Novel CRISPR-Cas system for genome editing

Abstract

Compositions and methods for genome modification of target sequences in the genome of cells using novel Cas endonucleases are provided. The methods and compositions employ a guiding polynucleotide / endonuclease system to provide an effective system for modifying or changing a target sequence in the genome of a cell or an organism. Novel effector and endonuclease systems and elements comprising such systems are also provided, such as guiding polynucleotide / endonuclease systems comprising endonucleases. Compositions and methods for guiding polynucleotide / endonuclease systems are also provided, the systems comprising at least one endonuclease, the at least one endonuclease being optionally covalently or non-covalently attached to at least one additional protein subunit or assembled with at least one additional protein subunit; and compositions and methods for directly delivering endonucleases as ribonucleotide proteins are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 779989, filed December 14, 2018, U.S. Provisional Application No. 62 / 794427, filed January 18, 2019, U.S. Provisional Application No. 62 / 819409, filed March 15, 2019, U.S. Provisional Application No. 62 / 852788, filed May 24, 2019, and U.S. Provisional Application No. 62 / 913492, filed October 10, 2019, all of which are incorporated herein by reference in their entireties.

[0003] References to electronically submitted sequence listings

[0004] An official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing (whose file name is RTS21920B_SequenceListing_ST25.txt, created on December 9, 2019, and is 714,386 bytes in size) and is submitted simultaneously with the specification. The sequence listing contained in the ASCII formatted document is part of the specification and is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure relates to the field of molecular biology, and in particular to compositions of novel RNA-guided Cas endonuclease systems, as well as compositions and methods for editing or modifying cellular genomes. Background Art

[0006] Recombinant DNA technology makes it possible to insert DNA sequences at target genomic locations and / or modify specific endogenous chromosomal sequences. Site-specific integration techniques using site-specific recombination systems and other types of recombination techniques have been used to produce targeted insertions of target genes in various organisms. Genome editing technologies such as designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or homing meganucleases can be used to produce targeted genomic perturbations, but these systems tend to have low specificity and use designed nucleases that need to be redesigned for each target site, making them expensive and time-consuming to prepare.

[0007] A newer technology that exploits the adaptive immune system of archaea or bacteria, called CRISPR (clustered regularly interspaced short palindromic repeats ( C lustered R egularly I Interspaced S horrP alindromic R epeats)), which contain different domains of effector proteins that encompass multiple activities (DNA recognition, binding and optionally cleavage).

[0008] Although some of these systems have been identified and characterized, there remains a need to identify novel effectors and systems, as well as demonstrate activity in eukaryotic organisms, particularly animals and plants, to achieve editing of endogenous and previously introduced heterologous polynucleotides.

[0009] Described herein are novel Cas endonucleases "Cas-α", exemplary proteins, and methods and compositions for their use. Summary of the invention

[0010] Disclosed herein are novel Cas endonucleases and methods of using the same. These novel Cas-α class endonucleases are capable of targeting and cleaving double-stranded DNA in a PAM-dependent manner under the guidance of a guide polynucleotide, as demonstrated in prokaryotes (Escherichia coli) and three different eukaryotic kingdoms (plants, animals, and fungi).

[0011] In one aspect, a synthetic composition is provided, comprising a CRISPR-Cas endonuclease comprising at least one zinc finger-like domain, at least one bridge-helix-like domain, a trisection RuvC domain (comprising a discontinuous RuvC-I domain, a RuvC-II domain, and a RuvC-III domain), optionally comprising a heterologous polynucleotide.

[0012] In any aspect, in any composition or method, at least one component is provided that has been optimized for expression in a eukaryotic cell, particularly a plant cell, a fungal cell, or an animal cell.

[0013] In one aspect, a synthetic composition is provided, the synthetic composition comprising a polynucleotide encoding a CRISPR-Cas effector protein derived from an organism selected from the group consisting of: Acidibacillus sulfuroxidans, Alicyclobacillus acidoterrestris, Aneurinibacillus danicus, Archaea, Bacillus, Bacillus cereus, Bacillus megaterium, Bacillus pseudomycoides, Bacillus sp., Bacillus thuringiensis, Bacillus toyonensis, Bacillus wiedmannii, Bacteroides plebeius, Bos taurus), Brevibacillus centrosporus, Candidatus Aureabacteria, Candidatus Levybacteria, Candidatus Micrarchaeota, Cellulosilyticum ruminicola, Clostridioides difficile, Clostridium botulinum, Clostridium fallax, Clostridium hiranonis, Clostridium ihumii, Clostridium novyi, Clostridium paraputrificum, Clostridium pasteurianum, Clostridium perfringens, Clostridium sp.), Clostridium tetani, Clostridium ventriculi, Desulfovibrio fructosivorans, Dorealongicatena, Eubacterium siraeum, Flavobacteriumthermophilum, Gallus gallus, Hepatitis D virus, Homo sapiens, Human betaherpesvirus 5, Hydrogenivirga sp., Mus musculus, Parageobacillusthermoglucosidasius, Peptoclostridium sp., Phascolarctobacterium sp., Prevotella copri, Ruminiclostridium hungatei, Ruminococcus albus, Ruminococcus sp.), Saccharomyces cerevisiae, Simian virus 40, Solanum tuberosum, Sulfurihydrogenibium azorense, Syntrophomonas palmitatica, tobacco etch virus, and Zea mays; and heterologous polynucleotides.

[0014] In one aspect, a synthetic composition is provided, comprising: a eukaryotic cell, a heterologous CRISPR-Cas effector; wherein the heterologous CRISPR-Cas effector protein comprises less than 800, 790 to 800, less than 790, 780 to 790, less than 780, 780 to 770, less than 770, 770 to 760, less than 760, 760 to 750, less than 750, 750 to 740, less than 740, 740 to 730, less than 730, 730 to 720, less than 720, 720 to 710, less than 710, 710 to 700, or even less than 700 amino acids, such as less than 700, less than 790, less than 780, less than 750, less than 700, less than 650, less than 600, less than 550, less than 500, less than 450, less than 400, less than 350, or even less than 350 amino acids.

[0015] In one aspect, a synthetic composition is provided, comprising a CRISPR-Cas endonuclease, wherein the CRISPR-Cas endonuclease comprises at least one, at least two, at least three, at least four, at least five, at least six, or seven of the following, numbered relative to the amino acid positions of SEQ ID NO: 17 when aligned with SEQ ID NO: 17: glycine (G) at position 337, glycine (G) at position 341, glutamate (E) at position 430, leucine (L) at position 432, cysteine ​​(C) at position 487, cysteine ​​(C) at position 490, cysteine ​​(C) at position 507, and / or cysteine ​​(C) or histidine (H) at position 512.

[0016] In one aspect, a synthetic composition is provided, comprising a CRISPR-Cas endonuclease, wherein the CRISPR-Cas endonuclease comprises one, two or three of the following motifs: GxxxG, ExL and / or one or more Cx n (C, H) (where n = one or more amino acids).

[0017] In one aspect the synthetic composition comprises a CRISPR-Cas endonuclease, wherein the CRISPR-Cas endonuclease comprises one or more zinc finger motifs.

[0018] In one aspect, a synthetic composition is provided, comprising a polypeptide selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294 , 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344 , 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, and 371, at least 250, 250 to 300, at least 300, 300 to 350, at least 350, 350 to 400, at least 400, or more than 400 consecutive amino acids having at least 50%, 50% to 55%, at least 55%, 55% or more In some embodiments, the present invention relates to a CRISPR-Cas effector protein having a sequence identity of at least 10%, 100%, 100%, 200%, 200%, 300%, 400%, 500%, 600%, 600%, 7 ...

[0019] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 3 00, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355 5, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370 and 371, at least 250, 250 to 500, at least 500, 500 to 600, at least 600, 600 to 700, at least 700, 700 to 750, at least 750, 750 to 800, at least 800, 800 to 850, at least 850, 850 to 900, at least 900, 900 to 950, at least 950, 950 to 1000, at least 1000 or more amino acids of the polypeptide of the group consisting of In some embodiments, the present invention relates to a CRISPR-Cas effector protein having 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity.

[0020] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding a CRISPR-Cas effector protein, wherein the polynucleotide is capable of interacting with a polypeptide selected from the group consisting of SEQ ID NO: 57, 58, 59, 64, 65, 66, 67, 68, 73, 74, 75, 76, 77, 102, 103, 104, 105, 177, 178, 179, 180, 181, 182, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 204, 205, 206 , 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 230, 231, 232, 233, 234, 238, 240, 241, 245, 246, 247, 248, 252, and 253. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2728, 29, 30 or more consecutive nucleotides have at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% In some embodiments, the present invention hybridizes to a polynucleotide having a sequence identity of at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity.

[0021] Any method or composition herein may further comprise a heterologous polynucleotide. The heterologous polynucleotide may be selected from the group consisting of: a non-coding regulatory expression element, such as a promoter, intron, enhancer, or terminator; a donor polynucleotide; a polynucleotide modification template, optionally comprising at least one nucleotide modification compared to a polynucleotide sequence in a cell; a transgene; a guide RNA; a guide DNA; a guide RNA-DNA hybrid; an endonuclease; a nuclear localization signal; and a cellular transit peptide.

[0022] On the one hand, a method for using any composition disclosed herein is provided. In some embodiments, a method for binding a Cas-α endonuclease to a polynucleotide target sequence (e.g., in a cell genome or in vitro) is provided. In some embodiments, the Cas-α endonuclease forms a complex with a guide polynucleotide such as a guide RNA. In some embodiments, the complex recognizes and binds to a polynucleotide at or near the target sequence and optionally produces a nick (one chain) or a break (two chains) in a polynucleotide at or near the target sequence. In some embodiments, the nick or break is repaired by non-homologous end joining (NHEJ). In some embodiments, the nick or break is repaired by homology-directed repair (HDR) or by homologous recombination (HR) using a polynucleotide modified template or donor DNA molecule.

[0023] In any prokaryotic or eukaryotic cell, the novel Cas endonucleases described herein are capable of generating double-strand breaks in or near a target polynucleotide comprising a suitable PAM and are guided to the target polynucleotide by a guide polynucleotide. In some cases, the cell is a plant cell or an animal cell or a fungal cell. In some cases, the plant cell is selected from the group consisting of: maize, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower and tomato.

[0024] DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0025] The present disclosure can be more fully understood from the following detailed description and accompanying drawings and sequence listing, which form a part of this application.

[0026] Figures 1A-1D The complete CRISPR-Cas system is depicted, including all components required for capture and interference. These include genes that together encode all proteins required for capture and integration of spacers (Cas1 and Cas2) and a novel protein (containing the DNA cleavage domain Cas-alpha (α), located in an operator-like structure adjacent to the CRISPR array). In addition, a gene encoding a protein with homology to Cas4 is also included in the locus. Figure 1A The locus architecture of the Cas-α1, Cas-α3, and Cas-α4 systems is depicted. Figure 1B The locus architecture of the Cas-α2 system is depicted. Figure 1C The locus architecture of the Cas-α6 system is depicted. Figure 1D The locus architecture of Cas-α5, 7, 8, 9, 10, and 11 systems is depicted.

[0027] Figure 2A detailed structural examination of the Cas-α protein is shown, depicting distinct differences from previously described class 2 endonucleases. Conserved residues are shown. Key residues involved in DNA cleavage are indicated with asterisks. Numbers correspond to the Cas-α1 protein.

[0028] Figure 3 Methods for detecting double-stranded DNA target recognition and cleavage using cell lysates expressing Cas-α endonuclease are outlined.

[0029] Figures 4A-4E Cleavage of the target polynucleotide by Cas-α1 endonuclease at nucleotide position 21 is shown. Figure 4A Data for the Cas-α1 negative control are shown. Figure 4B Data are shown for Cas-α1 using an entire (complete) CRISPR locus whose CRISPR array is modified to direct cleavage to a target polynucleotide, Figure 4C Data are shown for the Cas-α1 intact locus + when using the T7 promoter to enhance expression, Figure 4D Data for the Cas-α1 minimal locus when expression was enhanced using the T7 promoter are shown, and Figure 4E Data are shown for the response in the absence of Cas-α1 but with the rest of the CRISPR locus when expression is enhanced using the T7 promoter.

[0030] Figure 5A-5B A schematic diagram depicting the orientation of PAM recognition relative to spacer recognition is shown, with one or more guide RNAs designed to base pair with the sense or antisense strand of a T2 target. If one or more guide RNAs designed to base pair with the sense strand results in restoration of PAM preference and generates a cleavage signal, then the prospacer is located on the antisense strand and PAM recognition occurs at the 3′ ( Figure 5A ). In contrast, if one or more guide RNAs are designed to base pair with the antisense strand to generate a PAM preference and cleavage signal, the protospacer is located on the sense strand and PAM recognition occurs in its 5′ direction ( Figure 5B ).

[0031] Figures 6A-6E Cleavage of the target polynucleotide by Cas-α4 endonuclease at nucleotide position 24 is shown. Fig. 6A Data for the Cas-α4 negative control are shown. Figure 6B Data for Cas-α4 plus T2-1 sgRNA are shown. Figure 6C Data for Cas-α4 plus T2-2 sgRNA are shown. Fig.6D Data for Cas-α4 plus T2-1 crRNA / tracrRNA are shown. Fig. 6EData for Cas-α4 plus T2-2 crRNA / tracrRNA are shown.

[0032] Figures 7A-7K Representative Cas-α loci, endonucleases, proteins, guide RNA components, and other sequences identified from a variety of bacterial and archaeal organisms are shown, including: Fig. 7A , 7B , 7E), Candidatus Aureabacteria ( Figure 7C ), various uncultured bacteria ( Fig.7D , 7F ), Geobacter thermoglucosidase ( Figure 7G ), Acidibacillussulfuroxidans( Figure 7H ), Ruminococcus species ( Fig.7I ), Synechotrophomonas palmi Figure 7J novyi ( Figure 7K ).

[0033] Figures 8A-8K Different structural features of representative Cas-α proteins are shown, with the protein sequence shown in bold. The non-bold characters below each amino acid residue represent possible secondary structural features, with C representing nonstructural elements or helices, E representing beta strands, and H representing alpha helices. The zinc finger domain is represented by a dotted box, and the asterisks represent the key amino acid residues involved in zinc ion binding. The RuvC subdomains of the split RuvC domain are depicted by solid boxes. The bridge helix is ​​depicted by a dotted box. The coiled coil is represented by a solid cylinder. The solid plus sign indicates the characteristic key catalytic residues of the RuvC domain motif. Fig. 8A depicts Cas-α1 (SEQ ID NO: 17) from the archaeon Candidatus Micrarchaeota, Figure 8B depicts Cas-α2 from the archaeon Candidatus Micrarchaeota (SEQ ID NO: 18), Figure 8C Depicted is Cas-α3 (SEQ ID NO: 19) from the bacterium Candidatus Aureabacteria, Fig.8D Depicted is Cas-α4 (SEQ ID NO: 20) from uncultured bacteria, Fig. 8E depicts Cas-α5 (SEQ ID NO: 32) from the archaeon Candidatus Micrarchaeota, Figure 8F depicts Cas-α6 (SEQ ID NO: 33) from uncultured bacteria, Figure 8Gdepicts Cas-α7 from Geobacter thermoglucosidase (SEQ ID NO: 34), Figure 8H depicts Cas-α8 from Acidibacillus sulfuroxidans (SEQ ID NO: 35), Figure 8I depicts Cas-α9 from Ruminococcus sp. (SEQ ID NO: 36), Figure 8J Depicted is Cas-α10 from Synechocystis palmi (SEQ ID NO: 37), which has a unique motif of three zinc finger domains, Figure 8K Cas-α11 from Clostridium novyi (SEQ ID NO: 38) is depicted. Whole genome sequencing of an organism containing Cas-α11 showed that the Cas-α locus is the only CRISPR system in this organism.

[0034] Fig.9A Depicted is how the Cas-α protein subunits interact with the hybrid duplex of target DNA and guide RNA. Fig. 9B Figure 3 is a three-dimensional model of the C-terminal half of Cas-α4, showing the regions identified as the helical hairpin / bridge helix region, the RuvC domain, and the zinc finger motif common to Cas proteins.

[0035] Figures 10A-10D Depicted are exemplary expression constructs for use of the Cas-α endonuclease in eukaryotic cells. Fig. 10A is an example of a Cas-α DNA expression construct for human cells. Fig. 10B is an example of a Cas-α DNA expression construct for plant cells. Fig. 10C is an example of a yeast (Saccharomyces cerevisiae) Cas-α DNA expression construct. Fig. 10D is an example of an inducible yeast (Saccharomyces cerevisiae) Cas-α DNA expression construct.

[0036] Figures 11A-11D Examples of eukaryotic optimized Cas-α guide RNA expression constructs are depicted. Fig.11A is an example of a single guide RNA (sgRNA) DNA expression construct for human cells. Fig. 11B is an example of a single guide RNA (sgRNA) DNA expression construct in plant cells. Fig. 11C is an example of a yeast (Saccharomyces cerevisiae) single guide RNA (sgRNA) DNA expression construct. Fig.11D is another example of a single guide RNA (sgRNA) DNA expression construct in plant cells.

[0037] Fig.12 Depicted are examples of engineered genes for recombinant expression and purification of Cas-α endonuclease in E. coli.

[0038] Fig.13 Double-strand break repair mutations from Cas-α endonuclease activity in plant cells are shown. Depicted are mutations generated by Cas-α4 in maize. WT reference is SEQ ID NO: 120, mutation 1 is SEQ ID NO: 121, mutation 2 is SEQ ID NO: 122, mutation 3 is SEQ ID NO: 123, and mutation 4 is SEQ ID NO: 124.

[0039] Figures 14A-14B Double-strand break repair mutations from Cas-α endonuclease activity in animal cells are shown. Fig.14A Indel mutations generated by Cas-α4 RNP electroporation are depicted (VEGFA target 2 mutations 1-5 given as SEQ ID NOs: 127-131, compared to WT reference SEQ ID NO: 126; VEGFA target 3 (mutation given as SEQ ID NO: 133, compared to WT reference SEQ ID NO: 132)). Fig. 14B Depicted are indel mutants of VEGFA target 3 (mutations 1 and 2 given as SEQ ID NOs: 134-135, compared to WT reference SEQ ID NO: 132) generated by lipid transfection of Cas-α4 and sgRNA DNA expression cassettes.

[0040] Figures 15A-15D Cas-α4 double-stranded DNA target cleavage is shown. Fig.15A The supercoiled (SC) plasmid DNA containing a guide RNA target (about 20 bp) immediately 3' (5'-TTTR-3', where R represents A or G bp) of the PAM was completely converted into a linear form (FLL), thereby demonstrating the formation of dsDNA breaks. In addition, the cleavage of the linear DNA resulted in DNA fragments of the expected size, further validating the formation of dsDNA breaks mediated by Cas-α4. Fig. 15B showed that Cas-α4 requires both a PAM and a guide RNA to cleave dsDNA targets. Fig. 15C Cas-α4 was shown to generate a 5′ staggered protruding DNA cleavage site, where cleavage occurred primarily around the position 20–24 bp relative to the PAM sequence. Fig.15D The trans-acting ssDNA enzyme activity of Cas-α4 was shown, which was activated by dsDNA only in the presence of guide RNA.

[0041] Figures 16A-16T Double-stranded DNA target cleavage activity was shown for all Cas-α endonucleases except Cas-α5. Fig.16A is the negative control (-IPTG). Fig. 16B is the negative control (+IPTG). Fig. 16C Cleavage of a double-stranded DNA target by Cas-α2 (-IPTG) at protospacer position 21 is shown. Fig.16D Cleavage of a double-stranded DNA target by Cas-α2 (+IPTG) at protospacer position 21 is shown. Fig.16E It is shown that Cas-α3(-IPTG) does not cleave the double-stranded DNA target. Fig.16F Cleavage of a double-stranded DNA target by Cas-α3 (+IPTG) at protospacer position 21 is shown. Figure 16G It is shown that Cas-α5(-IPTG) does not cleave the double-stranded DNA target. Fig.16H It is shown that Cas-α5(-IPTG) does not cleave the double-stranded DNA target. Fig.16I Cleavage of a double-stranded DNA target by Cas-α6 (-IPTG) is shown. Fig.16J Cleavage of a double-stranded DNA target by Cas-α6 (+IPTG) at protospacer position 24 is shown. Figure 16K Cleavage of a double-stranded DNA target by Cas-α7 (-IPTG) at protospacer position 24 is shown. Figure 16L Cleavage of a double-stranded DNA target by Cas-α7 (+IPTG) at protospacer position 24 is shown. Figure 16M It is shown that Cas-α8(-IPTG) does not cleave the double-stranded DNA target. Fig.16N Cleavage of a double-stranded DNA target by Cas-α8 (+IPTG) at protospacer position 24 is shown. Fig.16O Cleavage of a double-stranded DNA target by Cas-α9 (-IPTG) at protospacer position 24 is shown. Figure 16P Cleavage of a double-stranded DNA target by Cas-α9 (+IPTG) at protospacer position 24 is shown. Figure 16Q Cleavage of a double-stranded DNA target by Cas-α10 (-IPTG) at protospacer position 24 is shown. Figure 16R Cleavage of a double-stranded DNA target by Cas-α10 (+IPTG) at protospacer position 24 is shown. Figure 16S Cleavage of a double-stranded DNA target by Cas-α11 (-IPTG) at protospacer position 24 is shown. Figure 16T Cleavage of a double-stranded DNA target by Cas-α11 (+IPTG) at protospacer position 24 is shown.

[0042] Fig.17A Depicted is a method to assess double-stranded DNA target cleavage by Cas-α in Escherichia coli cells. Figures 17B-17EDouble-stranded DNA target cleavage in E. coli is shown. The "no target" experiment provides a baseline for transformation efficiency in the absence of double-stranded DNA target cleavage. The "target" experiment, PAM+T2, was performed with and without IPTG (0.5 mM) to examine target cleavage under different Cas-α endonuclease and guide RNA expression conditions. Fig. 17B Results for Cas-α2 and Cas-α3 are shown. Fig. 17C Results for Cas-α6 and Cas-α7 are shown. Fig.17D Results for Cas-α8 and Cas-α9 are shown. Fig.17E Results for Cas-α10 and Cas-α11 are shown.

[0043] Figures 18A-18B Depicted are double-strand break repair mutations from Cas-α endonuclease activity in plant cells, used in particle gun experiments to deliver Cas-α10 DNA expression constructs into maize immature embryos. Fig.18A Restoration of targeted deletions generated at or near the nuclease cleavage site of the nptII target site is shown. Fig.18B Restoration of targeted deletions generated at or near the nuclease cleavage site of the ms26 target site is shown.

[0044] Fig.19A Depicted is the experimental design of homology-directed repair in the eukaryotic cell Saccharomyces cerevisiae. An exogenously provided DNA repair template (double-stranded) with homology flanking the Cas-α10 target site is used to introduce one or two premature stop codons (depending on the DNA repair outcome) in the ade2 gene after a Cas-α10-induced double-strand break (DSB). To avoid targeting the repair template, it also contains a T to A change in the PAM region for Cas-α10. Fig.19B showed that the erythroid phenotype indicative of ade2 gene disruption was restored when both the repair template and Cas-α10 and sgRNA expression constructs were transformed, and double-strand breaks were generated by Cas-α endonuclease and repaired with the template (HDR). Fig.19CThe sequencing results of the Cas-α10 ade2 gene target site are shown, confirming that at least one stop codon was introduced in 3 independent red colonies (labeled "1", "2" and "3"). The stop codon was introduced in the antisense frame. SEQ ID NO: 170 The reference DNA sequence from Saccharomyces cerevisiae is given as SEQ ID NO: 170, the repair template DNA is SEQ ID NO: 171, the red colony 1 repair result 1 is SEQID NO: 172, the red colony 1 repair result 2 is SEQID NO: 173, the red colony 2 repair result 1 is SEQID NO: 174, the red colony 3 repair result 1 is SEQID NO: 175, and the red colony 3 repair result 2 is SEQID NO: 176.

[0045] Fig. 20 The phylogenetic relationships between some Cas-α orthologs are shown. Three supergroups (I, II, and III) were identified. Group I includes evolutionary branch 1 (Candidate Archaea and Aureabacteria (Cas1, Cas2, Cas4 usually encoded in the locus)). Group II includes evolutionary branch 2 (Aquabacteria (Sulfurihydrogenibium and Hydrogenivirga genera) and Deltaproteobacteria (Desulfovibrio genus), evolutionary branch 3 (Candidate Archaea (Cas1, Cas2, and Cas4 usually encoded in the locus)), evolutionary branch 4 (Bacteroidetes (Prevotella and Bacteroides genera)), evolutionary branch 5 (Candidate Group III includes clade 7 (Bacillus (Bacillus, Acidibacillus, Aneurinibacillus, Brevibacillus, Parageobacillus, Alicyclobacillus), clade 8 (Negativicutes (Phascolarctobacterium)) and clade 9 (Flavobacteriia (Flavobacterium)). The diamond symbol represents the Cas-α1-11 endonuclease described herein.

