycosylation inhibitor is a uracil glycosylation inhibitor (UGI).

16. The fusion protein according to any one of claims 1 to 15, further comprising reverse transcriptase.

17. The fusion protein of claim 16, wherein the reverse transcriptase is fused (directly or indirectly) to the N-terminus of the CRISPR-Cas effector protein.

18. The fusion protein of claim 16, wherein the reverse transcriptase is fused (directly or indirectly) to the C-terminus of the CRISPR-Cas effector protein.

19. The fusion protein according to any one of claims 1 to 18, wherein the CRISPR-Cas effector protein has a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, and 299-301.

20. The fusion protein according to any one of claims 1 to 19, wherein the CRISPR-Cas effector protein has the sequence of one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287 or 299-301.

21. The fusion protein according to any one of claims 1 to 20, wherein the polypeptide has the sequence of one of SEQ ID NO: 241-242.

22. The fusion protein according to any one of claims 1 to 21, further comprising a nuclear localization sequence, optionally wherein the fusion protein comprises two or more nuclear localization sequences.

23. The fusion protein of claim 22, wherein the nuclear localization sequence is fused (directly or indirectly) to the N-terminus of the CRISPR-Cas effector protein.

24. The fusion protein of claim 22 or 23, wherein the nuclear localization sequence is fused (directly or indirectly) to the C-terminus of the CRISPR-Cas effector protein.

25. A fusion protein comprising: adenosine deaminase; CRISPR-Cas effector proteins; and The polypeptide having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 241-242.

26. The fusion protein of claim 25, wherein the adenosine deaminase is fused (directly or indirectly) to the N-terminus of the CRISPR-Cas effector protein.

27. The fusion protein of claim 25 or 26, wherein the polypeptide is fused (directly or indirectly) to the C-terminus of the CRISPR-Cas effector protein.

28. The fusion protein according to any one of claims 25 to 27, further comprising a first linker between the adenosine deaminase and the CRISPR-Cas effector protein.

29. The fusion protein according to any one of claims 25 to 28, further comprising a second linker between the polypeptide and the CRISPR-Cas effector protein.

30. The fusion protein according to claim 28 or 29, wherein the first linker and / or the second linker are each independently a peptide linker of length from 1, 5, 10 or 15 amino acid residues to 20, 25, 30, 35 or 40 amino acid residues.

31. The fusion protein according to any one of claims 28 to 30, wherein the first linker and / or the second linker are each independently a peptide linker comprising glycine and / or serine.

32. The fusion protein according to any one of claims 28 to 31, wherein the first linker and / or the second linker are each independently a peptide linker having an amino acid sequence of one of SEQ ID NO: 1-36 or a sequence selected from CA, CF, (GGS). n , GS, SG, where n is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20).

33. The fusion protein according to any one of claims 25 to 32, wherein the CRISPR-Cas effector protein has a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 39-65, 70-72, 88-189, 280-287, and 299-301.

34. The fusion protein according to any one of claims 25 to 33, wherein the CRISPR-Cas effector protein has the sequence of one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287 or 299-301.

35. The fusion protein according to any one of claims 25 to 34, wherein the polypeptide has the sequence of one of SEQ ID NO: 241-242.

36. The fusion protein according to any one of claims 25 to 35, further comprising a nuclear localization sequence, optionally wherein the fusion protein comprises two or more nuclear localization sequences.

37. The fusion protein of claim 36, wherein the nuclear localization sequence is fused (directly or indirectly) to the N-terminus of the CRISPR-Cas effector protein.

38. The fusion protein of claim 36 or 37, wherein the nuclear localization sequence is fused (directly or indirectly) to the C-terminus of the CRISPR-Cas effector protein.

39. A fusion protein having an amino acid sequence that contains at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with one or more of SEQ ID NO: 243-256, 288-292, and / or 308.

40. The fusion protein according to any one of claims 1 to 39, wherein the fusion protein has at least one improvement (e.g., increased activity and / or increased binding) compared to a protein having an amino acid sequence having any one of SEQ ID NO: 39-65, 70-72, 88-189, 280-287 or 299-301.

41. A complex comprising: The fusion protein according to any one of claims 1 to 40; and Guide nucleic acid.

42. The complex of claim 41, wherein the guide nucleic acid is an extended guide nucleic acid or further comprises an extended guide nucleic acid.

43. The complex according to claim 41 or 42, wherein it is contained in an expression cassette, optionally wherein the expression cassette is contained in a carrier.

44. A nucleic acid encoding a fusion protein according to any one of claims 1 to 40, wherein optionally the nucleic acid is codon-optimized for expression in an organism (e.g., a plant or a mammal).

45. An expression cassette, codon-optimized for expression in an organism, the expression cassette comprising: Polynucleotides encoding promoter sequences, and The polynucleotide encoding the fusion protein according to any one of claims 1 to 40 Optionally, the polynucleotide encoding the fusion protein is codon-optimized for expression in the organism.

46. ​​The expression cassette of claim 45, wherein the organism is an animal, plant, fungus, archaeon, or bacterium.

47. A method for modifying a target nucleic acid in a cell, the method comprising: The expression cassette according to claim 45 or 46 is introduced into the cell to modify the target nucleic acid in the cell.

48. The method of claim 47, wherein the cell is a plant cell (e.g., a plant cell including a cell wall), and the method further comprises regenerating the plant cell containing the modified target nucleic acid to produce a plant containing the modified target nucleic acid.

49. The method according to any one of claims 47 or 48, wherein the introduction is performed at a temperature of about 20°C to about 42°C.

50. A method for producing a fusion protein, the method comprising: Cultivating cells or cell populations transformed with nucleic acids encoding the fusion protein according to any one of claims 1 to 40; and The polypeptide is separated (e.g., by dialysis, centrifugation, column purification, etc.) to produce the polypeptide.

51. A method for modifying a target nucleic acid, the method comprising: Contact the target nucleic acid with the following: The fusion protein according to any one of claims 1 to 40; and Guide nucleic acids, thereby modifying the target nucleic acid.

52. The method of claim 51, wherein the target nucleic acid is present in a cell (e.g., a eukaryotic cell), optionally wherein the target nucleic acid is present in a plant cell or a human cell.

53. The method of claim 51 or 52, further comprising introducing a nucleic acid molecule encoding the fusion protein into the cell, and generating the fusion protein in the cell.

Images