[0046] Fig.21AIllustration of the transposase (Tnp)-associated Cas-α CRISPR system. In both cases, Tnp-like proteins are encoded upstream of the Cas-α endonuclease and the CRISPR array. Fig. 21B The Cas-α endonuclease and guide RNA are shown in complex with their target site and a Tnp-like protein that is configured to integrate a DNA payload (dashed circle) within or near the Cas-α double-stranded DNA target site.

[0047] These sequence descriptions and the accompanying sequence listing comply with the rules governing disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 CFR §§1.821 and 1.825. These sequence descriptions contain the three-letter codes for amino acids as defined in 37 CFR §§1.821 and 1.825, which are incorporated herein by reference.

[0048] SEQ ID NO: 1 is the Cas1 encoded in the Cas-α1 locus PRT sequence from the archaeon Candidatus Micrarchaeota.

[0049] SEQ ID NO: 2 is Cas1 encoded in the Cas-α2 locus PRT sequence from the archaeon Candidatus Micrarchaeota.

[0050] SEQ ID NO: 3 is the Cas1 encoded in the Cas-α3 locus PRT sequence from the bacterium Candidatus Aureabacteria.

[0051] SEQ ID NO: 4 is the Cas1 encoded in the PRT sequence of the Cas-α4 locus from an uncultured archaea.

[0052] SEQ ID NO: 5 is the Cas2 encoded in the Cas-α1 locus PRT sequence from the archaeon Candidatus Micrarchaeota.

[0053] SEQ ID NO: 6 is the Cas2 encoded in the Cas-α2 locus PRT sequence from the archaeon Candidatus Micrarchaeota.

[0054] SEQ ID NO: 7 is the Cas2 encoded in the Cas-α3 locus PRT sequence from the bacterium Candidatus Aureabacteria.

[0055] SEQ ID NO: 8 is the Cas2 encoded in the Cas-α4 locus PRT sequence from an uncultured archaea.

[0056] SEQ ID NO: 9 is the Cas4 encoded in the Cas-α1 locus PRT sequence from the archaeon Candidatus Micrarchaeota.

[0057] SEQ ID NO: 10 is the Cas4 encoded in the Cas-α2 locus PRT sequence from the archaeon Candidatus Micrarchaeota.

[0058] SEQ ID NO: 11 is the Cas4 encoded in the Cas-α3 locus PRT sequence from the bacterium Candidatus Aureabacteria.

[0059] SEQ ID NO: 12 is the Cas4 encoded in the Cas-α4 locus PRT sequence from an uncultured archaea.

[0060] SEQ ID NO: 13 is the DNA sequence of the Cas-α1 endonuclease gene from the archaeon Candidatus Micrarchaeota.

[0061] SEQ ID NO: 14 is the DNA sequence of the Cas-α2 endonuclease gene from the archaeon Candidatus Micrarchaeota.

[0062] SEQ ID NO: 15 is the DNA sequence of the Cas-α3 endonuclease gene from the bacterium Candidatus Aureabacteria.

[0063] SEQ ID NO: 16 is the DNA sequence of the Cas-α4 endonuclease gene from an uncultured archaea.

[0064] SEQ ID NO: 17 is the Cas-α1 endonuclease (Cas14b4) PRT sequence from the archaeon Candidatus Mictratrchaeota.

[0065] SEQ ID NO: 18 is the Cas-α2 endonuclease PRT sequence from the archaeon Candidatus Micrarchaeota.

[0066] SEQ ID NO: 19 is the Cas-α3 endonuclease PRT sequence from the bacterium Candidatus Aureabacteria.

[0067] SEQ ID NO: 20 is the Cas-α4 endonuclease (Cas14a1) PRT sequence from an uncultured archaea.

[0068] SEQ ID NO: 21 is the Cas-α1 locus DNA sequence from the archaeon Candidatus Micrarchaeota.

[0069] SEQ ID NO: 22 is the Cas-α2 locus DNA sequence from the archaeon Candidatus Micrarchaeota.

[0070] SEQ ID NO: 23 is the Cas-α3 locus DNA sequence from the bacterium Candidatus Aureabacteria.

[0071] SEQ ID NO: 24 is the Cas-α4 locus DNA sequence from an uncultured archaea.

[0072] SEQ ID NO: 25 is the DNA sequence of the Cas-α5 endonuclease gene from the archaeon Candidatus Micrarchaeota.

[0073] SEQ ID NO: 26 is the DNA sequence of the Cas-α6 endonuclease gene from an uncultured archaea.

[0074] SEQ ID NO: 27 is the DNA sequence of the Cas-α7 endonuclease gene from Parageobacillus thermoglucosidasius.

[0075] SEQ ID NO: 28 is the DNA sequence of the Cas-α8 endonuclease gene from Acidibacillus sulfuroxidans.

[0076] SEQ ID NO: 29 is the DNA sequence of the Cas-α9 endonuclease gene from Ruminococcus sp.

[0077] SEQ ID NO: 30 is the DNA sequence of the Cas-α10 endonuclease gene from Synechocystis palmi.

[0078] SEQ ID NO: 31 is the DNA sequence of the Cas-α11 endonuclease gene from Clostridium novyi.

[0079] SEQ ID NO: 32 is the Cas-α5 endonuclease PRT sequence from the archaeon Candidatus Micrarchaeota.

[0080] SEQ ID NO: 33 is the Cas-α6 endonuclease PRT sequence from an uncultured archaea.

[0081] SEQ ID NO: 34 is the Cas-α7 endonuclease PRT sequence from Geobacter thermoglucosidase.

[0082] SEQ ID NO: 35 is the Cas-α8 endonuclease PRT sequence from Acidibacillus sulfuroxidans.

[0083] SEQ ID NO: 36 is the Cas-α9 endonuclease PRT sequence from Ruminococcus sp.

[0084] SEQ ID NO: 37 is the Cas-α10 endonuclease PRT sequence from Synechomonas palmi.

[0085] SEQ ID NO: 38 is the Cas-α11 endonuclease PRT sequence from Clostridium novyi.

[0086] SEQ ID NO: 39 is the Cas-α5 locus DNA sequence from the archaeon Candidatus Micrarchaeota.

[0087] SEQ ID NO: 40 is the Cas-α6 locus DNA sequence from an uncultured archaea.

[0088] SEQ ID NO: 41 is the Cas-α7 locus DNA sequence from Geobacter thermoglucosidase.

[0089] SEQ ID NO: 42 is the Cas-α8 locus DNA sequence from Acidibacillus sulfuroxidans.

[0090] SEQ ID NO: 43 is the Cas-α9 locus DNA sequence from Ruminococcus sp.

[0091] SEQ ID NO: 44 is the Cas-α10 locus DNA sequence from Synechocystis palmi.

[0092] SEQ ID NO: 45 is the Cas-α11 locus DNA sequence from Clostridium novyi.

[0093] SEQ ID NO: 46 is the Cas-α1 repeat consensus DNA sequence from the archaeon Candidatus Micrarchaeota.

[0094] SEQ ID NO: 47 is the Cas-α2 repeat consensus DNA sequence from the archaeon Candidatus Micrarchaeota.

[0095] SEQ ID NO: 48 is the Cas-α3 repeat consensus DNA sequence from the bacterium Candidatus Aureabacteria.

[0096] SEQ ID NO: 49 is the Cas-α4 repeat consensus DNA sequence from an uncultured archaea.

[0097] SEQ ID NO: 50 is the Cas-α5 repeat consensus DNA sequence from the archaeon Candidatus Micrarchaeota.

[0098] SEQ ID NO: 51 is the Cas-α6 repeat consensus DNA sequence from an uncultured archaea.

[0099] SEQ ID NO: 52 is the Cas-α7 repeat consensus DNA sequence from Geobacter thermoglucosidase.

[0100] SEQ ID NO: 53 is the Cas-α8 repeat consensus DNA sequence from Acidibacillus sulfuroxidans.

[0101] SEQ ID NO: 54 is the Cas-α9 repeat consensus DNA sequence from Ruminococcus sp.

[0102] SEQ ID NO: 55 is the Cas-α10 repeat consensus DNA sequence from Synechotropha palmi.

[0103] SEQ ID NO: 56 is the Cas-α11 repeat consensus DNA sequence from Clostridium novyi.

[0104] SEQ ID NO: 57 is an artificial Cas-α1 crRNA (wherein N represents any nucleotide) RNA sequence.

[0105] SEQ ID NO: 58 is an artificial Cas-α2 crRNA (wherein N represents any nucleotide) RNA sequence.

[0106] SEQ ID NO: 59 is an artificial Cas-α4 crRNA (wherein N represents any nucleotide) RNA sequence.

[0107] SEQ ID NO: 60 is the Cas-α1 tracrRNA version 1 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0108] SEQ ID NO: 61 is the Cas-α1 tracrRNA version 2 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0109] SEQ ID NO: 62 is the Cas-α1 tracrRNA version 3 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0110] SEQ ID NO: 63 is the Cas-α1 tracrRNA version 4 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0111] SEQ ID NO: 64 is the Cas-α2 tracrRNA version 1 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0112] SEQ ID NO: 65 is the Cas-α2 tracrRNA version 2 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0113] SEQ ID NO: 66 is the Cas-α2 tracrRNA version 3 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0114] SEQ ID NO: 67 is the Cas-α2 tracrRNA version 4 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0115] SEQ ID NO: 68 is the Cas-α4 tracrRNA version 1 RNA sequence from an uncultured archaea.

[0116] SEQ ID NO: 69 is the artificial Cas-α1 sgRNA version 1 RNA sequence.

[0117] SEQ ID NO: 70 is the artificial Cas-α1 sgRNA version 2 RNA sequence.

[0118] SEQ ID NO: 71 is the artificial Cas-α1 sgRNA version 3 RNA sequence.

[0119] SEQ ID NO: 72 is the artificial Cas-α1 sgRNA version 4 RNA sequence.

[0120] SEQ ID NO: 73 is the artificial Cas-α2 sgRNA version 1 RNA sequence.

[0121] SEQ ID NO: 74 is the artificial Cas-α2 sgRNA version 2 RNA sequence.

[0122] SEQ ID NO: 75 is the artificial Cas-α2 sgRNA version 3 RNA sequence.

[0123] SEQ ID NO: 76 is the artificial Cas-α2 sgRNA version 4 RNA sequence.

[0124] SEQ ID NO: 77 is the artificial Cas-α4 sgRNA version 1 RNA sequence.

[0125] SEQ ID NO: 78 is an artificial T2 spacer DNA sequence.

[0126] SEQ ID NO: 79 is the complete Cas-α1 locus engineered to target an artificially derived T2 DNA sequence.

[0127] SEQ ID NO: 80 is a minimized Cas-α1 locus engineered to target an artificially derived T2 DNA sequence.

[0128] SEQ ID NO: 81 is an artificial 10X histidine tag PRT sequence.

[0129] SEQ ID NO: 82 is an artificial 6X histidine tag PRT sequence.

[0130] SEQ ID NO: 83 is the sequence of an artificial maltose binding protein tag PRT.

[0131] SEQ ID NO: 84 is the tobacco etch virus cleavage site PRT sequence from tobacco etch virus.

[0132] SEQ ID NO: 85 is an artificial A1 oligonucleotide DNA sequence.

[0133] SEQ ID NO: 86 is an artificial A2 oligonucleotide DNA sequence.

[0134] SEQ ID NO: 87 is an artificial R0 oligonucleotide DNA sequence.

[0135] SEQ ID NO: 88 is an artificial C0 oligonucleotide DNA sequence.

[0136] SEQ ID NO: 89 is an artificial F1 oligonucleotide DNA sequence.

[0137] SEQ ID NO: 90 is an artificial R1 oligonucleotide DNA sequence.

[0138] SEQ ID NO: 91 is the bridge amplification portion from the artificial F1 oligonucleotide DNA sequence.

[0139] SEQ ID NO: 92 is the bridge amplification portion from the artificial R1 oligonucleotide DNA sequence.

[0140] SEQ ID NO: 93 is an artificial F2 oligonucleotide DNA sequence.

[0141] SEQ ID NO: 94 is an artificial R2 oligonucleotide DNA sequence.

[0142] SEQ ID NO: 95 is an artificial C1 oligonucleotide DNA sequence.

[0143] SEQ ID NO: 96 is the sequence resulting from cleavage at position 21 of an artificial target DNA sequence and adaptor ligation.

[0144] SEQ ID NO: 97 is the adaptor portion from the artificial DNA sequence of SEQ ID NO. 96.

[0145] SEQ ID NO: 98 is the target portion from the artificial DNA sequence of SEQ ID NO. 96.

[0146] SEQ ID NO: 99 is the sequence 5' from the artificial PAM DNA sequence.

[0147] SEQ ID NO: 100 is a target DNA sequence derived from an artificial fixed double-stranded DNA.

[0148] SEQ ID NO: 101 is a DNA sequence derived from an artificial T2 target sequence.

[0149] SEQ ID NO: 102 is an artificial Cas-α4 T2-1 sgRNA RNA sequence.

[0150] SEQ ID NO: 103 is an artificial Cas-α4 T2-2sgRNA RNA sequence.

[0151] SEQ ID NO: 104 is an artificial Cas-α4 T2-1 crRNA RNA sequence.

[0152] SEQ ID NO: 105 is an artificial Cas-α4 T2-2crRNA RNA sequence.

[0153] SEQ ID NO: 106 is the ST-LS1 intron 2 DNA sequence from potato.

[0154] SEQ ID NO: 107 is the SV40 NLS PRT sequence from Simian Virus 40.

[0155] SEQ ID NO: 108 is the Nuc NLS PRT sequence from Mus musculus.

[0156] SEQ ID NO: 109 is the maize UBI promoter DNA sequence from maize.

[0157] SEQ ID NO: 110 is the chicken β-actin promoter DNA sequence from Gallus gallus.

[0158] SEQ ID NO: 111 is the CMV enhancer DNA sequence from human beta-herpesvirus 5.

[0159] SEQ ID NO: 112 is the Zea mays UBI 5 primer untranslated region DNA sequence from Zea mays.

[0160] SEQ ID NO: 113 is the Zea mays UBI intron 1 DNA sequence from Zea mays.

[0161] SEQ ID NO: 114 is an artificial hybrid intron DNA sequence.

[0162] SEQ ID NO: 115 is the maize U6 polymerase III promoter DNA sequence from Zea mays.

[0163] SEQ ID NO: 116 is the human U6 polymerase III promoter DNA sequence from Homo sapiens.

[0164] SEQ ID NO: 117 is an artificial Strep II tag PRT sequence.

[0165] SEQ ID NO: 118 is the bGH poly(A) terminator DNA sequence from bovine.

[0166] SEQ ID NO: 119 is the potato proteinase inhibitor II (Pin II) terminator DNA sequence from potato.

[0167] SEQ ID NO: 120 is the Zea mays Wt reference (no ligule targets 2 and 3) DNA sequence from Zea mays.

[0168] SEQ ID NO: 121 is the Mutation 1 (No Ligule Target 2 and 3 - DNA Exp.) DNA sequence from Zea mays.

[0169] SEQ ID NO: 122 is the Mutation 2 (Linguole Target 2 and 3-DNA Exp.) DNA sequence from Zea mays.

[0170] SEQ ID NO: 123 is the Mutation 3 (No Ligule Target 2 and 3 - DNA Exp.) DNA sequence from Zea mays.

[0171] SEQ ID NO: 124 is the Mutation 4 (No Ligule Target 2 and 3 - DNA Exp.) DNA sequence from Zea mays.

[0172] SEQ ID NO: 125 is the Mutation 5 (No Ligule Target 2 and 3 - DNA Exp.) DNA sequence from Zea mays.

[0173] SEQ ID NO: 126 is the HEK293 Wt reference (VEGFA target 2) DNA sequence from Homo sapiens.

[0174] SEQ ID NO: 127 is the Mutation 1 (VEGFA target 2-RNP) DNA sequence from Homo sapiens.

[0175] SEQ ID NO: 128 is the Mutation 2 (VEGFA target 2-RNP) DNA sequence from Homo sapiens.

[0176] SEQ ID NO: 129 is the mutation 3 (VEGFA target 2-RNP) DNA sequence from Homo sapiens.

[0177] SEQ ID NO: 130 is the mutation 4 (VEGFA target 2-RNP) DNA sequence from Homo sapiens.

[0178] SEQ ID NO: 131 is the mutation 5 (VEGFA target 2-RNP) DNA sequence from Homo sapiens.

[0179] SEQ ID NO: 132 is the HEK293 Wt reference (VEGFA target 3) DNA sequence from Homo sapiens.

[0180] SEQ ID NO: 133 is the Mutation 1 (VEGFA target 3-RNP) DNA sequence from Homo sapiens.

[0181] SEQ ID NO: 134 is the Mutation 1 (VEGFA Target 3 -DNA Exp) DNA sequence from Homo sapiens.

[0182] SEQ ID NO: 135 is the Mutation 2 (VEGFA Target 3 -DNA Exp) DNA sequence from Homo sapiens.

[0183] SEQ ID NO: 136 is the ROX3 promoter DNA sequence from Saccharomyces cerevisiae.

[0184] SEQ ID NO: 137 is the GAL promoter DNA sequence from Saccharomyces cerevisiae.

[0185] SEQ ID NO: 138 is a DNA sequence from an artificial HH ribozyme (wherein N represents a nucleotide complementary to the 6 nucleotides at the 3' end of the ribozyme).

[0186] SEQ ID NO: 139 is the HDV ribozyme DNA sequence from hepatitis delta virus.

[0187] SEQ ID NO: 140 is the SNR52 promoter DNA sequence from Saccharomyces cerevisiae.

[0188] SEQ ID NO: 141 is the SUP4 terminator DNA sequence from Saccharomyces cerevisiae.

[0189] SEQ ID NO: 142 is from artificial Fig. 15C Top sequence DNA sequence.

[0190] SEQ ID NO: 143 is from artificial Fig. 15C Bottom sequence DNA sequence.

[0191] SEQ ID NO: 144 is from Zea mays Fig.18A Reference DNA sequence.

[0192] SEQ ID NO: 145 is the Mutation 1 DNA sequence from Zea mays.

[0193] SEQ ID NO: 146 is the Mutant 2 DNA sequence from Zea mays.

[0194] SEQ ID NO: 147 is the mutant 3 DNA sequence from Zea mays.

[0195] SEQ ID NO: 148 is the Mutant 4 DNA sequence from Zea mays.

[0196] SEQ ID NO: 149 is the mutation 5 DNA sequence from Zea mays.

[0197] SEQ ID NO: 150 is the mutation 6 DNA sequence from Zea mays.

[0198] SEQ ID NO: 151 is the mutation 7 DNA sequence from Zea mays.

[0199] SEQ ID NO: 152 is the Mutation 8 DNA sequence from Zea mays.

[0200] SEQ ID NO: 153 is the Mutation 9 DNA sequence from Zea mays.

[0201] SEQ ID NO: 154 is the mutant 10 DNA sequence from Zea mays.

[0202] SEQ ID NO: 155 is the mutant 11 DNA sequence from Zea mays.

[0203] SEQ ID NO: 156 is the mutant 12 DNA sequence from Zea mays.

[0204] SEQ ID NO: 157 is the mutation 13 DNA sequence from Zea mays.

[0205] SEQ ID NO: 158 is the mutant 14 DNA sequence from Zea mays.

[0206] SEQ ID NO: 159 is the mutation 15 DNA sequence from Zea mays.

[0207] SEQ ID NO: 160 is the mutation 16 DNA sequence from Zea mays.

[0208] SEQ ID NO: 161 is the mutation 17 DNA sequence from Zea mays.

[0209] SEQ ID NO: 162 is the mutation 18 DNA sequence from Zea mays.

[0210] SEQ ID NO: 163 is the mutation 19 DNA sequence from Zea mays.

[0211] SEQ ID NO: 164 is from Zea mays Fig.18B Reference DNA sequence.

[0212] SEQ ID NO: 165 is the Mutation 1 DNA sequence from Zea mays.

[0213] SEQ ID NO: 166 is the Mutant 2 DNA sequence from Zea mays.

[0214] SEQ ID NO: 167 is the mutant 3 DNA sequence from Zea mays.

[0215] SEQ ID NO: 168 is the Mutant 4 DNA sequence from Zea mays.

[0216] SEQ ID NO: 169 is the mutation 5 DNA sequence from Zea mays.

[0217] SEQ ID NO: 170 is from Saccharomyces cerevisiae Fig.19C Reference DNA sequence.

[0218] SEQ ID NO: 171 is an artificial repair template DNA sequence.

[0219] SEQ ID NO: 172 is the repair result 1 DNA sequence from Saccharomyces cerevisiae.

[0220] SEQ ID NO: 173 is the repair result 2 DNA sequence from Saccharomyces cerevisiae.

[0221] SEQ ID NO: 174 is the repair result 1 DNA sequence from Saccharomyces cerevisiae.

[0222] SEQ ID NO: 175 is the repair result 1 DNA sequence from Saccharomyces cerevisiae.

[0223] SEQ ID NO: 176 is the repair result 2 DNA sequence from Saccharomyces cerevisiae.

[0224] SEQ ID NO: 177 is an artificial Cas-α3 crRNA (wherein N represents any nucleotide) RNA sequence.

[0225] SEQ ID NO: 178 is an artificial Cas-α5 crRNA (wherein N represents any nucleotide) RNA sequence.

[0226] SEQ ID NO: 179 is an artificial Cas-α6 crRNA (wherein N represents any nucleotide) RNA sequence.

[0227] SEQ ID NO: 180 is an artificial Cas-α7 crRNA (wherein N represents any nucleotide) RNA sequence.

[0228] SEQ ID NO: 181 is an artificial Cas-α8 crRNA (wherein N represents any nucleotide) RNA sequence.

[0229] SEQ ID NO: 182 is an artificial Cas-α9 crRNA (wherein N represents any nucleotide) RNA sequence.

[0230] SEQ ID NO: 183 is an artificial Cas-α10 crRNA (wherein N represents any nucleotide) RNA sequence.

[0231] SEQ ID NO: 184 is an artificial Cas-α11 crRNA (wherein N represents any nucleotide) RNA sequence.

[0232] SEQ ID NO: 185 is the Cas-α2 tracrRNA version 5 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0233] SEQ ID NO: 186 is the Cas-α2 tracrRNA version 6 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0234] SEQ ID NO: 187 is the Cas-α2 tracrRNA version 7 RNA sequence from the archaeon Candidatus Micrarchaeota.

[0235] SEQ ID NO: 188 is the Cas-α6 tracrRNA version 1 RNA sequence from an uncultured archaea.

[0236] SEQ ID NO: 189 is the Cas-α6 tracrRNA version 2 RNA sequence from an uncultured archaea.

[0237] SEQ ID NO: 190 is the Cas-α6 tracrRNA version 3 RNA sequence from an uncultured archaea.

[0238] SEQ ID NO: 191 is the Cas-α6 tracrRNA version 4 RNA sequence from an uncultured archaea.

[0239] SEQ ID NO: 192 is the Cas-α7 tracrRNA version 1 RNA sequence from Geobacter thermoglucosidase.

[0240] SEQ ID NO: 193 is the Cas-α7 tracrRNA version 2 RNA sequence from Geobacter thermoglucosidase.

[0241] SEQ ID NO: 194 is the Cas-α8 tracrRNA version 1 RNA sequence from Acidibacillus sulfuroxidans.

[0242] SEQ ID NO: 195 is the Cas-α8 tracrRNA version 2 RNA sequence from Acidibacillus sulfuroxidans.

[0243] SEQ ID NO: 196 is the Cas-α8 tracrRNA version 3 RNA sequence from Acidibacillus sulfuroxidans.

[0244] SEQ ID NO: 197 is the Cas-α9 tracrRNA version 1 RNA sequence from Ruminococcus sp.

[0245] SEQ ID NO: 198 is the Cas-α9 tracrRNA version 2 RNA sequence from Ruminococcus sp.

[0246] SEQ ID NO: 199 is the Cas-α10 tracrRNA version 1 RNA sequence from Synechotropha palmi.

[0247] SEQ ID NO: 200 is the Cas-α10 tracrRNA version 2 RNA sequence from Synechotropha palmi.

[0248] SEQ ID NO: 201 is the Cas-α10 tracrRNA version 3 RNA sequence from Synechotropha palmi.

[0249] SEQ ID NO: 202 is the Cas-α10 tracrRNA version 4 RNA sequence from Synechotropha palmi.

[0250] SEQ ID NO: 203 is the Cas-α10 tracrRNA version 5 RNA sequence from Synechotropha palmi.

[0251] SEQ ID NO: 204 is the Cas-α11 tracrRNA version 1 RNA sequence from Clostridium novyi.

[0252] SEQ ID NO: 205 is the Cas-α11 tracrRNA version 2 RNA sequence from Clostridium novyi.

[0253] SEQ ID NO: 206 is the Cas-α11 tracrRNA version 3 RNA sequence from Clostridium novyi.

[0254] SEQ ID NO: 207 is the Cas-α11 tracrRNA version 4 RNA sequence from Clostridium novyi.

[0255] SEQ ID NO: 208 is the artificial Cas-α2 sgRNA version 5 RNA sequence.

[0256] SEQ ID NO: 209 is the artificial Cas-α2 sgRNA version 6 RNA sequence.

[0257] SEQ ID NO: 210 is the artificial Cas-α2 sgRNA version 7 RNA sequence.

[0258] SEQ ID NO: 211 is the artificial Cas-α6 sgRNA version 1 RNA sequence.

[0259] SEQ ID NO: 212 is the artificial Cas-α6 sgRNA version 2 RNA sequence.

[0260] SEQ ID NO: 213 is the artificial Cas-α6 sgRNA version 3 RNA sequence.

[0261] SEQ ID NO: 214 is the artificial Cas-α6 sgRNA version 4 RNA sequence.

[0262] SEQ ID NO: 215 is the artificial Cas-α7 sgRNA version 1 RNA sequence.

[0263] SEQ ID NO: 216 is the artificial Cas-α7 sgRNA version 2 RNA sequence.

[0264] SEQ ID NO: 217 is the artificial Cas-α7 sgRNA version 3 RNA sequence.

[0265] SEQ ID NO: 218 is the artificial Cas-α8 sgRNA version 1 RNA sequence.

[0266] SEQ ID NO: 219 is the artificial Cas-α8 sgRNA version 2 RNA sequence.

[0267] SEQ ID NO: 220 is the artificial Cas-α8 sgRNA version 3 RNA sequence.

[0268] SEQ ID NO: 221 is the artificial Cas-α8 sgRNA version 4 RNA sequence.

[0269] SEQ ID NO: 222 is the artificial Cas-α9 sgRNA version 1 RNA sequence.

[0270] SEQ ID NO: 223 is the artificial Cas-α9 sgRNA version 2 RNA sequence.

[0271] SEQ ID NO: 224 is an artificial Cas-α9 sgRNA version 3 RNA sequence.

[0272] SEQ ID NO: 225 is the artificial Cas-α10 sgRNA version 1 RNA sequence.

[0273] SEQ ID NO: 226 is an artificial Cas-α10 sgRNA version 2 RNA sequence.

[0274] SEQ ID NO: 227 is the artificial Cas-α10 sgRNA version 3 RNA sequence.

[0275] SEQ ID NO: 228 is the artificial Cas-α10 sgRNA version 4 RNA sequence.

[0276] SEQ ID NO: 229 is the artificial Cas-α10 sgRNA version 5 RNA sequence.

[0277] SEQ ID NO: 230 is the artificial Cas-α11 sgRNA version 1 RNA sequence.

[0278] SEQ ID NO: 231 is the artificial Cas-α11 sgRNA version 2 RNA sequence.

[0279] SEQ ID NO: 232 is the artificial Cas-α11 sgRNA version 3 RNA sequence.

[0280] SEQ ID NO: 233 is the artificial Cas-α11 sgRNA version 4 RNA sequence.

[0281] SEQ ID NO: 234 is the artificial Cas-α11 sgRNA version 5 RNA sequence.

[0282] SEQ ID NO: 235 is the DNA sequence of the artificial Cas-α4 maize codon-optimized gene.

[0283] SEQ ID NO: 236 is the DNA sequence of the artificial Cas-α10 maize codon-optimized gene.

[0284] SEQ ID NO: 237 is the codon-optimized gene DNA sequence of Cas-α10 from Saccharomyces cerevisiae.

[0285] SEQ ID NO: 238 is an artificial Cas-α4 sgRNA backbone RNA sequence.

[0286] SEQ ID NO: 239 is an artificial Cas-α10 sgRNA backbone RNA sequence.

[0287] SEQ ID NO: 240 is the sgRNA target sequence RNA sequence from the artificial Cas-α4 liguleless 2.

[0288] SEQ ID NO: 241 is the sgRNA target sequence RNA sequence from the artificial Cas-α4 liguleless 3.

[0289] SEQ ID NO: 242 is the RNA sequence of the artificial Cas-α10 nptII sgRNA target sequence.

[0290] SEQ ID NO: 243 is the RNA sequence of the artificial Cas-α10 ms26 sgRNA target sequence.

[0291] SEQ ID NO: 244 is an artificial Cas-α10 ade2 sgRNA target sequence RNA sequence.

[0292] SEQ ID NO: 245 is the RNA sequence of the artificial Cas-α4 VEGFA 2sgRNA target sequence.

[0293] SEQ ID NO: 246 is the RNA sequence of the artificial Cas-α4 VEGFA 3sgRNA target sequence.

[0294] SEQ ID NO: 247 is an artificial Cas-α4 sgRNA targeting the leafless tongue 2 RNA sequence.

[0295] SEQ ID NO: 248 is an artificial Cas-α4 sgRNA targeting the leafless tongue 3 RNA sequence.

[0296] SEQ ID NO: 249 is an artificial Cas-α10 sgRNA targeting nptII RNA sequence.

[0297] SEQ ID NO: 250 is an artificial Cas-α10 sgRNA targeting ms26 RNA sequence.

[0298] SEQ ID NO: 251 is an artificial Cas-α10 sgRNA targeting ade2 RNA sequence.

[0299] SEQ ID NO: 252 is an artificial Cas-α4 sgRNA targeting VEGFA 2 RNA sequence.

[0300] SEQ ID NO: 253 is an artificial Cas-α4 sgRNA targeting VEGFA 3 RNA sequence.

[0301] SEQ ID NO: 254 is the Cas-α 12 endonuclease PRT sequence from Clostridium difficile.

[0302] SEQ ID NO: 255 is the Cas-α13 endonuclease PRT sequence from Clostridium purulentum.

[0303] SEQ ID NO: 256 is the Cas-α14 endonuclease PRT sequence from Clostridium novyi.

[0304] SEQ ID NO: 257 is the Cas-α15 endonuclease PRT sequence from Ruminococcus albus.

[0305] SEQ ID NO: 258 is the Cas-α16 endonuclease PRT sequence from Clostridium hiranonis.

[0306] SEQ ID NO: 259 is the Cas-α17 endonuclease PRT sequence from Clostridiidium ihumnii.

[0307] SEQ ID NO: 260 is the Cas-α18 endonuclease PRT sequence from CellulOsilyticum ruminicicola.

[0308] SEQ ID NO: 261 is the Cas-α19 endonuclease PRT sequence from Eubacterium iners.

[0309] SEQ ID NO: 262 is the Cas-α20 endonuclease PRT sequence from Clostridium botulinum.

[0310] SEQ ID NO: 263 is the Cas-α21 endonuclease PRT sequence from Clostridium botulinum.

[0311] SEQ ID NO: 264 is the Cas-α22 endonuclease PRT sequence from Clostridium ruminalis.

[0312] SEQ ID NO: 265 is the Cas-α23 endonuclease PRT sequence from Desulfovibrio fructosporum.

[0313] SEQ ID NO: 266 is the Cas-α24 endonuclease PRT sequence from Bacillus harvestmenis.

[0314] SEQ ID NO: 267 is the Cas-α25 endonuclease PRT sequence from Clostridium purulentum.

[0315] SEQ ID NO: 268 is the Cas-α26 endonuclease PRT sequence from Clostridium gastricis.

[0316] SEQ ID NO: 269 is the Cas-α27 endonuclease PRT sequence from Ruminococcus sp.

[0317] SEQ ID NO: 270 is the Cas-α28 endonuclease PRT sequence from Ruminococcus sp.

[0318] SEQ ID NO: 271 is the Cas-α29 endonuclease PRT sequence from Peptoclostridium species.

[0319] SEQ ID NO: 272 is the Cas-α30 endonuclease PRT sequence from Bacillus sp.

[0320] SEQ ID NO: 273 is the Cas-α31 endonuclease PRT sequence from Clostridium difficile.

[0321] SEQ ID NO: 274 is the Cas-α32 endonuclease PRT sequence from Clostridium difficile.

[0322] SEQ ID NO: 275 is the Cas-α33 endonuclease PRT sequence from an uncultured archaea.

[0323] SEQ ID NO: 276 is the Cas-α34 endonuclease PRT sequence from an uncultured archaea.

[0324] SEQ ID NO: 277 is the Cas-α35 endonuclease PRT sequence from an uncultured archaea.

[0325] SEQ ID NO: 278 is the Cas-α36 endonuclease PRT sequence from an uncultured archaea.

[0326] SEQ ID NO: 279 is the Cas-α37 endonuclease PRT sequence from an uncultured archaea.

[0327] SEQ ID NO: 280 is the Cas-α38 endonuclease PRT sequence from an uncultured archaea.

[0328] SEQ ID NO: 281 is the Cas-α39 endonuclease PRT sequence from an uncultured archaea.

[0329] SEQ ID NO: 282 is the Cas-α40 endonuclease PRT sequence from an uncultured archaea.

[0330] SEQ ID NO: 283 is the Cas-α41 endonuclease PRT sequence from an uncultured archaea.

[0331] SEQ ID NO: 284 is the Cas-α42 endonuclease PRT sequence from Clostridium difficile.

[0332] SEQ ID NO: 285 is the Cas-α43 endonuclease PRT sequence from Desulfovibrio fructosporum.

[0333] SEQ ID NO: 286 is the Cas-α44 endonuclease PRT sequence from Clostridium botulinum.

[0334] SEQ ID NO: 287 is the Cas-α45 endonuclease PRT sequence from Clostridium difficile.

[0335] SEQ ID NO: 288 is the Cas-α46 endonuclease PRT sequence from Clostridium difficile.

[0336] SEQ ID NO: 289 is the Cas-α47 endonuclease PRT sequence from Clostridium difficile.

[0337] SEQ ID NO: 290 is the Cas-α48 endonuclease PRT sequence from Clostridium difficile.

[0338] SEQ ID NO: 291 is the Cas-α49 endonuclease PRT sequence from Clostridium difficile.

[0339] SEQ ID NO: 292 is the Cas-α50 endonuclease PRT sequence from Clostridium difficile.

[0340] SEQ ID NO: 293 is the Cas-α51 endonuclease PRT sequence from Clostridium difficile.

[0341] SEQ ID NO: 294 is the Cas-α52 endonuclease PRT sequence from Clostridium difficile.

[0342] SEQ ID NO: 295 is the Cas-α53 endonuclease PRT sequence from Clostridium difficile.

[0343] SEQ ID NO: 296 is the Cas-α54 endonuclease PRT sequence from Clostridium difficile.

[0344] SEQ ID NO: 297 is the Cas-α55 endonuclease PRT sequence from Clostridium heranioides.

[0345] SEQ ID NO: 298 is the Cas-α56 endonuclease PRT sequence from Clostridium difficile.

[0346] SEQ ID NO: 299 is the Cas-α57 endonuclease PRT sequence from Bacillus thiamineolyticus from Denmark.

[0347] SEQ ID NO: 300 is the Cas-α58 endonuclease PRT sequence from Geobacter thermoglucosidase.

[0348] SEQ ID NO: 301 is the Cas-α59 endonuclease PRT sequence from Brevibacillus centosporus.

[0349] SEQ ID NO: 302 is the Cas-α60 endonuclease PRT sequence from Clostridium pasteurianum.

[0350] SEQ ID NO: 303 is the Cas-α61 endonuclease PRT sequence from Eubacterium iners.

[0351] SEQ ID NO: 304 is the Cas-α62 endonuclease PRT sequence from Bacillus harvestmenis.

[0352] SEQ ID NO: 305 is the Cas-α63 endonuclease PRT sequence from Ruminococcus sp.

[0353] SEQ ID NO: 306 is the Cas-α64 endonuclease PRT sequence from Ruminococcus sp.

[0354] SEQ ID NO: 307 is the Cas-α65 endonuclease PRT sequence from Clostridium perfringens.

[0355] SEQ ID NO: 308 is the Cas-α66 endonuclease PRT sequence from Bacillus thuringiensis.

[0356] SEQ ID NO: 309 is the Cas-α67 endonuclease PRT sequence from Clostridium perfringens.

[0357] SEQ ID NO: 310 is the Cas-α68 endonuclease PRT sequence from Bacillus cereus.

[0358] SEQ ID NO: 311 is the Cas-α69 endonuclease PRT sequence from Bacillus harvestmenis.

[0359] SEQ ID NO: 312 is the Cas-α70 endonuclease PRT sequence from Bacillus harvestmenis.

[0360] SEQ ID NO: 313 is the Cas-α71 endonuclease PRT sequence from Bacillus harvestmenis.

[0361] SEQ ID NO: 314 is the Cas-α72 endonuclease PRT sequence from Alicyclobacillus acidoterrestris.

[0362] SEQ ID NO: 315 is the Cas-α73 endonuclease PRT sequence from Clostridium tetani.

[0363] SEQ ID NO: 316 is the Cas-α74 endonuclease PRT sequence from Candidatus Levybacteria.

[0364] SEQ ID NO: 317 is the Cas-α75 endonuclease PRT sequence from Bacillus cereus.

[0365] SEQ ID NO: 318 is the Cas-α76 endonuclease PRT sequence from Bacillus cereus.

[0366] SEQ ID NO: 319 is the Cas-α77 endonuclease PRT sequence from Bacillus cereus.

[0367] SEQ ID NO: 320 is the Cas-α78 endonuclease PRT sequence from Clostridium purulentum.

[0368] SEQ ID NO: 321 is the Cas-α79 endonuclease PRT sequence from Bacillus cereus.

[0369] SEQ ID NO: 322 is the Cas-α80 endonuclease PRT sequence from Bacillus thuringiensis.

[0370] SEQ ID NO: 323 is the Cas-α81 endonuclease PRT sequence from Bacillus cereus.

[0371] SEQ ID NO: 324 is the Cas-α82 endonuclease PRT sequence from Bacillus harvestmenis.

[0372] SEQ ID NO: 325 is the Cas-α83 endonuclease PRT sequence from Bacillus cereus.

[0373] SEQ ID NO: 326 is the Cas-α84 endonuclease PRT sequence from Bacillus harvestmenis.

[0374] SEQ ID NO: 327 is the Cas-α85 endonuclease PRT sequence from Bacillus vietnamiensis.

[0375] SEQ ID NO: 328 is the Cas-α86 endonuclease PRT sequence from Bacillus cereus.

[0376] SEQ ID NO: 329 is the Cas-α87 endonuclease PRT sequence from Bacillus cereus.

[0377] SEQ ID NO: 330 is the Cas-α88 endonuclease PRT sequence from Bacillus harvestmenis.

[0378] SEQ ID NO: 331 is the Cas-α89 endonuclease PRT sequence from Bacillus cereus.

[0379] SEQ ID NO: 332 is the Cas-α90 endonuclease PRT sequence from Bacillus harvestmenis.

[0380] SEQ ID NO: 333 is the Cas-α91 endonuclease PRT sequence from Bacillus thuringiensis.

[0381] SEQ ID NO: 334 is the Cas-α92 endonuclease PRT sequence from Bacillus cereus.

[0382] SEQ ID NO: 335 is the Cas-α93 endonuclease PRT sequence from Bacillus cereus.

[0383] SEQ ID NO: 336 is the Cas-α94 endonuclease PRT sequence from Bacillus cereus.

[0384] SEQ ID NO: 337 is the Cas-α95 endonuclease PRT sequence from Bacillus thuringiensis.

[0385] SEQ ID NO: 338 is the Cas-α96 endonuclease PRT sequence from Bacillus sp.

[0386] SEQ ID NO: 339 is the Cas-α97 endonuclease PRT sequence from Bacillus cereus.

[0387] SEQ ID NO: 340 is the Cas-α98 endonuclease PRT sequence from Bacillus cereus.

[0388] SEQ ID NO: 341 is the Cas-α99 endonuclease PRT sequence from Bacillus thuringiensis.

[0389] SEQ ID NO: 342 is the Cas-α100 endonuclease PRT sequence from Bacillus sp.

[0390] SEQ ID NO: 343 is the Cas-α101 endonuclease PRT sequence from Prevotella hominis.

[0391] SEQ ID NO: 344 is the Cas-α102 endonuclease PRT sequence from Prevotella hominis.

[0392] SEQ ID NO: 345 is the Cas-α103 endonuclease PRT sequence from Clostridium difficile.

[0393] SEQ ID NO: 346 is the Cas-α104 endonuclease PRT sequence from Clostridium difficile.

[0394] SEQ ID NO: 347 is the Cas-α105 endonuclease PRT sequence from Clostridium difficile.

[0395] SEQ ID NO: 348 is the Cas-α106 endonuclease PRT sequence from Clostridium difficile.

[0396] SEQ ID NO: 349 is the Cas-α107 endonuclease PRT sequence from Clostridium difficile.

[0397] SEQ ID NO: 350 is the Cas-α108 endonuclease PRT sequence from Clostridium difficile.

[0398] SEQ ID NO: 351 is the Cas-α109 endonuclease PRT sequence from Clostridium difficile.

[0399] SEQ ID NO: 352 is the Cas-α110 endonuclease PRT sequence from Flavobacterium thermophilum.

[0400] SEQ ID NO: 353 is the Cas-α111 endonuclease PRT sequence from Phascolarctosporium sp.

[0401] SEQ ID NO: 354 is the Cas-α112 endonuclease PRT sequence from Bacillus pseudomycoides.

[0402] SEQ ID NO: 355 is the Cas-α113 endonuclease PRT sequence from Bacteroides vulgaris.

[0403] SEQ ID NO: 356 is the Cas-α114 endonuclease PRT sequence from Clostridium botulinum.

[0404] SEQ ID NO: 357 is the Cas-α115 endonuclease PRT sequence from Bacillus pseudomycoides.

[0405] SEQ ID NO: 358 is the Cas-α116 endonuclease PRT sequence from Bacillus pseudomycoides.

[0406] SEQ ID NO: 359 is the Cas-α117 endonuclease PRT sequence from Clostridium botulinum.

[0407] SEQ ID NO: 360 is the Cas-α118 endonuclease PRT sequence from Clostridium botulinum.

[0408] SEQ ID NO: 361 is the Cas-α119 endonuclease PRT sequence from Clostridium botulinum.

[0409] SEQ ID NO: 362 is the Cas-α120 endonuclease PRT sequence from Hydrogenivlrga sp.

[0410] SEQ ID NO: 363 is the Cas-α121 endonuclease PRT sequence from Bacillus megaterium.

[0411] SEQ ID NO: 364 is the Cas-α122 endonuclease PRT sequence from Clostridium fragilis.

[0412] SEQ ID NO: 365 is the Cas-α123 endonuclease PRT sequence from Bacteroides vulgaris.

[0413] SEQ ID NO: 366 is the Cas-α124 endonuclease PRT sequence from Bacillus thuringiensis.

[0414] SEQ ID NO: 367 is the Cas-α125 endonuclease PRT sequence from Bacillus cereus.

[0415] SEQ ID NO: 368 is the Cas-α126 endonuclease PRT sequence from Clostridium sp.

[0416] SEQ ID NO: 369 is the Cas-α127 endonuclease PRT sequence from Bacteroides vulgaris.

[0417] SEQ ID NO: 370 is the Cas-α128 endonuclease PRT sequence from Dorealongicatena.

[0418] SEQ ID NO: 371 is the Cas-α129 endonuclease PRT sequence from Sulfurihydrogenibium azorense. DETAILED DESCRIPTION

[0419] Compositions and methods are provided for novel CRISPR effector systems and elements comprising such systems, including but not limited to novel guide polynucleotide / endonuclease complexes, guide polynucleotides, guide RNA elements, Cas proteins and endonucleases, and proteins comprising endonuclease functions (domains). Compositions and methods for directly delivering endonucleases, cutting ready complexes, guide RNAs and guide RNA / Cas endonuclease complexes are also provided. The present disclosure further includes genome modification for target sequences in cell genomes, for gene editing, and for inserting target polynucleotides into cell genomes Compositions and methods.

[0420] Unless otherwise specified, the terms used in the claims and specification are defined as set forth below. It must be noted that, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0421] definition

[0422] As used herein, "nucleic acid" means polynucleotides, and includes a single-stranded or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases. Nucleic acids can also include fragments and modified nucleotides. Therefore, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" and "nucleic acid fragment" are used interchangeably to represent single-stranded or double-stranded RNA and / or DNA and / or RNA-DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0423] The term "genome" when applied to a prokaryotic or eukaryotic cell or cell of an organism encompasses not only the chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular components of the cell (eg, mitochondria, or plastids).

[0424] "Open reading frame" is abbreviated as ORF.

[0425] The term "selectively hybridizes" includes reference to hybridization of a nucleic acid sequence to a specific nucleic acid target sequence under stringent hybridization conditions to a detectably greater extent (e.g., at least 2 times background) than hybridization to non-target nucleic acid sequences and substantially to the exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) to each other.

[0426] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize with its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different situations. By controlling the stringency of hybridization conditions and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequence so that a lower degree of similarity is detected (heterologous detection). Typically, the probe length is less than about 1000 nucleotides, optionally less than 500 nucleotides in length. Typically, stringent conditions will be conditions where the salt concentration is less than about 1.5 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other one or more salts) at pH 7.0 to 8.3, and is at least about 30°C for short probes (e.g., 10 to 50 nucleotides), and is at least about 60°C for long probes (e.g., more than 50 nucleotides). Adding a destabilizing agent such as formamide can also achieve stringent conditions. Exemplary low stringency conditions include hybridization at 37° C. with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1X to 2X SSC (20X SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary medium stringency conditions include hybridization at 37° C. in 40% to 45% formamide, 1 M NaCl, 1% SDS, and washing in 0.5X to 1X SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization at 37° C. in 50% formamide, 1 M NaCl, 1% SDS, and washing in 0.1X SSC at 60° C. to 65° C.

[0427] "Homologous" means that the DNA sequences are similar. For example, a "region homologous to a genomic region" found on a donor DNA is a region of DNA having a similar sequence to a given "genomic sequence" in the genome of a cell or organism. A homologous region can have any length sufficient to promote homologous recombination at the target site of cleavage. For example, the length of a homologous region can include at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800, 5-2900, 5-3000 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-210, 1-220, 1-230, 1-240, 1-250, 1-260, 1-270, 1-280, 1-290, 1-300, 1-310 or more bases, so that the homologous region has enough homology to carry out homologous recombination with the corresponding genomic region." enough homology" means that two polynucleotide sequences have enough structural similarities to serve as substrates for homologous recombination reactions. Structural similarity includes the total length of each polynucleotide fragment and the sequence similarity of the polynucleotides. Sequence similarity can be described by percent sequence identity over the entire length of the sequence and / or by conserved regions that include local similarity (eg, contiguous nucleotides with 100% sequence identity) as well as percent sequence identity over a portion of the length of the sequence.

[0428] As used herein, a "genomic region" is a segment of a chromosome in the genome of a cell present at any one of the target sites, or alternatively, also comprises a portion of the target site. A genomic region may comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-120 0, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900, 5-3000, 5-3100 or more bases, so that the genomic region has sufficient homology to undergo homologous recombination with the corresponding homologous region.

[0429] As used herein, "homologous recombination (HR)" includes the exchange of DNA fragments between two DNA molecules at homologous sites. The frequency of homologous recombination is affected by multiple factors. Different organisms vary with respect to the amount of homologous recombination and the relative proportion of homologous and non-homologous recombination. Generally, the length of the homologous region can affect the frequency of homologous recombination events: the longer the homologous region, the higher the frequency. The length of the homologous region required for observing homologous recombination also varies with species. In many cases, at least 5kb of homology has been utilized, but homologous recombination with only 25-50bp of homology has been observed. See, for example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. Natl. Acad. Sci. USA 82:4768-72, Sugawara and Haber, (1992) Mol Cell Biol 12:563-75, Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay et al., (1987) Genetics 115:161-7.

[0430] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0431] "Percentage of sequence identity" refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein when comparing the optimal alignment of two sequences with a reference sequence (which does not include additions or deletions), the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., spaces). The percentage is calculated in the following manner: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window, and the result is then multiplied by 100 to produce the percentage of sequence identity. Useful examples of percentage sequence identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from 50% to 100%. These identities can be determined using any program described herein.

[0432] Sequence alignments and percent identity or similarity calculations can be determined using a variety of comparison methods designed to detect homologous sequences, including but not limited to MegAlign® of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin). TM Program. In the context of this application, it will be understood that, where sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" will mean any set of values ​​or parameters that are initially loaded into the software when it is first initialized.

[0433] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci [Computer Applications in Biological Sciences] 8:189-191) and found in MegAlign of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin). TMProgram. For multiple alignments, the default values ​​correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for pairwise alignments and percentage identity calculations of protein sequences using the Clustal method are KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5. For nucleic acids, these parameters are KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4, and DIAGONALS SAVED = 4. After aligning sequences using the Clustal V program, it is possible to obtain "percent identity" by viewing the "sequence distance" table in the same program. The "Clustal W alignment method" corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci [Computer Applications in Biological Sciences] 8:189-191) and found in MegAlign of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin). TMv6.1 program. Default parameters for multiple alignment (Gap Penalty = 10, Gap Length Penalty = 0.2, Delay Divergen Seqs (%) = 30, DNA Transformation Weight = 0.5, Protein Weight Matrix = Gonnet Series, DNA Weight Matrix = IUB). After aligning the sequences using the Clustal W program, it is possible to obtain the "Percent Identity" by viewing the "Sequence Distance" table in the same program. Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the values ​​obtained using GAP version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: % identity and % similarity for nucleotide sequences using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a gap creation penalty weight of 8 and a gap length extension penalty weight of 2 and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89: 10915). GAP uses the algorithm of Needleman and Wunsch (1970) J Mol Biol 48: 443-53 to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible comparisons and gap positions, and uses the gap generation penalty and gap extension penalty in the unit of matching bases to produce the comparison with the maximum number of matching bases and the minimum gaps. "BLAST" is a search algorithm for finding the region of similarity between biological sequences provided by the National Center for Biotechnology Information (NCBI). The program compares nucleotide or protein sequences with sequence databases, and calculates the statistical significance of the match to identify sequences with enough similarity to the query sequence, so that the similarity will not be predicted to have occurred randomly. The sequences identified by BLAST report and their local comparison with the query sequence. It is well understood by those skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or modified natural or synthetic polypeptides, wherein such polypeptides have the same or similar function or activity. Useful examples of percentage identity include but are not limited to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from 50% to 100%.In fact, any amino acid identity from 50% to 100% will be useful in describing the present disclosure, such as 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% or 99%.

[0434] Polynucleotide and polypeptide sequences, variants thereof, and the structural relationships of these sequences can be described by the terms "homology", "homologous", "substantially identical", "substantially similar", and "substantially corresponding", which are used interchangeably herein. These refer to polypeptide or nucleic acid sequences in which changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as the ability to mediate gene expression or produce a certain phenotype. These terms also refer to one or more modifications of a nucleic acid sequence that do not substantially change the functional properties of the resulting nucleic acid relative to the initial unmodified nucleic acid. These modifications include deletions, substitutions, and / or insertions of one or more nucleotides in a nucleic acid fragment. Substantially similar nucleic acid sequences encompassed can be defined by the ability of these nucleic acid sequences to hybridize with the sequences exemplified herein, or to hybridize with any portion of a nucleotide sequence disclosed herein and functionally equivalent to any nucleic acid sequence disclosed herein (under moderate stringency conditions, e.g., 0.5X SSC, 0.1% SDS, 60°C). Stringent conditions can be adjusted to screen for moderately similar fragments (e.g., homologous sequences from distantly related organisms) to highly similar fragments (e.g., genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions.

[0435] A "centimorgan" (cM) or "map distance unit" is the distance between two polynucleotide sequences, linked genes, markers, target sites, loci, or any pair thereof, at which 1% of the products of meiosis are recombinant. Thus, one centimorgan is equivalent to a distance equal to the 1% average recombination frequency between two linked genes, markers, target sites, loci, or any pair thereof.

[0436] An "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide or protein, or a biologically active portion thereof, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or polypeptide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide does not contain sequences that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived (optimally protein coding sequences). For example, in various embodiments, the isolated polynucleotide may comprise a nucleotide sequence of less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb, which naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Isolated polynucleotides may be purified from cells in which they naturally occur. Conventional nucleic acid purification methods known to the skilled artisan may be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

[0437] The term "fragment" refers to a continuous set of nucleotides or amino acids. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 continuous nucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 continuous amino acids. A fragment may or may not exhibit the function of a sequence that shares a certain percentage of identity over the length of the fragment.

[0438] The terms "functionally equivalent fragments" and "functionally equivalent fragments" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment or polypeptide that exhibits the same activity or function as the longer sequence from which it is derived. In one example, whether or not the fragment encodes an active protein, the fragment retains the ability to alter gene expression or produce a certain phenotype. For example, fragments can be used to design genes to produce a desired phenotype in a modified plant. A gene can be designed for use in inhibition, whether or not the gene encodes an active enzyme, by connecting its nucleic acid fragment in a sense or antisense orientation relative to a plant promoter sequence.

[0439] "Gene" includes a nucleic acid fragment that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene found in its natural endogenous location with its own regulatory sequences.

[0440] The term "endogenous" refers to a sequence or other molecule that is naturally present in a cell or organism. In one aspect, an endogenous polynucleotide is typically found in the genome of a cell; that is, not heterologous.

[0441] "Allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all alleles present at a given locus on a chromosome are identical, the plant is homozygous at that locus. If the alleles present at a given locus on a chromosome are different, the plant is heterozygous at that locus.

[0442] "Coding sequence" refers to a polynucleotide sequence that encodes a specific amino acid sequence. "Regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding sequence, and which affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5' non-translated sequences, 3' non-translated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0443] A "mutant gene" is a gene that has been altered by human intervention. Such a "mutant gene" has a sequence that differs from the sequence of the corresponding non-mutant gene by at least one nucleotide addition, deletion or substitution. In certain embodiments of the present disclosure, the mutated gene comprises an alteration caused by a guide polynucleotide / Cas endonuclease system as disclosed herein. A mutated plant is a plant comprising a mutated gene.

[0444] As used herein, the term "targeted mutation" is a mutation in a gene (referred to as a target gene), including a natural gene, generated by altering a target sequence within a target gene using any method known to those skilled in the art, including methods involving a guided Cas endonuclease system as disclosed herein.

[0445] The terms "knockout", "gene knockout" and "genetic knockout" are used interchangeably herein. Knockout means that a DNA sequence of a cell has been partially or completely ineffective by targeting with a Cas protein; for example, such a DNA sequence may have encoded an amino acid sequence before knockout, or may have had a regulatory function (e.g., a promoter).

[0446] The terms "knock-in", "gene knock-in", "gene insertion" and "genetic knock-in" are used interchangeably herein. Knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with a Cas protein (e.g., by homologous recombination (HR), wherein a suitable donor DNA polynucleotide is also used). Examples of knock-ins are the specific insertion of a heterologous amino acid coding sequence in the coding region of a gene, or the specific insertion of a transcriptional regulatory element in a genetic locus.

[0447] "Domain" means a continuous stretch of nucleotides (which may be RNA, DNA and / or RNA-DNA combination sequences) or amino acids.

[0448] The term "conserved domain" or "motif" refers to a group of polynucleotides or amino acids that are conserved at specific positions along aligned sequences of evolutionarily related proteins. Although amino acids at other positions may vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential to the structure, stability, or activity of the protein. Because they are identified by their high conservation in aligned sequences of a family of protein homologs, they can be used as identifiers or "signatures" to determine whether a protein with a newly determined sequence belongs to a previously identified protein family.

[0449] A "codon-modified gene" or "codon-biased gene" or "codon-optimized gene" is a gene whose frequency of codon usage has been designed to mimic the frequency of preferred codon usage of the host cell.

[0450] An "optimized" polynucleotide is a sequence that has been optimized for improved expression in a particular heterologous host cell.

[0451] A "plant optimized nucleotide sequence" is a nucleotide sequence optimized for expression in plants, particularly for increased expression in plants. Plant optimized nucleotide sequences include codon optimized genes. Plant preferred nucleotide sequences can be synthesized by modifying the nucleotide sequence encoding a protein, such as the Cas endonucleases disclosed herein, using one or more plant preferred codons to improve expression. See, e.g., Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host preferred codon usage.

[0452] "Promoter" is a DNA region that participates in the recognition and combination of RNA polymerase and other proteins to initiate transcription. The promoter sequence is composed of a proximal element and a more distal upstream element, and the latter element is generally referred to as an enhancer. "Enhancer" is a DNA sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. Promoters can all be derived from natural genes, or are composed of different elements derived from different promoters existing in nature, and / or comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may guide the expression of genes in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. It is further recognized that, since the exact boundaries of regulatory sequences are not yet fully defined in most cases, some DNA fragments of variations may have the same promoter activity.

[0453] Promoters that cause gene expression in most cell types under most circumstances are generally referred to as "constitutive promoters". The term "inducible promoter" refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of endogenous or exogenous stimuli, such as by chemical compounds (chemical inducers), or to environmental, hormone, chemical, and / or developmental signals. Inducible or regulated promoters include, for example, promoters that are induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounds, or chemicals (such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners).

[0454] "Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present in the mRNA upstream of the translation initiation sequence. The translation leader sequence can affect the processing of the mRNA by the primary transcript, mRNA stability, or translation efficiency. Examples of translation leader sequences have been described (e.g., Turner and Foster, (1995) Mol Biotechnol [Molecular Biotechnology] 3: 225-236).

[0455] "3' non-coding sequence", "transcription terminator", or "termination sequence" refers to a DNA sequence located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are generally characterized as affecting the addition of polyadenylic acid tracts to the 3' end of an mRNA precursor. The use of various 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1: 671-680.

[0456] "RNA transcript" refers to the product produced by the transcription catalyzed by the RNA polymerase of the DNA sequence. When the RNA transcript is a completely complementary copy of the DNA sequence, the RNA transcript is referred to as a primary transcript or pre-mRNA. When the RNA transcript is an RNA sequence derived from the post-transcriptional processing of the pre-mRNA of the primary transcript, the RNA transcript is referred to as a mature RNA or mRNA. "Messenger RNA" or "mRNA" refers to an RNA that does not contain introns and can be translated into protein by a cell. "cDNA" refers to a DNA that is complementary to an mRNA template and synthesized from an mRNA template using a reverse transcriptase. The cDNA can be single-stranded or can be converted into a double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to an RNA transcript that contains mRNA and can be translated into protein in a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and blocks the expression of a target gene (see, e.g., U.S. Patent No. 5,107,065). Antisense RNA can be complementary to any part of a particular gene transcript, i.e., 5' non-coding sequence, 3' non-coding sequence, intron or coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but still has an effect on cellular processes. The terms "complementary sequence" and "reverse complementary sequence" are used interchangeably herein with respect to mRNA transcripts, and are intended to define the antisense RNA of the messenger.

[0457] The term "genome" refers to the entire complementary sequence of genetic material (genes and non-coding sequences) present in every cell of an organism or virus or organelle; and / or the complete set of chromosomes inherited as a (haploid) unit from one parent.

[0458] The term "operably connected" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one of the nucleic acid sequences is regulated by another nucleic acid sequence. For example, when a promoter is capable of regulating the expression of a coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter), the promoter is operably connected to the coding sequence. The coding sequence can be operably connected to a regulatory sequence in a sense or antisense orientation. In another example, the complementary RNA region can be directly or indirectly operably connected to the 5' or 3' of the target mRNA, or in the target mRNA, or the first complementary region is 5' and its complementary sequence is 3' of the target mRNA.

[0459] Typically, "host" refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced. As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell) in vivo or in vitro, or a cell (e.g., a cell line) from a multicellular organism cultured as a unicellular entity, into which a heterologous polynucleotide or polypeptide has been introduced. In some embodiments, the cell is selected from the group consisting of a primitive cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algae cell, an animal cell, an invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, an insect cell, a mammalian cell, a pig cell, a cattle cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell. In some cases, the cell is an in vitro cell. In some cases, the cell is an in vivo cell.

[0460] The term "recombinant" refers to the artificial combination of two originally separate sequence segments, for example, by chemical synthesis or by the manipulation of separate nucleic acid segments through genetic engineering techniques.

[0461] The terms "plasmid", "vector" and "box" refer to linear or circular extrachromosomal elements, which usually carry genes that are not part of the central metabolism of the cell, and are usually in the form of double-stranded DNA. Such elements can be autonomous replication sequences, genome integration sequences, phages, or nucleotide sequences derived from any source, single-stranded or double-stranded DNA or RNA, in linear or circular form, many of which have been connected or recombined into unique structures that can introduce the polynucleotide of interest into cells." transformation box" refers to a specific vector that contains genes and has elements other than genes that promote transformation of specific host cells. "expression cassette" refers to a specific vector that contains genes and has elements other than genes that allow expression of the gene in a host.

[0462] The terms "recombinant DNA molecule", "recombinant DNA construct", "expression construct", "construct", and "recombinant construct" are used interchangeably herein. A recombinant DNA construct comprises an engineered combination of nucleic acid sequences, such as regulatory sequences and coding sequences that are not all found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that which occurs naturally. Such constructs may be used alone or in combination with a vector. If a vector is used, the choice of vector depends on the method that will be used to introduce the vector into a host cell, as is well known to those skilled in the art. For example, a plasmid vector may be used. The skilled artisan is fully aware of the genetic elements that must be present on the vector in order to successfully transform, select, and propagate the host cell. Those skilled in the art will also recognize that different independent transformation events may result in different expression levels and patterns (Jones et al., (1985) EMBO J [European Molecular Biology Organization Journal] 4: 2411-2418; De Almeida et al., (1989) Mol Gen Genetics [Molecular Genetics and General Genetics] 218: 78-86), and therefore typically multiple events are screened to obtain lines that display the desired expression levels and patterns. Such screening can be done by standard molecular biology assays, biochemical assays, and other assays, including Southern blot analysis, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzyme assays or activity assays, and / or phenotypic analysis.

[0463] The term "heterologous" refers to the difference between the original environment, location or composition of a particular polynucleotide or polypeptide sequence and its current environment, location or composition. Non-limiting examples include taxonomically derived differences (e.g., if a polynucleotide sequence obtained from Zea mays is inserted into the genome of a rice (Oryza sativa) plant or a different variant or cultivar of maize, the polynucleotide sequence is heterologous; or a polynucleotide obtained from bacteria is introduced into a plant's cell, the polynucleotide sequence is heterologous) or differences in sequence (e.g., a polynucleotide sequence obtained from maize is isolated, modified, and reintroduced into a maize plant). As used herein, "heterologous" with respect to a sequence may refer to a sequence that is derived from a different species, variant, alien species, or, if derived from the same species, a sequence that is substantially modified from its native form in a composition and / or genomic locus by deliberate human intervention. For example, the promoter effectively connected to the heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or, if from the same / similar species, one or both parties are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter of the polynucleotide effectively connected. Alternatively, one or more regulatory regions and / or polynucleotides provided herein can be synthesized as a whole. In another example, the target polynucleotide for being cut by the Cas endonuclease can belong to an organism different from the Cas endonuclease. In another example, the Cas endonuclease and the guide RNA can be introduced into the target polynucleotide together with another polynucleotide used as a template or donor for inserting the target polynucleotide, wherein the other polynucleotide is heterologous to the target polynucleotide and / or the Cas endonuclease.

[0464] As used herein, the term "expression" refers to the production of a functional end product (e.g., mRNA, guide RNA, or protein) in either precursor or mature form.

[0465] A "mature" protein refers to a post-translationally processed polypeptide (ie, a polypeptide from which any pre-peptide or propeptide present in the primary translation product has been removed).

[0466] "Precursor" protein refers to the primary product of translation of mRNA (ie, pre- or propeptides are still present). Pre- or propeptides may be, but are not limited to, intracellular localization signals.

[0467] “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats ( C lustered R egularly I Interspaced Short P alindromic R CRISPR loci refer to certain genetic loci encoding components of DNA cleavage systems, such as those used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327: 167-170; WO 2007 / 025097 published March 1, 2007). CRISPR loci can consist of a CRISPR array, comprising short direct repeat sequences (CRISPR repeats) separated by short variable DNA sequences (called 'spacers'), which can be flanked by different Cas (CRISPR associated) genes.

[0468] As used herein, an "effector" or "effector protein" is a protein having an activity that includes recognition, binding and / or cleavage of a polynucleotide target or nicking of a polynucleotide target. An effector or effector protein may also be an endonuclease. The "effector complex" of the CRISPR system includes Cas proteins that participate in crRNA and target recognition and binding. Some component Cas proteins may additionally include domains that participate in target polynucleotide cleavage.

[0469] The term "Cas protein" refers to a polypeptide encoded by a Cas (CRISPR-related) gene. Cas proteins include proteins encoded by genes in the cas locus, and include adaptation molecules and interfering molecules. Interfering molecules of bacterial adaptive immune complexes include endonucleases. The Cas endonucleases described herein include one or more nuclease domains. Cas endonucleases include, but are not limited to, novel Cas-α proteins disclosed herein, Cas9 proteins, Cpf1 (Cas12) proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or combinations or complexes of these. When complexed with a suitable polynucleotide component, the Cas protein can be a "Cas endonuclease" or "Cas effector protein" that can recognize, bind to all or part of a specific polynucleotide target sequence, and optionally cause all or part of a specific polynucleotide target sequence to produce an incision or cut all or part of a specific polynucleotide target sequence. The Cas-α endonucleases disclosed herein may include those endonucleases having one or more RuvC nuclease domains. The Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a functional fragment or functional variant of a native Cas protein having at least 50%, 50% to 55%, at least 55% to 56%, or more than 500 contiguous amino acids of at least 50, 50 to 100, at least 100, 100 to 150, at least 150, 150 to 200, at least 200, 200 to 250, at least 250, 250 to 300, at least 300, 300 to 350, at least 350, 350 to 400, at least 400, 400 to 450, at least 500, or more than 500 contiguous amino acids of a native Cas protein. %, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity to the native sequence and retains at least some of the activity of the native sequence.

[0470] "Functional fragments", "functionally equivalent fragments" and "functionally equivalent fragments" of Cas endonucleases are used interchangeably herein and refer to a portion or subsequence of the Cas endonucleases of the present disclosure, wherein the ability to recognize, bind to and optionally nick or cleave (introduce single-strand or double-strand breaks) a target site is retained. A portion or subsequence of a Cas endonuclease may comprise a complete peptide or a partial (functional) peptide of any one of its domains, such as, but not limited to, a complete functional portion of the Cas3 HD domain, a complete functional portion of the Cas3 helicase domain, a complete functional portion of a protein (such as, but not limited to, Cas5, Cas5d, Cas7 and Cas8b1).

[0471] The terms "functional variant", "functionally equivalent variant" and "functionally equivalent variant" of a Cas endonuclease or Cas effector protein (including Cas-α described herein) are used interchangeably herein and refer to variants of the Cas effector proteins disclosed herein in which the ability to recognize, bind and optionally unwind, nick or cut all or part of a target sequence is retained.

[0472] Cas endonucleases may also include multifunctional Cas endonucleases. The terms "multifunctional Cas endonucleases" and "multifunctional Cas endonuclease polypeptides" are used interchangeably herein and include reference to a single polypeptide having a Cas endonuclease function (comprising at least one protein domain that can be used as a Cas endonuclease) and at least another function, such as but not limited to, a function of forming a complex (comprising at least a second protein domain that can form a complex with other proteins). In one aspect, the multifunctional Cas endonuclease comprises at least one additional protein domain relative to those typical domains of the Cas endonuclease (inside upstream (5') or downstream (3', or in both 5' and 3', or any combination thereof).

[0473] The terms "cascade" and "cascade complex" are used interchangeably herein and include reference to a multi-subunit protein complex that can be assembled with a polynucleotide to form a polynucleotide-protein complex (PNP). A cascade is a polynucleotide-dependent PNP to achieve complex assembly and stability and to identify target nucleic acid sequences. The cascade is used as a monitoring complex that finds and optionally binds to a target nucleic acid that is complementary to the variable targeting domain of a guide polynucleotide.

[0474] The terms "cleavage-ready Cascade", "crCascade", "cleavage-ready Cascade complex", "crCascade complex", "cleavage-ready Cascade system", "CRC" and "crCascade system" are used interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP), wherein one of the cascade proteins is a Cas endonuclease that is capable of recognizing, binding to, and optionally unwinding, nicking or cleaving all or part of a target sequence.

[0475] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. The 7-methylguanylate residue is located at the 5' end of messenger RNA (mRNA) in eukaryotes. In eukaryotes, RNA polymerase II (Pol II) transcribes mRNA. Messenger RNA capping is generally performed as follows: the very terminal 5' phosphate group of the mRNA transcript is removed with an RNA terminal phosphatase, leaving two terminal phosphates. Guanosine monophosphate (GMP) is added to the terminal phosphate of the transcript with a guanylyltransferase, leaving a 5'-5' triphosphate-linked guanine at the end of the transcript. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyltransferase.

[0476] The term "without a 5'-cap" and the like is used herein to refer to RNAs that have, for example, a 5'-hydroxyl group instead of a 5'-cap. For example, such RNAs may be referred to as "uncapped RNAs". Because 5'-capped RNAs tend to be exported from the nucleus, uncapped RNAs may accumulate better in the nucleus after transcription. One or more RNA components herein are uncapped.

[0477] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease (including a Cas endonuclease described herein) and enable the Cas endonuclease to recognize, optionally bind, and optionally cleave a DNA target site. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence).

[0478] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a guide RNA, crRNA, or tracrRNA are used interchangeably herein and refer to a portion or subsequence of a guide RNA, crRNA, or tracrRNA of the present disclosure, respectively, in which the ability to function as a guide RNA, crRNA, or tracrRNA, respectively, is retained.

[0479] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" of a guide RNA, crRNA, or tracrRNA (respectively) are used interchangeably herein and refer to variants of a guide RNA, crRNA, or tracrRNA, respectively, of the present disclosure, in which the ability to function as a guide RNA, crRNA, or tracrRNA, respectively, is retained.

[0480] The terms "single guide RNA" and "sgRNA" are used interchangeably herein and refer to the synthetic fusion of two RNA molecules, wherein a crRNA (CRISPR RNA) containing a variable targeting domain (connected to a tracr pairing sequence hybridized to tracrRNA) is fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA may comprise a crRNA or crRNA fragment of a type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease and a tracrRNA or tracrRNA fragment, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, such that the Cas endonuclease can recognize, optionally bind to, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0481] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and include a nucleotide sequence that can hybridize (be complementary) to one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 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%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a continuous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0482] The terms "Cas endonuclease recognition domain" or "CER domain" (of a guide polynucleotide) are used interchangeably herein and include nucleotide sequences that interact with a Cas endonuclease polypeptide. A CER domain comprises a (trans-acting) tracr nucleotide chaperone sequence followed by a tracr nucleotide sequence. A CER domain may be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., US 20150059010A1, published on February 26, 2015), or any combination thereof.

[0483] As used herein, the terms "guide polynucleotide / Cas endonuclease complex", "guide polynucleotide / Cas endonuclease system", "guide polynucleotide / Cas complex", "guide polynucleotide / Cas system" and "guide Cas system", "polynucleotide-guided endonuclease", "PGEN" are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease capable of forming a complex, wherein the guide polynucleotide / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site. The guide polynucleotide / Cas endonuclease complex herein may comprise one or more Cas proteins and one or more suitable polynucleotide components of any known CRISPR system (Horvath and Barrangou, 2010, Science 327: 167-170; Makarova et al., 2015, Nature Reviews Microbiology 13: 1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13).

[0484] The terms "guide RNA / Cas endonuclease complex", "guide RNA / Cas endonuclease system", "guide RNA / Cas complex", "guide RNA / Cas system", "gRNA / Cas complex", "gRNA / Cas system", "RNA-guided endonuclease", "RGEN" are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease capable of forming a complex, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cut (introduce single-stranded or double-stranded breaks) the DNA target site.

[0485] The terms "target site", "target sequence", "target site sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", "genomic target locus" and "protospacer" are used interchangeably herein and refer to polynucleotide sequences, for example, but not limited to, nucleotide sequences on a chromosome, episome, locus or any other DNA molecule in the genome of a cell (including chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA), at which the guide polynucleotide / Cas endonuclease complex can recognize, bind and optionally nick or cut. The target site can be an endogenous site in the genome of the cell, or alternatively, the target site can be heterologous to the cell and thus not naturally present in the genome of the cell, or the target site can be found in a heterogeneous genomic location compared to the location where it occurs in nature. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of the cell and is located at the endogenous or native location of the target sequence in the genome of the cell. "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the cell, but located at a different position in the genome of the cell (i.e., a non-endogenous or non-native position).

[0486] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to the (targeted) target sequence (pre-spacer sequence) recognized by the guide polynucleotide / Cas endonuclease system described herein. If the target DNA sequence is not behind the PAM sequence, the Cas endonuclease may not successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0487] "Altered target site", "altered target sequence", "modified target site", "modified target sequence" are used interchangeably herein and refer to a target sequence as disclosed herein, which comprises at least one change when compared to a non-altered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0488] "Modified nucleotides" or "edited nucleotides" refer to a nucleotide sequence of interest that contains at least one change when compared to its non-modified nucleotide sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0489] Methods for "modifying a target site" and "altering a target site" are used interchangeably herein and refer to methods for generating an altered target site.

[0490] As used herein, "donor DNA" is a DNA construct that includes a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.

[0491] The term "polynucleotide modification template" includes a polynucleotide comprising at least one nucleotide modification when compared to a nucleotide sequence to be edited. The nucleotide modification can be at least one nucleotide substitution, addition or deletion. Optionally, the polynucleotide modification template may further comprise homologous nucleotide sequences flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0492] The term "plant-optimized Cas endonuclease" herein refers to a Cas protein encoded by a nucleotide sequence that has been optimized for expression in a plant cell or plant, including a multifunctional Cas protein.

[0493] "Plant optimized nucleotide sequence encoding a Cas endonuclease," "plant optimized construct encoding a Cas endonuclease," and "plant optimized polynucleotide encoding a Cas endonuclease" are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, which has been optimized for expression in a plant cell or plant. Plants comprising a plant optimized Cas endonuclease include: plants comprising a nucleotide sequence encoding a Cas sequence, and / or plants comprising a Cas endonuclease protein. In one aspect, the plant optimized Cas endonuclease nucleotide sequence is a maize optimized, rice optimized, wheat optimized, soybean optimized, cotton optimized, or canola optimized Cas endonuclease.

[0494] The term "plant" generally includes whole plant, plant organ, plant tissue, seed, plant cell, seed and plant offspring. The plant is a monocot or dicot. Plant cells include but are not limited to cells derived from the following materials: seed, suspension culture, embryo, meristematic region, callus, leaf, root, bud, gametophyte, sporophyte, pollen and microspore. "Plant element" is intended to refer to whole plant or plant component, and can include differentiation and / or undifferentiated tissue, such as but not limited to plant tissue, part and cell type. In one embodiment, plant element is one of the following: whole plant, seedling, meristematic tissue, basic tissue, vascular tissue, epidermis tissue, seed, leaf, root, bud, stem, flower, fruit, runner, bulb, tuber, corm, asexual terminal branch, bud, young shoot, tumor tissue, and various forms of cell and culture (for example, unicellular, protoplast, embryo and callus). It should be noted that protoplasts are not technically "complete" plant cells (all components are naturally present) because protoplasts do not have cell walls. The term "plant organ" refers to a group of tissues that form or constitute different parts of a plant in terms of morphology and function. As used herein, "plant element" is a synonym for "part" of a plant, refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably with the term "tissue" in the full text. Similarly, "plant propagation element" is intended to generally refer to any plant part that can create other plants through the sexual or asexual propagation of the plant, such as but not limited to: seed, seedling, root, bud, cutting, scion, grafted seedling, runner, bulb, tuber, corm, asexual terminal branch or young shoot. Plant elements may be present in a plant or in a plant organ, tissue culture or cell culture.

[0495] "Progeny" includes any subsequent generation of a plant.

[0496] As used herein, the term "plant part" refers to plant cells, plant protoplasts, plant cell tissue cultures of regenerable plants, plant calli, plant pieces, and intact plant cells in plants or plant parts (such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, spikes, cobs, shells, stems, roots, root tips, anthers, etc.), together with these parts themselves. Grains mean mature seeds produced by commercial growers for purposes other than cultivation or propagation of species. The offspring, variants, and mutants of these regenerated plants are also included within the scope of the present invention, provided that these parts contain introduced polynucleotides.

[0497] The term "monocotyledonous" or "monocotyledonous plant" refers to a subclass of angiosperms, also known as the "monocots," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0498] The term "dicotyledonous" or "dicots" refers to a subclass of angiosperms, also known as the "dicots," whose seeds typically contain two embryonic leaves or cotyledons. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0499] As used herein, a "male sterile plant" is a plant that does not produce viable or otherwise fertilized male gametes. As used herein, a "female sterile plant" is a plant that does not produce viable or otherwise fertilized female gametes. It should be appreciated that male sterile plants and female sterile plants can be female fertile and male fertile, respectively. It should be further appreciated that male fertile (but female sterile) plants can produce viable offspring when crossed with female fertile plants, and female fertile (but male sterile) plants can produce viable offspring when crossed with male fertile plants.

[0500] As used herein, the term "non-conventional yeast" refers to any yeast that is not a species of yeast of the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or the genus Schizosaccharomyces. (See "Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols", edited by K. Wolf, KD Breunig, G. Barth, Springer-Verlag, Berlin, Germany, 2003).

[0501] In the context of the present disclosure, the term "hybridized" or "cross" refers to the fusion of gametes via pollination to produce offspring (i.e., cells, seeds, or plants). The term encompasses sexual crossing (one plant being pollinated by another plant) and selfing (self-pollination, i.e., when the pollen and ovules (or microspores and megaspores) are from the same plant or genetically identical plants).

[0502] The term "infiltration" refers to the phenomenon that the desired allele of the locus is transferred to another genetic background from a genetic background. For example, the infiltration of the desired allele at the designated locus can be passed to at least one offspring plant via the sexual hybridization between two parental plants, wherein at least one parental plant has a desired allele in its genome. Alternatively, for example, allelic transmission can occur by the reorganization between two donor genomes, such as in fusion protoplasts, wherein at least one of the donor protoplasts has a desired allele in its genome. Desired allele can be, for example, the allele of the selection of natural allele (mutated or edited) or marker or QTL of transgenic, modified.

[0503] The term "homologous" is a comparative term referring to organisms that are genetically identical but have different treatments. In one example, two genetically identical maize plant embryos can be divided into two different groups, one group receiving treatment (such as the introduction of CRISPR-Cas effector endonucleases), and one group not receiving such treatment as a control. Therefore, any phenotypic differences between the two groups may be attributed only to the treatment, rather than to any inherent nature of the endogenous gene composition of the plant.

[0504] "Introduced" is intended to mean providing a polynucleotide or polypeptide or polynucleotide-protein complex to a target, such as a cell or organism, in such a way that the component or components gain access to the interior of a cell of the organism or into the cell itself.

[0505] "Purpose polynucleotides" include any nucleotide sequence encoding a protein or polypeptide that improves the desirability of a crop, i.e., agronomical purpose traits. Purpose polynucleotides include, but are not limited to, polynucleotides encoding important traits for agronomy, herbicide-resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial products, phenotypic markers, or any other traits of important agronomic or commercial significance. Purpose polynucleotides may additionally be utilized in sense or antisense orientations. In addition, more than one purpose polynucleotide may be utilized together or "stacked" to provide additional benefits.

[0506] A "complex trait" locus includes a genomic locus with multiple transgenes that are genetically linked to each other.

[0507] The compositions and methods herein can provide plants with improved "agronomic traits" or "traits of agronomic importance" or "traits of agronomic significance", which traits can include, but are not limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen use efficiency, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, improved yield, enhanced health, improved vigor, improved growth, improved photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root structure, modulation of metabolites, modulation of the proteome, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient content compared to a homologous plant that does not contain the modifications derived from the methods and compositions herein.

[0508] "Agronomic potential" is intended to mean the ability of a plant element to express a phenotype, preferably an improved agronomic trait, at some point in its life cycle, or to transmit said phenotype to another plant element with which it is associated in the same plant.

[0509] As used herein, the terms "reduce," "less," "slower," and "increase," "faster," "enhanced," "greater" refer to a decrease or increase in a characteristic of a modified plant element or resulting plant compared to an unmodified plant element or resulting plant. For example, the reduction in a characteristic can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400%, or more below an untreated control. More, the increase can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5% to 10%, at least 10%, 10% to 20%, at least 15%, at least 20%, 20% to 30%, at least 25%, at least 30%, 30% to 40%, at least 35%, at least 40%, 40% to 50%, at least 45%, at least 50%, 50% to 60%, at least about 60%, 60% to 70%, 70% to 80%, at least 75%, at least about 80%, 80% to 90%, at least about 90%, 90% to 100%, at least 100%, 100% and 200%, at least 200%, at least about 300%, at least about 400% or more above an untreated control.

[0510] As used herein, the term "preceded by" when referring to sequence position means that one sequence occurs upstream or 5' to another sequence.

[0511] The meanings of the abbreviations are as follows: “sec” means second, “min” means minute, “h” means hour, “d” means day, “μL” means microliter, “mL” means milliliter, “L” means liter, “μM” means micromole, “mM” means millimole, “M” means mole, “mmol” means millimole, “μmole” or “umole” micromole, “g” means gram, “μg” or “ug” means microgram, “ng” means nanogram, “U” means unit, “bp” means base pair, and “kb” means kilobase.

[0512] Classification of CRISPR-Cas systems

[0513] CRISPR-Cas systems have been classified based on sequence and structural analysis of the components. A variety of CRISPR / Cas systems have been described, including class 1 systems (including types I, III, and IV) with multi-subunit effector complexes, and class 2 systems (including types II, V, and VI) with single protein effectors (Makarova et al., 2015, Nature Reviews Microbiology 13: 1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13; Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6): e60; and Koonin et al., 2017, Curr Opinion Microbiology 37: 67-78).

[0514] The CRISPR-Cas system includes at least one CRISPR RNA (crRNA) molecule and at least one (Cas) protein associated with CRISPR to form a crRNA ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus includes a series of identical repetitive sequences interspersed with DNA targeting spacers encoding crRNA components and operon-like units of cas genes encoding Cas protein components. The generated ribonucleoprotein complex recognizes polynucleotides in a sequence-specific manner (Jore et al., Nature Structural & Molecular Biology [Natural Structure and Molecular Biology] 18, 529-536 (2011)). The crRNA forms a base pair with a complementary DNA strand while displacing a non-complementary strand to form a so-called R loop, thereby acting as an effector (protein or complex) and a double-stranded DNA sequence for sequence-specific binding to a guide RNA. (Jore et al., 2011. Nature Structural & Molecular Biology [Natural Structure and Molecular Biology] 18, 529-536).

[0515] The RNA transcripts (pre-crRNA) of the CRISPR loci are specifically cleaved at repetitive sequences by CRISPR-associated (Cas) endonucleases in type I and III systems or by RNase III in type II systems. The number of CRISPR-associated genes at a given CRISPR locus can vary between species.

[0516] There are different cas genes in different CRISPR systems, which encode proteins with different domains. The cas operon contains genes encoding one or more effector endonucleases and other Cas proteins. Protein subunits include those described in: Makarova et al. 2011, Nat Rev Microbiol. [Nature Review Microbiology] 20119 (6): 467-477; Makarova et al. 2015, Nature Reviews Microbiology [Nature Review Microbiology] Volume 13: 1-15; and Koonin et al. 2017, Current Opinion Microbiology [Current View Microbiology] 37: 67-78). The types of domains include domains involved in expression (pre-crRNA processing, such as Cas 6 or RNase III), interference (including effector modules for crRNA and target binding, and one or more domains for target cleavage), adaptation (spacer insertion, such as Cas1 or Cas2) and auxiliary (regulatory or auxiliary or unknown function). Some domains can serve more than one role, for example Cas9 includes domains for endonuclease function as well as for target cleavage, etc.

[0517] Cas endonucleases are guided by a single CRISPR RNA (crRNA) and recognize DNA target sites immediately adjacent to the protospacer adjacent motif (PAM) by direct RNA-DNA base pairing (Jore, MM et al., 2011, Nat. Struct. Mol. Biol. 18:529-536, Westra, ER et al., 2012, Molecular Cell 46:595-605, and Sinkunas, T. et al., 2013, EMBO J. 32:385-394).

[0518] Class I CRISPR-Cas systems

[0519] Class I CRISPR-Cas systems include Type I, Type III, and Type IV. Class I systems are characterized by the presence of an effector endonuclease complex rather than a single protein. The Cascade complex includes an RNA recognition motif (RRM) and a nucleic acid binding domain, which is the core fold of a diverse RAMP (repeat sequence associated mysterious protein) superfamily of proteins (Makarova et al. 2013, Biochem Soc Trans [Biochemical Society Bulletin] 41, 1392-1400); Makarova et al. 2015, Nature Reviews Microbiology [Nature Review Microbiology] Vol. 13: 1-15). RAMP protein subunits include Cas5 and Cas7 (which comprise the backbone of the crRNA-effector complex), where the Cas5 subunit binds to the 5′ handle of the crRNA and interacts with the large subunit, and typically include Cas6, which is loosely associated with the effector complex and typically functions as a repeat-specific RNase in pre-crRNA processing (Charpentier et al., FEMS Microbiol Rev [FEMS Microbiology Review] 2015, 39:428-441; Niewoehner et al., RNA 2016, 22:318-329).

[0520] The type I CRISPR-Cas system comprises an effector protein complex, called Caseade (CRISPR-associated complex for antiviral defense), which comprises at least Cas5 and Cas7. The effector complex functions together with a single CRISPR RNA (crRNA) and Cas3 to defend against invading viral DNA (Brouns, SJJ et al. Science [Science] 321: 960-964; Makarova et al. 2015, Nature Reviews Microbiology [Nature Review Microbiology] Volume 13: 1-15). The type I CRISPR-Cas locus comprises a signature gene cas3 (or variant cas3′ or cas3′′), which encodes a metal-dependent nuclease having a single-stranded DNA (ssDNA)-stimulated superfamily 2 helicase, which has the ability to unwind double-stranded DNA (dsDNA) and RNA-DNA duplexes (Makarova et al. 2015, Nature Reviews; Microbiology [Nature Review Microbiology] Volume 13: 1-15). After target recognition, the Cas3 endonuclease is recruited to the Caseade-crRNA-target DNA complex to cleave and degrade the DNA target (Westra, ER et al. (2012) Molecular Cell 46:595-605, Sinkunas, T. et al. (2011) EMBO J. 30:1335-1342, and Sinkunas, T. et al. (2013) EMBO J. 32:385-394). In some type I systems, Cas6 is the active endonuclease responsible for crRNA processing, and Cas5 and Cas7 function as non-catalytic RNA binding proteins; although in type IC systems, crRNA processing can be catalyzed by Cas5 (Makarova et al. 2015, Nature Reviews Microbiology 13:1-15). Type I systems are divided into seven subtypes (Makarova et al. 2011, Nat Rev Microbiol. 20119(6):467-477; Koonin et al. 2017, Curr Opinion Microbiology 37:67-78).A modified type I CRISPR-associated complex (Cascade) for adaptive antiviral defense has been described, comprising at least the protein subunits Cas7, Cas5 and Cas6, wherein one of these subunits is synthetically fused to the Cas3 endonuclease or the modified restriction endonuclease FokI (WO2013098244, published on July 4, 2013).

[0521] Type III CRISPR-Cas systems (including multiple cas7 genes) target ssRNA or ssDNA and function as RNases and targeted RNA-activated DNA nucleases (Tamulaitis et al., Trends in Microbiology 25(10)49-61, 2017). Csm (Type III-A) and Cmr (Type III-B) complexes function as RNA-activated single-stranded (ss) DNA enzymes, coupling target RNA binding / cleavage with ssDNA degradation. Upon infection with exogenous DNA, CRISPR RNA (crRNA)-guided Csm or Cmr complexes bind to emerging transcripts and recruit Cas10 DNA enzymes to actively transcribed phage DNA, resulting in degradation of transcripts and phage DNA, rather than host DNA. The Cas10 HD-domain is responsible for ssDNA enzyme activity, and the Csm3 / Cmr4 subunits are responsible for the endoribonuclease activity of the Csm / Cmr complex. The 3′ flanking sequence of the target RNA is crucial for the ssDNA enzyme activity of Csm / Cmr: base pairing with the 5′-handle of crRNA protects the host DNA from degradation.

[0522] Type IV systems, although including the typical type I Cas5 and Cas7 domains and a Cas8-like domain, may lack the CRISPR array that is characteristic of most other CRISPR-Cas systems.

[0523] Class II CRISPR-Cas systems

[0524] Class II CRISPR-Cas systems include types II, V, and VI. Class II systems are characterized by the presence of a single Cas effector protein rather than an effector complex. Type II and V Cas proteins contain a RuvC endonuclease domain that adopts an RNase H fold.

[0525] Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNA contains a spacer region complementary to one strand of the double-stranded DNA target and a region base-paired with tracrRNA (trans-activating CRISPR RNA), which forms an RNA duplex that guides the Cas endonuclease to cut the DNA target, leaving a blunt end. The spacer is obtained by a process involving Cas1 and Cas2 proteins that is not fully understood. Type II CRISPR / Cas loci typically include cas1 and cas2 genes and cas9 genes (Chylinski et al., 2013, RNA Biology [RNA Biology] 10: 726-737; Makarova et al. 2015, Nature Reviews MiCrobiology [Natural Reviews Microbiology] Volume 13: 1-15). Type II CRISR-Cas loci can encode tracrRNA, which is partially complementary to the repeat sequence in the corresponding CRISPR array, and can contain other proteins (such as Csn1 and Csn2). The presence of cas9 near cas1 and cas2 genes is a hallmark of type II loci (Makarova et al. 2015, Nature Reviews Microbiology [Microbial Nature Review] Vol. 13: 1-15).

[0526] Type V CRISPR / Cas systems contain a single Cas endonuclease, including Cpfl (Casl2) (Koonin et al., Curr Opinion Microbiology 37:67-78, 2017), which is an active RNA-guided endonuclease that does not necessarily require additional trans-activating CRISPR (tracr) RNA for target cleavage, unlike Cas9.

[0527] Type VI CRISPR-Cas systems include cas13 genes encoding nucleases with two HEPN (higher eukaryotic and prokaryotic nucleotide binding) domains but without HNH or RuvC domains, and are independent of tracrRNA activity. Most HEPN domains contain conserved motifs that constitute metal-independent endo-RNase active sites (Anantharam et al., Biol Direct [Biological Guidance] 8: 15, 2013). Due to this feature, it is believed that type VI systems act on RNA targets rather than DNA targets common to other CRISPR-Cas systems.

[0528] Novel CRISPR-Cas system

[0529] Disclosed herein are novel CRISPR-Cas systems, components thereof, and methods of using the components. The systems comprise a novel Cas effector protein, Cas-α.

[0530] The novel CRISPR-Cas system components described herein may include one or more subunits from different Cas systems, subunits derived from or modified from more than one different bacterial or archaeal prokaryotes, and / or synthetic or engineered components.

[0531] Described herein is a newly identified CRISPR-Cas system comprising a novel arrangement of cas genes. Further described are novel cas genes and proteins.

[0532] A feature of some novel Cas-α systems is that Figures 1A-1D The locus architecture shown. In some aspects, the Cas-α genomic locus comprises a cas1 gene, a cas2 gene, a cas4 gene, and a cas-α gene encoding an effector protein Cas-α. A CRISPR array comprising a repeat sequence of a nucleotide sequence may be found before or after the gene encoding the Cas-α endonuclease. In some aspects, the cas-α locus may comprise a cas-α gene encoding an effector protein and a CRISPR array comprising a repeat sequence, but does not comprise any one or more of the cas1 gene, the cas2 gene, and / or the cas4 gene.

[0533] Components of the CRISPR-Cas system

[0534] Cas proteins

[0535] Many proteins can be encoded in a CRISPR cas operon, including those involved in adaptation (spacer insertion), interference (effector module target binding, target nicking or cleavage - e.g., endonuclease activity), expression (pre-crRNA processing), regulation, or others.

[0536] The two proteins, Cas1 and Cas2, are conserved in many CRISPR systems (e.g., as described in Koonin et al., Curr Opinion Microbiology 37:67-78, 2017). Cas1 is a metal-dependent DNA-specific endonuclease that generates double-stranded DNA fragments. In some systems, Cas1 forms a stable complex with Cas2, which is essential for spacer acquisition and insertion in CRISPR systems ( et al., Nature Str Mol Biol [Nature Structural Molecular Biology] 21: 528-534, 2014).

[0537] Many other proteins have been identified in different systems, including Cas4, which may have similarities to the RecB nuclease and is thought to play a role in capturing new viral DNA sequences for integration into the CRISPR array (Zhang et al., PLOS One 7(10):e47232, 2012).

[0538] Some proteins may contain multiple functions. For example, Cas9, the signature protein of the class II system, has been shown to be involved in pre-crRNA processing, target binding, and target cleavage.

[0539] The novel Cas-α proteins disclosed herein include effector proteins (endonucleases) and adaptor proteins. Cas endonucleases have been identified from a variety of bacterial and archaeal sources, including Figures 7A-7K Those shown in .

[0540] Cas endonucleases and effectors

[0541] Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains and include restriction endonucleases that cleave DNA at specific sites without damaging bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas ( C RISPR- as societal) effector endonucleases.

[0542] Cas endonucleases (as single effector proteins or effector complexes with other components) unwind DNA duplexes at target sequences and optionally cut at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (e.g., but not limited to crRNA or guide RNA) complexed with the Cas effector protein. If the correct protoplast adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence, such recognition and cutting of the target sequence by the Cas endonuclease typically occurs. Alternatively, the Cas endonucleases herein may lack DNA cutting or nicking activity, but when complexed with a suitable RNA component, can still specifically bind to the DNA target sequence. (See also U.S. patent application US 20150082478 published on March 19, 2015 and US 20150059010 published on February 26, 2015).

[0543] Cas endonucleases can occur as single effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).

[0544] Cas endonucleases that have been described include, but are not limited to, for example: Cas3 (characteristic of 1 class, type I systems), Cas9 (characteristic of 2 class, type II systems), and Cas12 (Cpf1) (characteristic of 2 class, type V systems).

[0545] Cas3 (and its variants Cas3′ and Cas3″) function as a single-stranded DNA nuclease (HD domain) and an ATP-dependent helicase. Variants of Cas3 endonucleases can be obtained by disabling the functional activity of one or both domains of the Cas3 endonuclease polypeptide. Disabling the ATPase-dependent helicase activity (by deleting, knocking out the Cas3-helicase domain, or by mutating key residues, or by assembling the reaction in the absence of ATP, as previously described (Sinkunas, T. et al., 2013, EMBO J. [Journal of the European Molecular Biology Association] 32: 385-394)) can convert a cleavage-ready Cascade comprising a modified Cas3 endonuclease into a nickase (because the HD domain is still functional). Disabling the HD endonuclease activity can be accomplished by any method known in the art, such as, but not limited to, mutation of key residues of the HD domain, which can convert a cleavage-ready Cascade comprising a modified Cas3 endonuclease into a helicase. Disabling the Cas helicase and Cas3 Disabling the HD endonuclease activity can be accomplished by any method known in the art, such as, but not limited to, mutation of key residues in both the helicase and HD domains, which can convert a cleavage-ready Cascade comprising a modified Cas3 endonuclease into a binding protein that binds to the target sequence.

[0546] Cas9 (formerly known as Cas5, Csn1, or Csx12) is a Cas endonuclease that forms a complex with cr nucleotides and tracr nucleotides or with a single guide polynucleotide, which is used to specifically recognize and cut all or part of a DNA target sequence. Cas9 recognizes the 3′ GC-rich PAM sequence on the target dsDNA. The Cas9 protein contains a RuvC nuclease, and an HNH (HNH) nuclease adjacent to the RuvC-II resultant domain. The RuvC nuclease and the HNH nuclease can each cut a single DNA chain at the target sequence (the synergistic action of the two domains results in DNA double-stranded cutting, while the activity of one domain results in a nick). Typically, the RuvC domain contains subdomains I, II, and III, wherein subdomain I is located near the N-terminus of Cas9, and subdomains II and III are located in the middle of the protein, i.e., flanking the HNH domain (Hsu et al., 2013, Cell [Cell] 157: 1262-1278). Cas9 endonucleases are generally derived from type II CRISPR systems, which include DNA cleavage systems utilizing Cas9 endonucleases complexed with at least one polynucleotide component. For example, Cas9 can be complexed with CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In another example, Cas9 can be complexed with a single guide RNA (Makarova et al., 2015, Nature Reviews Microbiology [Nature Review Microbiology] Vol. 13: 1-15).

[0547] Cas12 (formerly known as Cpfl, and variants c2c1, c2c3, CasX, and CasY) contains a RuvC nuclease domain and produces staggered 5' overhangs on dsDNA targets. Unlike the function of Cas9, some variants do not require tracrRNA. Cas12 and its variants recognize 5' AT-rich PAM sequences on target dsDNA. An insertion domain of the Cas12a protein called Nuc has been shown to be responsible for the cutting of the target chain (Yamano et al., Cell [Cell] 2016, 165: 949-962). Other mutation studies in other Cas12 proteins have shown that the Nuc domain contributes to the binding of the guide and target, while the RuvC domain is responsible for cutting (Swarts et al., Mol Cell [Molecular Cell] 2017, 66: 221-233e224).

[0548] Cas endonucleases and effector proteins can be used for targeted genome editing (via single and multiple double-strand breaks and gaps) and targeted genome regulation (via tethering epigenetic effector domains to Cas proteins or sgRNAs). Cas endonucleases can also be engineered to function as RNA-guided recombinases and can serve as scaffolds for assembling multiprotein and nucleic acid complexes via RNA tethers (Mali et al., 2013 Nature Methods [Natural Methods] Vol. 10: 957-963).

[0549] Cas-α endonuclease

[0550] Cas-α endonucleases are defined as functional RNA-guided, PAM-dependent dsDNA cleavage proteins with less than 800 amino acids, comprising: a C-terminal RuvC catalytic domain (split into three subdomains) and further comprising a bridge-helix and one or more zinc finger motifs; and an N-terminal Rec subunit with a helical bundle, a WED wedge (or "oligonucleotide binding domain", OBD) domain, and optionally a zinc finger motif.

[0551] The CRISPR-Cas endonuclease comprises, relative to the amino acid position numbering of SEQ ID NO: 17 when aligned with SEQ ID NO: 17, at least one, at least two, at least three, at least four, at least five, at least six, or seven of the following: glycine (G) at position 337, glycine (G) at position 341, glutamic acid (E) at position 430, leucine (L) at position 432, cysteine ​​(C) at position 487, cysteine ​​(C) at position 490, cysteine ​​(C) at position 507, and / or cysteine ​​(C) or histidine (H) at position 512. The Cas-α endonuclease comprises, the following motifs: GxxxG, ExL, Cx n C, Cx n (C or H) (where n = one or more amino acids).

[0552] It has been documented that the RuvC domain contains endonuclease function. The Cas-α endonuclease can be isolated or identified from a locus comprising a cas-α gene encoding an effector protein and an array comprising multiple repeat sequences. In some aspects, the cas-α locus may also comprise a partial or complete cas1 gene, a cas2 gene, and / or a cas4 gene.

[0553] Zinc finger motifs are domains that coordinate one or more zinc ions, usually by cysteine ​​and histidine side chains to stabilize their folding. Zinc fingers are named after the pattern of cysteine ​​and histidine residues that coordinate the zinc ion (e.g., C4 indicates that the zinc ion is coordinated by four cysteine ​​residues; C3H indicates that the zinc ion is coordinated by three cysteine ​​residues and one histidine residue).

[0554] The Cas-α protein contains one or more zinc finger (ZFN) coordination motifs that can form a zinc binding domain. The zinc finger-like motif can help separate the target and non-target strands and load the guide RNA into the DNA target. The Cas-α protein containing one or more zinc finger motifs can provide additional stability to the ribonucleoprotein complex on the target polynucleotide. The Cas-α protein contains a C4 or C3H zinc binding domain.

[0555] Some Cas-α proteins and polynucleotides are Figures 7A-7K Given in Figures 8A-8K The key structural motifs of endonuclease proteins are depicted separately.

[0556] Cas-α endonucleases are RNA-guided endonucleases that are capable of binding and cleaving a double-stranded DNA target comprising: (1) a sequence having homology to the nucleotide sequence of the guide RNA, and (2) a PAM sequence. In some aspects, the PAM is T-rich. In some aspects, the PAM is C-rich.

[0557] Cas-α endonucleases have the function of double-strand break inducers, and may also be nickases or single-strand break inducers. In some aspects, catalytically inactive Cas-α endonucleases can be used to target or recruit to target DNA sequences but do not induce cleavage. In some aspects, catalytically inactive Cas-α proteins can be used together with functional endonucleases to cut target sequences. In some aspects, catalytically inactive Cas-α proteins can be combined with base editing molecules, such as deaminases. In some aspects, the deaminase can be a cytidine deaminase. In some aspects, the deaminase can be an adenine deaminase. In some aspects, the deaminase can be ADAR-2.

[0558] Cas-alpha endonuclease is further defined as an RNA-guided double-stranded DNA cleavage protein corresponding to SEQ ID NO: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 99, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353 , 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370 and 371 or a functional fragment thereof, or a functional variant thereof that retains at least some of the activity, at least 50, 50 to 100, at least 100, 100 to 150, at least 150, 150 to 200, at least 200, 200 to 250, at least 250, 250 to 300, at least 300, 300 to 350, at least 350, 350 to 400, at least 400, 400 to 450, at least 500 or more %, 500 contiguous amino acids have at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity. A "functional fragment" of a Cas-α endonuclease retains the ability to recognize, bind to, or nick a single strand of a double-stranded polynucleotide, or to cleave both strands of a double-stranded polynucleotide, or any combination of the foregoing.

[0559] The Cas-α endonuclease may be encoded by a polynucleotide having the same sequence as SEQ ID at least 50, 50 to 100, at least 100, 100 to 150, at least 150, 150 to 200, at least 200, 200 to 250, at least 250, 250 to 300, at least 300, 300 to 350, at least 350, 350 to 400, at least 400, 400 to 450, at least 500, 500 to 550, at least 600, 600 to 650, at least 650, 650 to 700, at least 700, 700 to 750, at least 750, 750 to 800, at least 800, 800 to 850, at least 850, 850 to 900, at least 900 %, 900 to 950, at least 950, 950 to 1000, at least 1000 or even more than 1000 consecutive nucleotides having at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity,or encoding SEQ ID NO: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37, 38, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, Any one of 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, and 371. ,

[0560] The Cas endonucleases, effector proteins or functional fragments thereof used in the disclosed methods can be isolated from natural sources or recombinant sources, in which genetically modified host cells are modified to express nucleic acid sequences encoding the proteins. Alternatively, Cas proteins can be produced using cell-free protein expression systems or synthetically produced. Effector Cas nucleases can be isolated and introduced into heterologous cells, or can be modified from their native form to exhibit an activity type or size different from that of their natural source. Such modifications include, but are not limited to, fragments, variants, substitutions, deletions and insertions.

[0561] Fragments and variants of Cas endonucleases and Cas effector proteins can be obtained via methods such as site-directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art, such as, but not limited to, WO 2013166113 published on November 7, 2013, WO 2016186953 published on November 24, 2016, and WO 2016186946 published on November 24, 2016.

[0562] Cas endonucleases may include modified forms of Cas polypeptides. Modified forms of Cas polypeptides may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the naturally occurring nuclease activity of Cas proteins. For example, in some cases, the modified form of the Cas protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas polypeptide (US 20140068797 disclosed on March 6, 2014). In some cases, the modified form of the Cas polypeptide has no substantial nuclease activity and is referred to as catalytically "inactivated Cas" or "inactivated Cas (dCas)". Inactivated Cas / inactivated Cas includes inactivated Cas endonucleases (dCas). Catalytically inactive Cas effector proteins may be fused to heterologous sequences to induce or modify activity.

[0563] The Cas endonuclease can be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1, 2, 3 or more domains other than the Cas protein). Such a fusion protein can include any additional protein sequences, and optionally a linker sequence between any two domains (e.g., between the Cas and the first heterologous domain). Examples of protein domains that can be fused to the Cas proteins herein include, but are not limited to, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]); reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], β-galactosidase, β-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]); and domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity (e.g., VP16 or VP64), transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also be fused to proteins that bind to DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

[0564] The Cas endonuclease of catalytic activity and / or inactivation can be fused to heterologous sequences (US 20140068797 disclosed on March 6, 2014). Suitable fusion partners include, but are not limited to providing active polypeptides, which increase transcription indirectly by acting directly on the target DNA or on a polypeptide (e.g., histone or other DNA-binding proteins) related to the target DNA. Other suitable fusion partners include, but are not limited to providing methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinase activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or a polypeptide of demyristoylation activity. In addition, suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription of the target nucleic acid (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecule / drug-responsive transcriptional regulators, etc.). Partially active or catalytically inactive Cas-α endonucleases can also be fused to another protein or domain, such as Clo51 or FokI nucleases, to produce double-strand breaks (Guilinger et al. Nature Biotechnology, Vol. 32, No. 6, June 2014).

[0565] Catalytically active or inactive Cas proteins, such as the Cas-α proteins described herein, can also be fused to molecules that direct single or multiple base editing in a polynucleotide sequence, such as a site-specific deaminase that can change the identity of a nucleotide, for example, from C·G to T·A or from A·T to G·C (Gaudelli et al., Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al. "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353(6305) (2016); Komor et al. "Programmable editing of a target base in genomic DNA without double-stranded DNA." "Cleavage [Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage]." Nature [Nature] 533(7603) (2016): 420-4.). The base editing fusion protein can include, for example, an active (double-strand break generation), partially active (nickase) or inactive (catalytically inactive) Cas-α endonuclease and a deaminase (such as but not limited to cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). Base editing repair inhibitors and glycosylase inhibitors (e.g., in some embodiments, uracil glycosylase inhibitors (to prevent uracil removal)) are considered as other components of the base editing system.

[0566] The Cas endonucleases described herein can be expressed and purified by methods known in the art, for example as described in WO / 2016 / 186953 published on November 24, 2016.

[0567] To date, many Cas endonucleases have been described that can recognize specific PAM sequences (WO2016186953 published on November 24, 2016, WO 2016186946 published on November 24, 2016, and Zetsche B et al. 2015. Cell [Cell] 163, 1013) and cut target DNA at specific locations. It should be understood that based on the methods and embodiments described herein using the novel guided Cas system, those skilled in the art can now customize these methods so that they can utilize any guided endonuclease system.

[0568] Cas effector protein may include a heterologous nuclear localization sequence (NLS). For example, the heterologous NLS amino acid sequence herein may have sufficient strength to drive the accumulation of a detectable amount of Cas protein in the yeast cell nucleus herein. NLS may include one (monotype) or multiple (e.g., ditype) short sequences (e.g., 2 to 20 residues) of basic, positively charged residues (e.g., lysine and / or arginine), and may be located anywhere in the Cas amino acid sequence, but exposed on the surface of the protein. For example, NLS may be effectively connected to the N-terminus or C-terminus of the Cas protein herein. Two or more NLS sequences may be connected to the Cas protein, for example, at both the N-terminus and the C-terminus of the Cas protein. The Cas endonuclease gene may be operably connected to the SV40 nuclear targeting signal upstream of the Cas codon region and the ditype VirD2 nuclear localization signal downstream of the Cas codon region (Tinland et al., (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 89: 7442-6). Non-limiting examples of suitable NLS sequences herein include those disclosed in US Pat. Nos. 6,660,830 and 7,309,576.

[0569] Guide polynucleotide

[0570] The guidance polynucleotide enables the Cas endonuclease to perform target recognition, binding and optionally cutting, and can be a single molecule or a double molecule. The guidance polynucleotide sequence can be an RNA sequence, a DNA sequence or a combination thereof (RNA-DNA combination sequence). Optionally, the guidance polynucleotide can include at least one nucleotide, a phosphodiester bond or a connection modification, such as but not limited to a locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, a thiophosphate bond, a connection with a cholesterol molecule, a connection with a polyethylene glycol molecule, a connection with a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent connection resulting in cyclization. The guidance polynucleotide comprising only ribonucleic acid is also referred to as "guidance RNA" or "gRNA" (US 20150082478 disclosed on March 19, 2015 and US 20150059010 disclosed on February 26, 2015). The guidance polynucleotide can be engineered or synthesized.

[0571] The guide polynucleotide includes a chimeric non-naturally occurring guide RNA that includes regions that are not found together in nature (i.e., they are heterologous to each other). For example, the chimeric non-naturally occurring guide RNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA, the first nucleotide sequence domain being linked to a second nucleotide sequence that can recognize a Cas endonuclease, such that the first and second nucleotide sequences are not found linked together in nature.

[0572] The guiding polynucleotide can be a double molecule (also referred to as a duplex guiding polynucleotide) comprising a cr nucleotide sequence (e.g., crRNA) and a tracr nucleotide (e.g., tracrRNA) sequence. In some cases, there is a linker polynucleotide, such as an sgRNA, connecting crRNA and tracrRNA to form a single guiding object.

[0573] The cr nucleotide includes a first nucleotide sequence region (referred to as a variable targeting domain or VT domain) that can be hybridized with a nucleotide sequence in a target DNA and a second nucleotide sequence (also referred to as a tracr pairing sequence) that is a part of a Cas endonuclease recognition (CER) domain. The tracr pairing sequence can hybridize with the tracr nucleotide along the complementary region and form a Cas endonuclease recognition domain or a CER domain together. The CER domain can interact with the Cas endonuclease polypeptide. The cr nucleotides and tracr nucleotides of the duplex guidance polynucleotide can be RNA, DNA and / or RNA-DNA combination sequences. In certain embodiments, the cr nucleotide molecules of the duplex guidance polynucleotide are referred to as "crDNA" (when composed of a continuous extension of DNA nucleotides) or "crRNA" (when composed of a continuous extension of RNA nucleotides) or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The cr nucleotides may be included in fragments of crRNA naturally occurring in bacteria and archaea. The size of the crRNA fragments naturally occurring in bacteria and archaea that may be present in the cr nucleotides disclosed herein may be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the crRNA molecule is selected from the group consisting of: SEQ ID NO: 57, 58 and 59.

[0574] In some embodiments, the tracr nucleotides are referred to as "tracrRNA" (when composed of a continuous stretch of RNA nucleotides) or "tracrDNA" (when composed of a continuous stretch of DNA nucleotides) or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In one embodiment, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. In the 5'- to -3' direction, the tracrRNA (trans-activating CRISPR RNA) comprises (i) a sequence that anneals to the repeat region of the CRISPR type II crRNA and (ii) a stem-loop-containing portion (Deltcheva et al., Nature 471: 602-607). The duplex guide polynucleotide can form a complex with a Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cut (introduce a single-strand or double-strand break) the target site. (US 20150082478 published on March 19, 2015 and US 20150059010 published on February 26, 2015).

[0575] In some embodiments, the tracrRNA molecule is selected from the group consisting of: SEQ ID NOs: 60-68.

[0576] In one aspect, the guiding polynucleotide is a guiding polynucleotide capable of forming the PGEN described herein, wherein the guiding polynucleotide comprises a first nucleotide sequence domain complementary to a nucleotide sequence in a target DNA and a second nucleotide sequence domain interacting with the Cas endonuclease polypeptide.

[0577] In one aspect, the guide polynucleotide is a targeting polynucleotide described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain are selected from the group consisting of: a DNA sequence, an RNA sequence, and a combination thereof.

[0578] In one aspect, the guide polynucleotide is a guide polynucleotide as described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain are selected from the group consisting of: RNA backbone modifications that enhance stability, DNA backbone modifications that enhance stability, and combinations thereof (see Kanasty et al., 2013, Common RNA-backbone modifications, Nature Materials 12:976-977; US 20150082478 published on March 19, 2015 and US 20150059010 published on February 26, 2015)

[0579] The guide RNA includes a bimolecule comprising a chimeric non-natural crRNA connected to at least one tracrRNA. The chimeric non-natural crRNA includes a crRNA that is contained in a region that is not found together in nature (i.e., they are heterologous to each other). For example, the crRNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA, and the first nucleotide sequence domain is connected to a second nucleotide sequence (also referred to as a tracr pairing sequence) so that the first and second sequences are not found to be connected together in nature.

[0580] The guide polynucleotide can also be a single molecule comprising a cr nucleotide sequence linked to a tracr nucleotide sequence (also referred to as a single guide polynucleotide). The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as a variable targeting ( V ariable T argeting) domain or VT domain) and a Cas endonuclease recognition ( C as e ndonuclease r Ecognition) domain (CER domain). In some embodiments, the sgRNA molecule is selected from the group consisting of: SEQ ID NO: 69-77.

[0581] The VT domain and / or CER domain of a single guide polynucleotide may comprise an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. A single guide polynucleotide consisting of a sequence from a cr nucleotide and a tracr nucleotide may be referred to as a "single guide RNA" (when consisting of a continuous extension of RNA nucleotides) or a "single guide DNA" (when consisting of a continuous extension of DNA nucleotides) or a "single guide RNA-DNA" (when consisting of a combination of RNA and DNA nucleotides). A single guide polynucleotide may form a complex with a Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) may guide the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to the target site, and optionally cause the target site to produce a nick or cut (introduce a single-strand or double-strand break) target site. (US 20150082478, published on March 19, 2015, and US20150059010, published on February 26, 2015).

[0582] Chimeric non-naturally occurring single guide RNAs (sgRNAs) include sgRNAs that include regions that are not found together in nature (i.e., they are heterologous to each other). For example, an sgRNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA, which is linked to a second nucleotide sequence (also referred to as a tracr mate sequence) that is not found linked together in nature.

[0583] The nucleotide sequence connecting the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. In an embodiment, the nucleotide sequence connecting the cr nucleotides and the tracr nucleotides of the single guide polynucleotide (also referred to as a "loop") can be at least 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 98, 99, or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can include a tetraloop sequence, such as, but not limited to, a GAAA tetraloop sequence.

[0584] The guide polynucleotides can be produced by any method known in the art, including chemically synthesized guide polynucleotides (e.g., but not limited to, Hendel et al. 2015, Nature Biotechnology 33, 985-989), in vitro produced guide polynucleotides, and / or self-splicing guide RNAs (e.g., but not limited to, Xie et al. 2015, PNAS 112: 3570-3575).

[0585] Protospacer adjacent motif (PAM)

[0586] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to a (targeted) target sequence (pre-spacer) that can be recognized by a guide polynucleotide / Cas endonuclease system. If the target DNA sequence is not behind the PAM sequence, the Cas endonuclease may not successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0587] "Random PAM" and "random protospacer adjacent motif" are used interchangeably herein and refer to a random DNA sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide / Cas endonuclease system. The random PAM sequence can be of any length, but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. Random nucleotides include any of the nucleotides A, C, G, or T.

[0588] Guide polynucleotide / Cas endonuclease complex

[0589] The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or a portion of a target sequence.

[0590] The guiding polynucleotide / Cas endonuclease complex that can cut both chains of the DNA target sequence generally comprises a Cas protein with all its endonuclease domains in a functional state (e.g., a wild-type endonuclease domain or a variant thereof retains some or all activity in each endonuclease domain). Therefore, a wild-type Cas protein (e.g., a Cas protein disclosed herein) or a variant thereof that retains some or all activity in each endonuclease domain of the Cas protein is a suitable example of a Cas endonuclease that can cut both chains of the DNA target sequence.

[0591] The guiding polynucleotide / Cas endonuclease enzyme complex that can cut one strand of the DNA target sequence can be characterized as having nickase activity (e.g., partial cleavage ability) herein. Cas nickases generally include a functional endonuclease domain that allows Cas to cut only one strand of the DNA target sequence (i.e., form a nick). For example, the Cas9 nickase may include (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, the Cas9 nickase may include (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a mutated dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in US 20140189896, published on July 3, 2014. A pair of Cas nickases can be used to increase the specificity of DNA targeting. In general, this can be carried out by providing two Cas nickases, which are associated with RNA components with different guide sequences, and are targeted and nicked near the DNA sequence on the opposite strands in the region of the target. Such a nearby cutting of each DNA strand produces a double-strand break (i.e., a DSB with a single-stranded overhang), which is then identified as a substrate for non-homologous end joining (NHEJ) (tending to produce an imperfect repair causing a mutation) or homologous recombination (HR). Each nick in these embodiments can be separated from each other by, for example, at least about 5, 5 to 10, at least 10, 10 to 15, at least 15, 15 to 20, at least 20, 20 to 30, at least 30, 30 to 40, at least 40, 40 to 50, at least 50, 50 to 60, at least 60, 60 to 70, at least 70, 70 to 80, at least 80, 80 to 90, at least 90, 90 to 100 or 100 or more (or any integer of 5 to 100) bases. One or two Cas nickase proteins herein can be used for Cas nickase pairs. For example, a Cas9 nickase (i.e., Cas9 HNH+ / RuvC-) with a RuvC domain having a mutation but with a functional HNH domain (e.g., Streptococcus pyogenes Cas9HNH+ / RuvC-) can be used. By using a suitable RNA component herein (with a guide RNA sequence for targeting each specific DNA site to each nickase), each Cas9 nickase (e.g., Cas9HNH+ / RuvC-) is guided to a specific DNA site adjacent to each other (separating up to 100 base pairs).

[0592] In certain embodiments, the polynucleotide / Cas endonuclease complex can bind to the DNA target site sequence, but does not cut any chain at the target site sequence. Such a complex can include a Cas protein in which all nuclease domains are mutated and dysfunctional. For example, a Cas9 protein that can bind to a DNA target site sequence but does not cut any chain at the target site sequence can include a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. The Cas protein herein that binds but does not cut the target DNA sequence can be used to regulate gene expression, for example, in this case, the Cas protein can be fused to a transcription factor (or part thereof) (e.g., an inhibitor or activator, such as any of those disclosed herein).

[0593] In one aspect, the guide polynucleotide / Cas endonuclease complex (PGEN) described herein is a PGEN, wherein the Cas endonuclease is optionally covalently or non-covalently linked or assembled to at least one protein subunit or a functional fragment thereof.

[0594] In one embodiment of the present disclosure, the guide polynucleotide / Cas endonuclease complex is a guide polynucleotide / Cas endonuclease complex (PGEN) comprising at least one guide polynucleotide and at least one Cas endonuclease polypeptide, wherein the Cas endonuclease polypeptide comprises at least one protein subunit or a functional fragment thereof, wherein the guide polynucleotide is a chimeric non-naturally occurring guide polynucleotide, wherein the guide polynucleotide / Cas endonuclease complex is capable of recognizing, binding to all or part of a target sequence and optionally causing all or part of a target sequence to nick, unwind or cut all or part of the target sequence.

[0595] The Cas effector protein may be a Cas-α effector protein as disclosed herein.

[0596] In one embodiment of the present disclosure, the guide polynucleotide / Cas effector complex is a guide polynucleotide / Cas effector protease complex (PGEN) comprising at least one guide polynucleotide and a Cas-α effector protein, wherein the guide polynucleotide / Cas effector protein complex is capable of recognizing, binding to all or part of a target sequence and optionally nicking, unwinding or cleaving all or part of the target sequence.

[0597] The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises one or more copies of at least one protein subunit or its functional fragment. In some embodiments, the protein subunit is selected from the group consisting of Cas1 protein subunit, Cas2 protein subunit, Cas4 protein subunit and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises at least two different protein subunits selected from the group consisting of Cas1, Cas2 and Cas4.

[0598] The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises at least three different protein subunits or functional fragments thereof, the protein subunits being selected from the group consisting of Cas1, Cas2 and optionally an additional Cas protein comprising Cas4.

[0599] In one aspect, the guide polynucleotide / Cas effector protein complex (PGEN) described herein is a PGEN, wherein the Cas effector protein is covalently or non-covalently linked to at least one protein subunit or a functional fragment thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein polypeptide is covalently or non-covalently linked or assembled to one or more copies of at least one protein subunit or a functional fragment thereof, wherein the at least one protein subunit is selected from the group consisting of: Cas1 protein subunit, Cas2 protein subunit, optionally comprising a Cas4 protein subunit, and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein is covalently or non-covalently linked or assembled to at least two different protein subunits, wherein the at least two different protein subunits are selected from the group consisting of: Cas1, Cas2, and optionally comprising a Cas4. One additional Cas protein. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein is covalently or non-covalently linked to at least three different protein subunits or functional fragments thereof, and the at least three different protein subunits are selected from the group consisting of: Cas1, Cas2, and optionally one additional Cas protein including Cas4, and any combination thereof.

[0600] Any components of the guide polynucleotide / Cas effector protein complex, the guide polynucleotide / Cas effector protein complex itself, as well as one or more polynucleotide modification templates and / or one or more donor DNAs can be introduced into a heterologous cell or organism by any method known in the art.

[0601] Recombinant constructs for cell transformation

[0602] The guiding polynucleotides, Cas endonucleases, polynucleotide modification templates, donor DNA, guiding polynucleotide / Cas endonuclease systems, and any combination thereof (optionally further comprising one or more polynucleotides of interest) disclosed herein can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein.

[0603] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are more fully described in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described below.

[0604] Vectors and constructs include circular plasmids and linear polynucleotides comprising a polynucleotide of interest, and optionally include linkers, adapters, other components for regulation or analysis. In some examples, the recognition site and / or target site may be contained within an intron, coding sequence, 5'UTR, 3'UTR, and / or regulatory region.

[0605] Expression and utilization of components of novel CRISPR-Cas systems in prokaryotic and eukaryotic cells

[0606] The present invention also provides expression constructs for expressing a guide RNA / Cas system in a prokaryotic or eukaryotic cell / organism that is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or part of a target sequence.

[0607] In one embodiment, the expression construct of the present invention comprises a promoter operably connected to a nucleotide sequence encoding a Cas gene (or plant-optimized, including a Cas endonuclease gene as described herein) and a promoter operably connected to a guide RNA of the present disclosure. The promoter is capable of driving the expression of a nucleotide sequence operably connected in a prokaryotic or eukaryotic cell / organism.

[0608] The nucleotide sequence modifications of the guide polynucleotide, VT domain and / or CER domain can be selected from, but not limited to, the group consisting of: a 5′ cap, a 3′ poly(A) tail, a riboswitch sequence, a stability control sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a protein binding site, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2′-fluoro A nucleotides, 2′-fluoro U nucleotides, 2′-O-methyl RNA nucleotides, a phosphorothioate bond, a linkage to a cholesterol molecule, a linkage to a polyethylene glycol molecule, a linkage to a spacer 18 molecule, a 5′ to 3′ covalent linkage, or any combination thereof. The modifications can produce at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group consisting of: modified or regulated stability, subcellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity to complementary target sequences, modified resistance to cellular degradation and increased cellular permeability.

[0609] Methods for expressing RNA components (e.g., gRNA) in eukaryotic cells for Cas9-mediated DNA targeting have used RNA polymerase III (Pol III) promoters, which allow transcription of RNA with precisely defined unmodified 5′- and 3′-ends (DiCarlo et al., Nucleic Acids Res. 41:4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:el61). This strategy has been successfully applied in cells of several different species, including maize and soybean (US 20150082478, published on March 19, 2015). Methods for expressing RNA components that do not have a 5′ cap have been described (WO 2016 / 025131, published on February 18, 2016).

[0610] Different methods and compositions can be used to obtain cells or organisms having a target polynucleotide inserted into a target site for a Cas endonuclease. Such methods can use homologous recombination (HR) to provide integration of the target polynucleotide at the target site. In a method described herein, the target polynucleotide is introduced into an organism cell via a donor DNA construct.

[0611] The donor DNA construct further comprises a first region and a second region of homology flanking the target polynucleotide. The first region and the second region of homology of the donor DNA are homologous to the first and second genomic regions present in or flanking the target site of the cell or organism genome, respectively.

[0612] The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, which can be used for genome editing, gene insertion, and targeted genome regulation, and can also be used to target late mitotic cells, in which the function of the endogenous HR machinery is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Nature Methods] Vol. 10: 957-963).

[0613] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450 In some embodiments, the target site may be a target site having a length of 1-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or a region having a unit integer value in a range up to and including the total length of the target site. These ranges include each integer in the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology can also be described by the percent sequence identity over the entire compared length of the two polynucleotides, including 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%, 98% to 99%, 99%, 99% to 100% or 100% percent sequence identity. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example, sufficient homology can be described as a region of 75-150 bp having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be described by the predicted ability of two polynucleotides to hybridize specifically under high stringency conditions, see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., eds. (1994) Current Protocols (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes (Elsevier, New York).

[0614] The structural similarity between a given genomic region and the corresponding homologous region found on the donor DNA can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of homology or sequence identity shared by the "homology region" of the donor DNA and the "genomic region" of the organism's genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, so that the sequence undergoes homologous recombination.

[0615] The homologous regions on the donor DNA may have homology with any sequence flanking the target site. Although in some cases, the homologous regions share significant sequence homology with the genomic sequences immediately flanking the target site, it should be recognized that the homologous regions can be designed to have sufficient homology with regions that may be closer to the 5' or 3' of the target site. The homologous regions may also have homology with fragments of the target site and downstream genomic regions.

[0616] In one embodiment, the first region of homology further comprises a first segment in the target site, and the second region of homology comprises a second segment in the target site, wherein the first segment and the second segment are different.

[0617] Target polynucleotide

[0618] Further described in this article is the target polynucleotide, and comprises the polynucleotide of those commercial markets and interests that reflect and relate to crop development.Target crop and market change, and along with developing country open international market, new crop and technology also will appear.In addition, along with our understanding of agronomy character and characteristic (for example output and heterosis increase) gradually deepen, the selection of the gene that is used for genetic engineering will change accordingly.

[0619] General categories of polynucleotides of interest include, for example, those genes of interest involved in information (e.g., zinc fingers), those genes involved in communication (e.g., kinases), and those genes involved in housekeeping (e.g., heat shock proteins). More specific polynucleotides of interest include, but are not limited to, genes involved in traits of agronomic importance, such as, but not limited to, crop yield, grain quality, crop nutrient content, starch and carbohydrate quality and quantity, genes associated with and affecting grain size, sucrose loading, protein quality and quantity, nitrogen fixation and / or nitrogen utilization, fatty acid and oil composition, genes encoding proteins that confer resistance to abiotic stresses (e.g., drought, nitrogen, temperature, salinity, toxic metals, or trace elements), or those conferring resistance to toxins (e.g., pesticides and herbicides), genes encoding proteins that confer resistance to biotic stresses (e.g., fungal, viral, bacterial, insect and nematode attack and the development of diseases associated with these organisms).

[0620] In addition to using traditional breeding methods, agronomically important traits (e.g., oil, starch, and protein content) can also be altered genetically. Modifications include increasing the content of oleic acid, saturated and unsaturated oils, increasing the levels of lysine and sulfur, providing essential amino acids, and also modifications to starch. Protein modifications of hordothionin are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0621] The polynucleotide sequence of interest can encode proteins that are involved in providing disease or pest resistance. "Disease resistance" or "pest resistance" is intended to be a plant that avoids the occurrence of harmful symptoms that are a consequence of plant-pathogen interactions. Pest resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borers, etc. Disease resistance genes and insect resistance genes, such as lysozymes or cecropins for antibacterial protection, or proteins for antifungal protection, such as defensins, glucanases, or chitinases, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects, are examples of useful gene products. Genes encoding disease resistance traits include detoxification genes, such as resistance to fumonisins (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); etc. Insect resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borers, etc. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); etc.

[0622] "Herbicide resistance proteins" or proteins expressed by "herbicide resistance encoding nucleic acid molecules" include proteins that confer to cells the ability to tolerate higher concentrations of herbicides compared to cells that do not express the protein, or confer to cells the ability to tolerate a certain concentration of herbicides for a longer period of time compared to cells that do not express the protein. The herbicide resistance trait can be introduced into plants by genes encoding resistance to herbicides that inhibit acetolactate synthase (ALS, also known as acetohydroxyacid synthase, AHAS) (particularly sulfonylurea (UK: sulfonylurea (sulphonylurea)) herbicides), genes encoding resistance to herbicides that inhibit glutamine synthase (e.g., phosphinothricin or basta) (e.g., bar genes), genes encoding resistance to glyphosate (e.g., EPSP synthase genes and GAT genes), genes encoding resistance to HPPD inhibitors (e.g., HPPD genes), or other such genes known in the art. See, e.g., U.S. Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and 9,187,762. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutant encodes resistance to the herbicide chlorsulfuron.

[0623] In addition, it is recognized that the target polynucleotide can also include an antisense sequence complementary to at least a portion of the messenger RNA (mRNA) of the gene sequence targeted for the purpose. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. The antisense sequence can be modified as long as the sequence hybridizes with the corresponding mRNA and interferes with the expression of the corresponding mRNA. In this way, an antisense construct with 70%, 80% or 85% sequence identity to the corresponding antisense sequence can be used. In addition, the part of the antisense nucleotide can be used to destroy the expression of the target gene. Usually, a sequence of at least 50 nucleotides, 100 nucleotides, 200 nucleotides or more nucleotides can be used.

[0624] In addition, the target polynucleotide can also be used in a sense orientation to suppress the expression of endogenous genes in plants. The method of using polynucleotides in a sense orientation to suppress gene expression in plants is known in the art. These methods generally relate to transformed plants with a DNA construct comprising a promoter, which is operably connected to at least a portion of the nucleotide sequence of the transcript corresponding to the endogenous gene, driving the expression in the plant. Generally, such nucleotide sequences have substantial sequence identity with the sequence of the transcript of the endogenous gene, generally greater than about 65% sequence identity, about 85% sequence identity or greater than about 95% sequence identity. Referring to U.S. Patent Nos. 5,283,184 and 5,034,323.

[0625] The target polynucleotide can also be a phenotypic marker. Phenotypic markers are screenable or selectable markers, which include visual markers and selectable markers, whether it is a positive or negative selectable marker. Any phenotypic marker can be used. Specifically, selectable or screenable markers include DNA segments that allow people to identify or select molecules or cells that contain it or select it under specific conditions. These markers can encode activity, such as but not limited to the production of RNA, peptides or proteins, or can provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, etc.

[0626] Examples of selectable markers include, but are not limited to, DNA segments comprising restriction endonuclease sites; DNA segments encoding products that provide resistance to otherwise toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT); DNA segments encoding products that are inherently deficient in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that can be easily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and cell surface proteins); generating new primer sites for PCR (e.g., juxtaposition of two DNA sequences that were not previously juxtaposed), comprising DNA sequences that are inactivated or activated by restriction endonucleases or other DNA modifying enzymes, chemicals, etc.; and comprising DNA sequences required for specific modifications (e.g., methylation) that allow their identification.

[0627] Additional selectable markers include genes that confer resistance to herbicidal compounds such as sulfonylureas, glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See, for example, acetolactate synthase (ALS) for resistance to sulfonylureas, imidazolinones, triazolopyrimidinesulfonamides, pyrimidine salicylates, and sulfonylaminocarbonyl-triazolinones (Shaner and Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69-110); glyphosate resistance 5-enolpyruvylshikimate-3-phosphate (EPSPS) (Saroha et al., 1998, J. Plant Biochemistry & Biotechnology 7: 65-72);

[0628] Target polynucleotides include genes that are stacked or used in combination with other traits (such as, but not limited to, herbicide resistance or any other trait described herein). Target polynucleotides and / or traits can be stacked together in a complex trait locus, as described in US 20130263324, published on October 3, 2013, and WO / 2013 / 112686, published on August 1, 2013.

[0629] The polypeptide of interest includes a protein or polypeptide encoded by a polynucleotide of interest described herein.

[0630] Further provided is a method for identifying at least one plant cell, which contains a polynucleotide of interest integrated at the target site in its genome. A variety of methods can be used to identify those plant cells inserted into the genome at or near the target site. Such methods can be considered to directly analyze the target sequence to detect any change in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blotting, and any combination thereof. See, for example, US 20090133152 disclosed on May 21, 2009. The method also includes reobtaining plants from plant cells containing polynucleotides of interest integrated into its genome. The plant can be sterile or fertile. It should be appreciated that any polynucleotide of interest can be provided, which is integrated into the genome of the plant at the target site and expressed in the plant.

[0631] Optimization of sequences for expression in plants

[0632] Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498. Additional sequence modifications are known to enhance gene expression in plant hosts. For example, these sequence modifications include elimination of: one or more sequences encoding false polyadenylation signals, one or more exon-intron splice site signals, one or more transposon-like repeats, and other such well-characterized sequences that may be harmful to gene expression. The GC content of the sequence can be adjusted to an average level for a given plant host calculated by reference to known genes expressed in host plant cells. When possible, the sequence is modified to avoid the presence of one or more predicted hairpin secondary mRNA structures. Therefore, the "plant-optimized nucleotide sequence" of the present disclosure includes one or more such sequence modifications.

[0633] Expression elements

[0634] Any polynucleotide encoding Cas protein or other CRISPR system components herein can be functionally connected to a heterologous expression element to promote transcription or regulation in a host cell. Such expression elements include, but are not limited to, promoters, leaders, introns, and terminators. An expression element can be "minimal" - meaning a shorter sequence derived from a natural source that still acts as an expression regulator or modifier. Alternatively, an expression element can be "optimized" - meaning that its polynucleotide sequence has been changed from its native state so that it can function with more desirable features in a specific host cell (for example, but not limited to, a bacterial promoter can be "maize optimized" to improve its expression in corn plants). Alternatively, an expression element can be "synthetic" - meaning that it is designed with a computer and synthesized for use in a host cell. Synthetic expression elements can be completely synthetic or partially synthetic (fragments comprising naturally occurring polynucleotide sequences).

[0635] Certain promoters have been shown to be able to direct RNA synthesis at a higher rate than other promoters. These are referred to as "strong promoters". Certain other promoters have been shown to direct RNA synthesis only at higher levels in specific types of cells or tissues, and if the promoter preferentially directs RNA synthesis in certain tissues but also at reduced levels in other tissues, it is often referred to as a "tissue-specific promoter" or "tissue-preferential promoter".

[0636] Plant promoters include promoters that are capable of initiating transcription in plant cells. For a review of plant promoters, see Potenza et al., 2004 In vitro Cell Dev Biol 40: 1-22; Porto et al., 2014, Molecular Biotechnology (2014), 56(1), 38-49.

[0637] Constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al., (1989) Plant Mol Biol 12:619-32; ALS promoter (U.S. Pat. No. 5,659,026), etc.

[0638] Tissue-preferred promoters can be used to target enhanced expression in specific plant tissues. Tissue-preferred promoters include, for example, WO 2013103367 published on July 11, 2013, Kawamata et al., (1997) Plant Cell Physiol [Plant Cell Physiology] 38: 792-803; Hansen et al., (1997) Mol Gen Genet [Molecular and General Genetics] 254: 337-43; Russell et al., (1997) Transgenic Res [Transgenic Research] 6: 157-68; Rinehart et al., (1996) Plant Physiol [Plant Physiology] 112: 1331-41; Van Camp et al., (1996) Plant Physiol. [Plant Physiology] 112: 525-35; Canevascini et al., (1996) Plant Physiol. [Plant Physiology] 112: 525-35; Physiol. [Plant Physiology] 112: 513-524; Lam, (1994) Results Probl Cell Differ [Results and Problems in Cell Differentiation] 20: 181-96; and Guevara-Garcia et al., (1993) Plant J. [Plant Journal] 4: 495-505. Leaf-preferred promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18; Orozco et al., (1993) Plant Mol Biol 23:1129-38; Matsuoka et al., (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al., (1958) EMBO J [Journal of the European Molecular Biology Association] 4: 2723-9; Timko et al., (1988) Nature 318: 57-8.Root-preferred promoters include, for example, Hire et al., (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al., (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of mannopine synthase (MAS) from Agrobacterium tumefaciens); Bogusz et al., (1990) Plant Cell 3:11-23 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al., (1990) Plant Mol Biol 14:433-43 (root-specific promoter of mannopine synthase (MAS) from Agrobacterium tumefaciens); Cell 2:633-41 (root-specific promoter isolated from Parasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (rolC and rolD root-inducible genes of Agrobacterium rhizogenes); Teeri et al., (1989) EMBO J 8:343-50 (Agrobacterium wound-induced TR1′ and TR2′ genes); VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana et al., (1994) Plant Mol Biol Biol [Plant Molecular Biology] 25: 681-91); phaseolin gene (Murai et al., (1983) Science [Science] 23: 476-82; Sengopta-Gopalen et al., (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 82: 3320-4). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

[0639] Seed-preferred promoters include both seed-specific promoters active during seed development and seed germination promoters active during seed germination. See Thompson et al., (1989) BioEssays 10: 108. Seed-preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); and milps (inositol-1-phosphate synthase); and, for example, those disclosed in WO 2000011177 published on March 2, 2000 and U.S. Pat. No. 6,225,529. For dicotyledons, seed-preferred promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters include, but are not limited to, maize 15 kDa maize protein, 22 kDa maize protein, 27 kDa gamma maize protein, wax, contractin 1, contractin 2, globulin 1, oleosin, and nucl. See also WO 2000012733 published on March 9, 2000, which discloses seed-preferred promoters from END1 and END2 genes.

[0640] Chemically inducible (regulatable) promoters can be used to regulate gene expression in prokaryotic and eukaryotic cells or organisms by applying exogenous chemical regulators. The promoter can be a chemical-inducible promoter in the case where the application of a chemical induces gene expression, or a chemical-repressible promoter in the case where the application of a chemical represses gene expression. Chemically inducible promoters include, but are not limited to, the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter activated by hydrophobic electrophilic compounds used as pre-emergence herbicides (GST-II-27, WO 1993001294 published on January 21, 1993), and the tobacco PR-1a promoter activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Other chemically-regulated promoters include steroid-responsive promoters (see, e.g., glucocorticoid-inducible promoters (Schena et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al., (1998) Plant J 14:247-257); tetracycline-inducible promoters and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156).

[0641] Pathogen-inducible promoters that are induced upon infection by pathogens include, but are not limited to, promoters that regulate the expression of PR proteins, SAR proteins, β-1,3-glucanase, chitinase, and the like.

[0642] Stress-inducible promoters include the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17: 287-91). Those skilled in the art are familiar with protocols for simulating stress conditions (such as drought, osmotic stress, salt stress, and temperature stress) and evaluating stress tolerance in plants that have been subjected to simulated or naturally occurring stress conditions.

[0643] Another example of an inducible promoter useful in plant cells is the ZmCAS1 promoter, described in US 20130312137 published on November 21, 2013.

[0644] New promoters of different types that are useful in plant cells are continually being discovered; many examples can be found in the compilation by Okamuro and Goldberg, (1989) The Biochemistry of Plants, Vol. 115, Stumpf and Conn, eds. (New York, NY: Academic Press), pp. 1-82.

[0645] Genome modification using novel CRISPR-Cas system components

[0646] As described herein, the guided Cas endonuclease can recognize, bind to the DNA target sequence, and introduce single-strand (nick) or double-strand breaks. Once single-strand breaks or double-strand breaks are induced in DNA, the cell's DNA repair mechanism is activated to repair the break. Error-prone DNA repair mechanisms can produce mutations at the site of double-strand breaks. The most common repair mechanism used to combine the ends of the breaks is the non-homologous end joining (NHEJ) pathway (Bleuyard et al., (2006) DNA Repair [DNA repair] 5: 1-12). The structural integrity of chromosomes is typically preserved by repair, but deletions, insertions, or other rearrangements (such as chromosome translocations) are possible (Siebert and Puchta, 2002 Plant Cell [plant cells] 14: 1121-31; Pacher et al., 2007 Genetics [genetics] 175: 21-9).

[0647] DNA double-strand breaks appear to be potent factors in stimulating homologous recombination pathways (Puchta et al. (1995) Plant Mol Biol 28:281-92; Tzfira and White (2005) Trends Biotechnol 23:567-9; Puchta (2005) J Exp Bot 56:1-14). Using DNA-breaking agents, a two- to nine-fold increase in homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al. (1995) Plant Mol Biol 28:281-92). Experiments with linear DNA molecules in maize protoplasts demonstrated enhanced homologous recombination between plasmids (Lyznik et al. (1991) Mol Gen Genet 230:209-18).

[0648] Homologous-directed repair (HDR) is a mechanism used in cells to repair double-stranded and single-stranded DNA breaks. Homologous-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem [Annual Review of Biochemistry]. 79: 181-211). The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirement between donor and recipient DNA. Other forms of HDR include single-strand annealing (SSA) and break-induced replication, and these require shorter sequence homology relative to HR. Homologous-directed repair at gaps (single-strand breaks) can occur via a different mechanism from HDR at double-strand breaks (Davis and Maizels. PNAS [Proceedings of the National Academy of Sciences of the United States] (0027-8424), 111 (10), pages E924-E932).

[0649] The alteration of the genome of prokaryotic and eukaryotic cells or biological cells, for example by homologous recombination (HR), is a powerful tool for genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al., (1992) Mol Gen Genet [Molecular and General Genetics] 231: 186-93) and insects (Dray and Gloor, 1997, Genetics [Genetics] 147: 689-99). Homologous recombination can also be achieved in other organisms. For example, in the parasitic protozoan Leishmania, at least 150-200 bp of homology is required for homologous recombination (Papadopoulou and Dumas, (1997) Nucleic Acids Res [Nucleic Acids Research] 25: 4278-86). In the filamentous fungus Aspergillus nidulans, gene replacement has been achieved with only 50 bp of flanking homology (Chaveroche et al., (2000) Nucleic Acids Res 28:e97). Targeted gene replacement has also been demonstrated in the ciliate Tetrahymena thermophila (Gaertig et al., (1994) Nucleic Acids Res 22:5391-8). In mammals, homologous recombination has been most successful in mice using pluripotent embryonic stem (ES) stem cell lines that can be grown in culture, transformed, selected, and introduced into mouse embryos (Watson et al., (1992) Recombinant DNA, 2nd ed., Scientific American Books distributed by WH Freeman & Co.).

[0650] Gene targeting

[0651] The guide polynucleotide / Cas system described herein can be used for gene targeting.

[0652] Typically, DNA targeting can be performed by cutting one or both strands at a specific polynucleotide sequence in a cell with a Cas protein associated with an appropriate polynucleotide component. Once single-strand breaks or double-strand breaks are induced in the DNA, the cell's DNA repair mechanism is activated to repair the break via non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes that result in modifications at the target site.

[0653] The length of the DNA sequence at the target site can vary and includes, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 nucleotides in length. It is also possible that the target site can be palindromic, that is, the sequence on one strand is the same as reading in the opposite direction on the complementary strand. The nick / cleavage site can be within the target sequence, or the nick / cleavage site can be outside the target sequence. In another variation, the cleavage can occur at nucleotide positions that are exactly opposite to each other to produce a blunt-end cleavage, or in other cases, the nicks can be staggered to produce single-stranded overhangs, also known as "sticky ends", which can be 5' overhangs or 3' overhangs. Active variants of genomic targets can also be used. Such active variants may comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a given target site, wherein the active variant retains biological activity and is therefore capable of being recognized and cleaved by a Cas endonuclease.

[0654] Assays that measure single-strand or double-strand breaks at a target site caused by an endonuclease are known in the art, and generally measure the overall activity and specificity of the agent on a DNA substrate comprising the recognition site.

[0655] The targeting method herein can be carried out in such a way that two or more DNA target sites are targeted, for example, in the method. This method can optionally be characterized as a multiplex method. In certain embodiments, two, three, four, five, six, seven, eight, nine, ten or more target sites can be targeted simultaneously. Multiplexing methods are typically carried out by the targeting method herein, wherein a plurality of different RNA components are provided, each of which is designed to guide the polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0656] Gene Editing

[0657] The editing process of the genomic sequence of the combined DSB and modification template generally includes: introducing a DSB inducer or a nucleic acid encoding a DSB inducer (recognizing a target sequence in a chromosomal sequence and being able to induce a DSB in a genomic sequence) into a host cell, and at least one polynucleotide modification template comprising at least one nucleotide change when compared to the nucleotide sequence to be edited. The polynucleotide modification template may also include a nucleotide sequence flanking the at least one nucleotide change, wherein the flanking sequence is substantially homologous to the chromosomal region flanking the DSB. Genome editing using DSB inducers (such as Cas-gRNA complexes) has been described, for example, in US 20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and WO / 2016 / 025131 published on February 18, 2016.

[0658] Some uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US 20150082478A1 published on March 19, 2015, WO 2015026886 published on February 26, 2015, and US 20150059010 published on February 26, 2015) and include, but are not limited to, modification or replacement of a nucleotide sequence of interest (such as a regulatory element), insertion of a polynucleotide of interest, gene knockout, gene knock-in, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a protein of interest, amino acid and / or protein fusions, and gene silencing caused by expression of an inverted repeat sequence in a gene of interest.

[0659] Proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of one or more proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein. Guidance for amino acid substitutions that are unlikely to affect the biological activity of the protein is found, for example, in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC [National Biomedical Research Foundation, Washington, DC, USA]. Conservative substitutions, such as exchanging one amino acid for another with similar properties, are preferred. Conservative deletions, insertions, and amino acid substitutions are not expected to produce radical changes in protein characteristics, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by conventional screening assays. Assays for double-strand-break-inducing activity are known and generally measure the overall activity and specificity of an agent for a DNA substrate containing a target site.

[0660] Methods for genome editing with Cas endonucleases and complexes of Cas endonucleases and guide polynucleotides are described herein. After characterizing the guide RNA and PAM sequences, the components of the endonuclease complex and the associated CRISPR RNA (crRNA) can be used to modify chromosomal DNA in other organisms including plants. To promote optimal expression and nuclear localization (for eukaryotic cells), genes containing the complex can be optimized as described in WO 2016186953 published on November 24, 2016, and then delivered to cells as DNA expression cassettes by methods known in the art. Components that must contain active complexes can also be delivered as RNA (with or without modifications that protect RNA from degradation) or as capped or uncapped mRNA (Zhang, Y. et al., 2016, Nat. Commun. [Natural Communications] 7: 12617) or Cas protein guide polynucleotide complexes (published in WO 2017070032 on April 27, 2017), or any combination thereof. In addition, one or more parts of the complex and crRNA can be expressed from a DNA construct, while the other components are delivered as RNA (with or without modifications that protect the RNA from degradation) or as capped or uncapped mRNA (Zhang et al. 2016 Nat. Commun. [Nature Communication] 7: 12617) or Cas protein-guided polynucleotide complexes (published in WO 2017070032 on April 27, 2017), or any combination thereof. For in vivo production of crRNA, tRNA-derived elements can also be used to recruit endogenous RNases to cleave crRNA transcripts into mature forms that can guide the complex to its DNA target site, for example, as described in WO 2017105991 published on June 22, 2017. The nickase complex can be used alone or in concert to produce single or multiple DNA nicks on one or both DNA strands. In addition, the cleavage activity of Cas endonucleases can be inactivated by changing key catalytic residues in the cleavage domain (Sinkunas, T. et al., 2013, EMBO J [Journal of the European Molecular Biology Association]. 32: 385-394), thereby generating RNA-guided helicases that can be used to enhance homology-directed repair, induce transcriptional activation, or remodel local DNA structure. Moreover, the activities of both the Cas cleavage and helicase domains can be knocked out and used in combination with other DNA shearing, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancement, DNA integration, DNA inversion, and DNA repair agents.

[0661] The transcription direction of tracrRNA for the CRISPR-Cas system (if present) and other components of the CRISPR-Cas system (e.g., variable targeting domains, crRNA repeats, loops, anti-repeat sequences) can be derived as described in WO 2016186946 published on November 24, 2016 and WO 2016186953 published on November 24, 2016.

[0662] As described herein, once the appropriate guide RNA requirements are established, the PAM preference of each new system disclosed herein can be checked. If the cleavage complex results in degradation of the random PAM library, the complex can be converted into a nickase by mutagenesis of key residues or by inactivating the ATPase-dependent helicase activity by assembling the reaction in the absence of ATP, as previously described (Sinkunas, T. et al., 2013, EMBO J. [Journal of the European Molecular Biology Association] 32: 385-394). Double-stranded DNA breaks can be generated using two regions of PAM randomization separated by two pro-spacer targets, which can be captured and sequenced to check for PAM sequences that support cleavage by the respective complexes.

[0663] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein into a cell, and identifying at least one cell having a modification at the target, wherein the modification at the target site is selected from the group consisting of: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, chemical alteration of at least one nucleotide, and (v) any combination of (i)-(iv).

[0664] The nucleotide to be edited can be located inside or outside the target site recognized and cut by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification on the target site recognized and cut by the Cas endonuclease. In another embodiment, there are at least 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, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between the at least one nucleotide to be edited and the genomic target site.

[0665] Knockouts can be produced by indels (insertion or deletion of nucleotide bases in the target DNA sequence by NHEJ), or by specific removal of sequences that reduce or completely abolish sequence function at or near the targeted site.

[0666] Guide polynucleotide / Cas endonuclease-induced targeted mutations can occur in nucleotide sequences located inside or outside the genomic target site recognized and cleaved by the Cas endonuclease.

[0667] The method for editing a nucleotide sequence in the genome of a cell may be a method by restoring the function of a non-functional gene product without using an exogenous selectable marker.

[0668] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein and at least one donor DNA into the cell, wherein the donor DNA comprises a polynucleotide of interest, and optionally, the method further comprises identifying at least one cell that has integrated the polynucleotide of interest into or near the target site.

[0669] In one aspect, the methods disclosed herein can employ homologous recombination (HR) to provide for integration of a polynucleotide of interest at a target site.

[0670] A variety of methods and compositions can be used to produce cells or organisms with a polynucleotide of interest inserted into a target site through the activity of a CRISPR-Cas system component as described herein. In one method described herein, a polynucleotide of interest is introduced into an organism cell via a donor DNA construct. As used herein, "donor DNA" is a DNA construct comprising a polynucleotide of interest to be inserted into a target site of a Cas endonuclease. The donor DNA construct further comprises a first region and a second region of homology flanking the polynucleotide of interest. The first and second regions of homology of the donor DNA are respectively homologous to the first and second genomic regions present in the target site of a cell or organism genome or flanking the target site.

[0671] The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, which can be used for genome editing, gene insertion, and targeted genome regulation, and can also be used to target late mitotic cells, in which the function of the endogenous HR machinery is expected to be greatly reduced (Mali et al., 2013 Nature Methods [Nature Methods] Vol. 10: 957-963).

[0672] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450 In some embodiments, the target site may be a target site having a length of 1-900 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or a region having a unit integer value in a range up to and including the total length of the target site. These ranges include each integer in the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 bp. The amount of homology can also be described by the percentage sequence identity over the complete comparison length of two polynucleotides, which includes about at least 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 100% percentage sequence identity. Sufficient homology includes polynucleotide length, overall percentage sequence identity, and optionally any combination of conserved regions or local percentage sequence identity of continuous nucleotides, for example, enough homology can be described as a region of 75-150bp with at least 80% sequence identity to the region of the target locus.Sufficient homology can also be described by the predicted ability of two polynucleotides to hybridize specifically under high stringency conditions, see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., eds. (1994) Current Protocols (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes (Elsevier, New York).

[0673] Episomal DNA molecules can also be ligated into double-strand breaks, for example, T-DNA is integrated into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J. 17:6086-95). Once the sequence surrounding the double-strand break is altered, for example by mature exonuclease activity involved in double-strand breaks, the gene conversion pathway can restore the original structure if homologous sequences are available, such as homologous chromosomes in non-dividing somatic cells, or sister chromatids after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA repair templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0674] In one embodiment, the present disclosure includes a method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing at least one PGEN described herein, and a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification of the nucleotide sequence, and the method optionally further comprises selecting at least one cell comprising the edited nucleotide sequence.

[0675] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow editing (modification) of the target genomic nucleotide sequence. (See also US 20150082478 published on March 19, 2015 and WO 20]5026886 published on February 26, 2015).

[0676] The polynucleotides of interest and / or traits can be stacked together in a complex trait locus as described in WO 2012129373 published on September 27, 2012 and WO 2013112686 published on August 1, 2013. The guide polynucleotide / Cas9 endonuclease system described herein provides an efficient system for generating double strand breaks and allows for the stacking of traits in a complex trait locus.

[0677] The guide polynucleotide / Cas system for mediating gene targeting as described herein can be used in the following methods for guiding heterologous gene insertion and / or generating complex trait loci comprising multiple heterologous genes in a manner similar to that disclosed in WO 2012129373 published on September 27, 2012, wherein the guide polynucleotide / Cas system as disclosed herein is used to replace the use of double-strand break inducing agents to introduce the target gene. By inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or even 5 centimorgans (cM) of each other, these transgenes can be bred as a single genetic locus (e.g., see US 20130263324 published on October 3, 2013 or WO2012129373 published on March 14, 2013). After selecting plants comprising transgenes, plants comprising (at least) one transgene can be crossed to form F1 comprising both transgenes. In the offspring from these F1 (F2 or BC1), 1 / 500 of the offspring will have two different transgenes recombined on the same chromosome. The composite locus can then be bred to a single genetic locus with all two transgenic traits. This process can be repeated to stack as many traits as possible.

[0678] Further uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US 20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, US20150059010 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and PCT application WO2016025131 published on February 18, 2016) and include, but are not limited to, modification or substitution of a nucleotide sequence of interest (such as a regulatory element), insertion of a polynucleotide of interest, gene knockout, gene knockin, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a protein of interest, amino acid and / or protein fusions, and gene silencing caused by expression of an inverted repeat sequence in a gene of interest.

[0679] The characteristics produced by the gene editing compositions and methods described herein can be evaluated. Chromosome intervals associated with the target phenotype or trait can be identified. Various methods well known in the art can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are extended to cover markers that will be linked to genes controlling the target trait. In other words, the chromosomal interval is extended so that any marker located within the interval (including the end markers defining the boundaries of the interval) can be used as markers for specific traits. In one embodiment, the chromosomal interval contains at least one QTL, and in addition, more than one QTL can indeed be included. Multiple QTLs very close in the same interval can confuse the association of a specific marker with a specific QTL, because a marker can be shown to be linked to more than one QTL. On the contrary, for example, if two very close markers show co-segregation with the desired phenotypic trait, it is sometimes unclear whether each of those markers identifies the same QTL or two different QTLs. The term "quantitative trait locus" or "QTL" refers to a DNA region associated with the differential expression of a quantitative phenotypic trait in at least one genetic background (e.g., in at least one breeding population). The region of a QTL encompasses or is closely linked to one or more genes that affect the trait under consideration. "Alleles of a QTL" may include multiple genes or other genetic factors, such as haplotypes, in a continuous genomic region or linkage group. The alleles of a QTL may represent haplotypes within a specified window, wherein the window is a continuous genomic region that can be defined and tracked with a set of one or more polymorphic markers. A haplotype may be specified as being defined by a unique fingerprint of the alleles of each marker within the window.

[0680] Introducing CRISPR-Cas system components into cells

[0681] The methods described herein do not depend on a specific method for introducing a sequence into an organism or cell, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the organism. Introducing includes reference to incorporating nucleic acids into eukaryotic or prokaryotic cells, wherein the nucleic acid can be incorporated into the genome of the cell, and includes reference to nucleic acids, proteins, or polynucleotide-protein complexes (PGEN, RGEN) being provided to the cell instantaneously (directly).

[0682] Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated introduction, transfection, transduction, cell penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical application, sexual hybridization, sexual breeding, and any combination thereof.

[0683] For example, a guide polynucleotide (guide RNA, cr nucleotides + tracr nucleotides, guide DNA and / or guide RNA-DNA molecules) can be directly introduced into cells (transiently) as single-stranded or double-stranded polynucleotide molecules. Guide RNA (or crRNA + tracrRNA) can also be indirectly introduced into cells by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding guide RNA (or crRNA + tracrRNA), and the guide RNA is effectively connected to a specific promoter capable of transcribing the guide RNA (or crRNA + tracrRNA) in the cell. The specific promoter can be, but is not limited to, an RNA polymerase III promoter, which allows RNA transcription with precisely defined unmodified 5′- and 3′-ends (Ma et al., 2014, Mol. Ther. Nucleic Acids [Molecular Therapy - Nucleic Acids] 3: e161; DiCarlo et al., 2013, Nucleic Acids Res. [Nucleic Acids Research] 41: 4336-4343; WO 2015026887 published on February 26, 2015). Any promoter capable of transcribing the guide RNA in a cell can be used, and these include heat shock / heat-inducible promoters operably linked to the nucleotide sequence encoding the guide RNA.

[0684] Plant cells are distinct from animal cells (eg, human cells), fungal cells (eg, yeast cells), and protoplasts, including, for example, in that they contain a plant cell wall that can serve as a barrier to the delivery of components.

[0685] Cas endonucleases, and / or guide RNAs, and / or ribonucleoprotein complexes, and / or polynucleotides encoding any one or more of the foregoing can be delivered into plant cells by methods known in the art, such as, but not limited to, Rhizobium-mediated transformation (e.g., Agrobacterium, Ochrobacterium), particle-mediated delivery (particle bombardment), polyethylene glycol (PEG)-mediated transfection (e.g., to protoplasts), electroporation, cell penetrating peptides or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery.

[0686] Cas endonucleases herein, such as Cas endonucleases described herein, can be introduced into cells by directly introducing the Cas polypeptide itself (referred to as direct delivery of Cas endonucleases), mRNA encoding Cas proteins and / or guiding polynucleotide / Cas endonuclease complexes themselves, using any method known in the art. Cas endonucleases can also be introduced into cells indirectly by introducing recombinant DNA molecules encoding Cas endonucleases. Using any method known in the art, endonucleases can be introduced into cells instantaneously, or endonucleases can be incorporated into the genome of host cells. Cell penetrating peptides (CPPs) as described in WO 2016073433 disclosed on May 12, 2016 can be used to promote the uptake of endonucleases and / or guided polynucleotides into cells. Any promoter capable of expressing Cas endonucleases in cells can be used, and these promoters include heat shock / heat-inducible promoters operably linked to nucleotide sequences encoding Cas endonucleases.

[0687] Direct delivery of polynucleotide modified templates into plant cells can be achieved by particle-mediated delivery, and any other direct delivery method, such as but not limited to polyethylene glycol (PEG)-mediated protoplast transfection, whisker-mediated transformation, electroporation, particle bombardment, cell penetrating peptides or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery can be successfully used to deliver polynucleotide modified templates in eukaryotic cells (e.g., plant cells).

[0688] Donor DNA can be introduced by any means known in the art. Donor DNA can be provided by any transformation method known in the art (including, for example, Agrobacterium-mediated transformation or biolistic particle bombardment). Donor DNA can be transiently present in the cell, or can be introduced via a viral replicon. In the presence of Cas endonucleases and target sites, donor DNA is inserted into the genome of the transformed plant.

[0689] Direct delivery of any one of the guided Cas system components can be accompanied by direct delivery (co-delivery) of other mRNAs that can facilitate enrichment and / or visualization of cells receiving the guide polynucleotide / Cas endonuclease complex components. For example, direct co-delivery of mRNAs encoding phenotypic markers (such as, but not limited to, transcriptional activators such as CRC (Bruce et al. 2000 The Plant Cell [Plant Cell] 12: 65-79) can be achieved by restoring the function of non-functional gene products without using exogenous selectable markers. Cell selection and enrichment, as described in WO 2017070032 published on April 27, 2017.

[0690] Introducing the guide RNA / Cas endonuclease complex described herein (representing the cleavable complex described herein) into the cell includes introducing the components of the complex into the cell individually or in combination, and directly (delivered as RNA (for guides) and proteins (for Cas endonucleases and protein subunits or functional fragments thereof)) or via a recombinant construct expressing these components (guide RNA, Cas endonucleases, protein subunits or functional fragments thereof). Introducing the guide RNA / Cas endonuclease complex (RGEN) into the cell includes introducing the guide RNA / Cas endonuclease complex into the cell as a ribonucleotide-protein. The ribonucleotide-protein can be assembled prior to introduction into the cell as described herein. Components comprising guide RNA / Cas endonuclease ribonucleotide proteins (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) can be assembled in vitro or by any method known in the art prior to introduction into the cell (targeted for genome modification as described herein).

[0691] Direct delivery of RGEN ribonucleoprotein allows genome editing at the target site in the genome of the cell, after which the complex can be rapidly degraded and only the complex is allowed to exist briefly in the cell. This short-lived presence of the RGEN complex may result in reduced off-target effects. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can result in constant expression of RGEN from these plasmids, which can enhance off-target effects (Cradick, TJ et al. (2013) Nucleic Acids Res [Nucleic Acids Research] 41: 9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. [Natural Biotechnology] 31: 822-826).

[0692] Direct delivery can be achieved by combining any one of the components of a guide RNA / Cas endonuclease complex (RGEN) (standing for a cleavage-ready complex as described herein, such as at least one guide RNA, at least one Cas protein, and optionally at least one additional protein) with a delivery matrix comprising microparticles, such as but not limited to gold particles, tungsten particles, and silicon carbide whisker particles (see also WO 2017070032 published on April 27, 2017). The delivery matrix can comprise any of the components, such as a Cas endonuclease, attached to a solid matrix (e.g., a particle for bombardment).

[0693] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein forming the guide RNA / Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

[0694] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein forming the guide RNA / Cas endonuclease complex and at least one protein subunit of the complex are introduced into the cell as RNA and protein, respectively.

[0695] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and Cas endonuclease protein and at least one protein subunit of the complex that form the guide RNA / Cas endonuclease complex (cleavage-ready complex) are preassembled in vitro and introduced into a cell as a ribonucleotide-protein complex.

[0696] Protocols for introducing polynucleotides, polypeptides or polynucleotide-protein complexes (PGEN, RGEN) into eukaryotic cells such as plants or plant cells are known and include microinjection (Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543); meristem transformation (U.S. Pat. No. 5,736,369); electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6); Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840); whisker-mediated transformation (Ainley et al. 2013, Plant Biotechnology . Journal [Plant Biotechnology Journal] 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide [Properties and Applications of Silicon Carbide] (2011), 345-358, Editor: Gerhardt, Rosario., Publisher: InTech, Riieka, Croatia, Code: 69PQBP; ISBN: 978-953-307-201-2); direct gene transfer (Paszkowski et al., (1984) EMBO J [Journal of the European Molecular Biology Association] 3: 2717-22); and ballistic particle acceleration (U.S. Patents 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al., (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment" in Plant Cell, Tissue, and Organ Culture: Fundamental Methods [Plant Cell, Tissue and Organ Culture: Basic Methods], ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-6; Weissinger et al. (1988) Ann Rev Genet 22:421-77; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen (1991) In vitro Cell Dev Biol 27P:175-82 (soybean); Singh et al. (1998) Theor Appl Genet [Theoretical and Applied Genetics] 96: 319-24 (soybean); Datta et al., (1990) Biotechnology [Biotechnology] 8: 736-40 (rice); Klein et al., (1988) Proc. Natl. Acad. Sci.USA [Proceedings of the National Academy of Sciences of the United States of America] 85:4305-9 (maize); Klein et al., (1988) Biotechnology [Biotechnology] 6:559-63 (maize); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol [Plant Physiol] 91:440-4 (maize); Fromm et al., (1990) Biotechnology [Biotechnology] 8:833-9 (maize); Hooykaas-Van Slogteren et al., (1984) Nature 311:763-4; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) Theor Appl. Genet [Theoretical and Applied Genetics] 84:560-6 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell [Plant Cell] 4:1495-505 (electroporation); Li et al., (1993) Plant Cell Rep [Plant Cell Reports] 12:250-5; Christou and Ford (1995) Annals Botany [Annals Botany] 75:407-13 (rice) and Osjoda et al., (1996) Nat Biotechnol [Natural Biotechnology] 14:745-50 (maize transformed by Agrobacterium tumefaciens).

[0697] Alternatively, polynucleotides can be introduced into plants or plant cells by contacting cells or organisms with viruses or viral nucleic acids. Typically, such methods involve incorporating polynucleotides into viral DNA or RNA molecules. In some instances, the target polypeptide can be initially synthesized as a part of a viral polyprotein, and then the synthesized polypeptide is processed in vivo or in vitro by proteolytic processing to produce a desired recombinant protein. It is known to express polynucleotides in plants and to express proteins encoded therein (involving viral DNA or RNA molecules), see, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931.

[0698] A variety of transient transformation methods can be used to provide or introduce polynucleotides or recombinant DNA constructs into prokaryotic and eukaryotic cells or organisms. Such transient transformation methods include, but are not limited to, introducing polynucleotide constructs directly into plants.

[0699] Nucleic acids and proteins can be provided to cells by any method, including methods using molecules to promote the uptake of any or all components (e.g., cell penetrating peptides and nanocarriers) of the directed Cas system (proteins and / or nucleic acids). See also US 20110035836 published on February 10, 2011 and EP 2821486A1 published on January 7, 2015.

[0700] Other methods for introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts may be used, including plastid transformation methods, and methods for introducing polynucleotides into tissues from seedlings or mature seeds.

[0701] "Stable transformation" is intended to mean that a nucleotide construct introduced into an organism is incorporated into the genome of the organism and can be inherited by its progeny. "Transient transformation" is intended to mean that a polynucleotide is introduced into the organism and is not incorporated into the genome of the organism, or that a polypeptide is introduced into the organism. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

[0702] A variety of methods can be used to identify those cells with altered genomes at or near the target site without using a screenable marker phenotype. Such methods can be considered to be direct analysis of the target sequence to detect any changes in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blotting, and any combination thereof.

[0703] Cells and plants

[0704] The polynucleotides and polypeptides disclosed herein can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, mammalian, bacterial, fungal, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein. Any plant (including monocots and dicots and plant elements) can be used with the compositions and methods described herein.

[0705] Examples of monocots that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet, Pennisetum glaucum), broomcorn millet (Panicum miliaceum), foxtail millet (Setaria italica), foxtail millet (Eleusine coracana), wheat (Triticum species, e.g., Triticum aestivum, Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), bananas (Musa spp.), palms, ornamentals, turf grasses, and other grasses.

[0706] Examples of dicots that can be used include, but are not limited to, soybean (Glycine max), Brassica species (such as, but not limited to, rapeseed or canola) (Brassica napus and B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (A. thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum, Gossypium barbadense), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum), and the like.

[0707] Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musas spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamentals and conifers.

[0708] Vegetables that can be used include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumber such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamental plants include azaleas (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums.

[0709] Conifers that can be used include pines, such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); Western hemlock (Tsugacanadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs, such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars, such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis).

[0710] In certain embodiments of the present disclosure, fertile plants are plants that produce live male and female gametes and are self-fertile. Such self-fertilized plants can produce offspring plants without the contribution of gametes from any other plant and the genetic material contained therein. Other embodiments of the present disclosure may involve the use of non-self-fertile plants because the plant does not produce viable or otherwise fertile male or female gametes or both.

[0711] The present disclosure can be used for breeding of plants comprising one or more introduced traits or edited genomes.

[0712] A non-limiting example of how two traits can be stacked into a genome at a genetic distance of, for example, 5 cM from each other is described below: A first plant comprising a first transgenic target site integrated into a first DSB target site within a genomic window and without a first genomic locus of interest is crossed with a second transgenic plant, the second transgenic plant comprising a genomic locus of interest at a different genomic insertion site within the genomic window, and the second plant not comprising the first transgenic target site. Approximately 5% of the plant offspring from this cross will have within the genomic window the first transgenic target site integrated into the first DSB target site and the first genomic locus of interest integrated at a different genomic insertion site. Offspring plants with two sites in a defined genomic window can be further crossed with a third transgenic plant, the third transgenic plant comprising within the defined genomic window a second transgenic target site integrated into a second DSB target site, and / or a second genomic locus of interest and lacking the first transgenic target site and the first genomic locus of interest. Offspring with the first transgenic target site, the first genomic locus of interest, and the second genomic locus of interest integrated at different genomic insertion sites within the genomic window are then selected. Such methods can be used to generate plants comprising complex trait loci having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more transgenic target sites integrated into the DSB target site and / or genomic loci of interest integrated at different sites within the genomic window. In this way, a variety of complex trait loci can be generated.

[0713] Cells and Animals

[0714] The polynucleotides and polypeptides disclosed herein can be introduced into animal cells. Animal cells can include, but are not limited to, organisms of the following phyla, including Chordata, Arthropoda, Mollusca, Annelida, Coelenterate, or Echinodermata; organisms of the following classes, including mammals, insects, birds, amphibians, reptiles, or fish. In some aspects, the animal is a human, mouse, C.elegans, rat, fruit fly (Drosophila spp.), zebrafish, chicken, dog, cat, guinea pig, hamster, chicken, Japanese rice fish, sea lamprey, pufferfish, tree frog (e.g., Xenopus spp.), monkey, or chimpanzee. Specific cell types contemplated include haploid cells, diploid cells, germ cells, neurons, muscle cells, endocrine or exocrine cells, epithelial cells, muscle cells, tumor cells, embryonic cells, hematopoietic cells, osteocytes, germ plasm cells, somatic cells, stem cells, pluripotent stem cells, induced pluripotent stem cells, progenitor cells, meiotic cells, and mitotic cells. In some aspects, multiple cells from an organism can be used.

[0715] The disclosed novel Cas9 orthologs can be used to edit the genome of animal cells in various ways. In one aspect, it may be necessary to delete one or more nucleotides. On the other hand, it may be desirable to insert one or more nucleotides. In one aspect, it may be desirable to replace one or more nucleotides. On the other hand, it may be desirable to modify one or more nucleotides by covalent or non-covalent interaction with another atom or molecule.

[0716] Genome modification by Cas9 orthologs can be used to achieve genotypic and / or phenotypic changes in target organisms. Such changes are preferably related to the improvement of the target phenotype or physiologically important characteristics, the correction of endogenous defects, or the expression of a certain type of expression marker. In some aspects, the target phenotype or physiologically important characteristics are related to the overall health, adaptability or fertility of the animal, the ecological adaptability of the animal, or the relationship or interaction between the animal and other organisms in the environment.In some aspects, the phenotype of interest or physiologically important characteristic is selected from the group consisting of improved overall health, disease reversal, disease modification, disease stabilization, disease prevention, treatment of parasitic infections, treatment of viral infections, treatment of retroviral infections, treatment of bacterial infections, treatment of neurological disorders (such as but not limited to multiple sclerosis), endogenous genetic defects (such as but not limited to metabolic disorders, rickets, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Barth syndrome, breast cancer, Charcot-Marie-Tooth, colon cancer, Cri du chat, Crohn's disease, cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor V Leiden Thrombophilia Thrombophilia, Familial Hypercholesterolemia, Familial Mediterranean Fever, Fragile X Syndrome, Gaucher Disease, Hemochromatosis, Hemophilia, Holoprosencephaly, Huntington Disease, Klinefelter Syndrome, Marfan Syndrome, Myotonic Dystrophy, Neurofibromatosis, Noonan Syndrome, Osteogenesis Imperfecta, Parkinson Disease, Phenylketonuria, Poland Anomaly, Porphyria, Progeria, Prostate Cancer, Retinitis Pigmentosa, Severe Combined Immune Deficiency (SCID), Sickle Cell Disease, Skin Cancer, Spinal Muscular Atrophy, Tay-Sachs, Thalassemia, Trimethylaminuria, Turner Syndrome, Velocardiofacial Svndrome, WAGR Syndrome, and Wilson Disease. Disease), treatment of innate immune disorders (such as, but not limited to, immunoglobulin subclass deficiencies), treatment of acquired immune disorders (such as, but not limited to, AIDS and other HIV-related disorders), treatment of cancer, and treatment of diseases, including rare or "orphan" conditions, for which no effective treatment options are available by other means.

[0717] Cells genetically modified using the compositions or methods disclosed herein can be transplanted into a subject for purposes such as gene therapy, for example, to treat a disease or as an antiviral, antipathogen or anticancer therapeutic, for production of genetically modified organisms in agriculture, or for biological research.

[0718] In vitro polynucleotide detection, binding and modification

[0719] In some respects, compositions disclosed herein can be further used as compositions for (in some respects together with one or more separated polynucleotide sequences) in vitro methods. The one or more separated polynucleotide sequences can include one or more target sequences for modification. In some respects, the one or more separated polynucleotide sequences can be genomic DNA, PCR product or synthetic oligonucleotide.

[0720] Composition

[0721] Modification of the target sequence can be in the form of nucleotide insertion, nucleotide deletion, nucleotide substitution, addition of atomic molecules to existing nucleotides, nucleotide modification, or binding of heterologous polynucleotides or polypeptides to the target sequence. Insertion of one or more nucleotides can be accomplished by including a donor polynucleotide in the reaction mixture: the donor polynucleotide is inserted into the double-stranded break produced by the Cas-α ortholog polypeptide. Insertion can be via non-homologous end joining or via homologous recombination.

[0722] In one aspect, the sequence of the target polynucleotide is known prior to modification and is compared to one or more sequences of one or more polynucleotides produced by treatment with a Cas-α ortholog. In one aspect, the sequence of the target polynucleotide is unknown prior to modification and treatment with a Cas-α ortholog is used as part of a method to determine the sequence of the target polynucleotide.

[0723] Polynucleotide modifications with Cas-α orthologs can be accomplished by using a full-length polypeptide identified from the Cas locus, or a fragment, modification or variant of a polypeptide identified from the Cas locus. In some aspects, the Cas-α ortholog is obtained or derived from an organism listed in Table 1. In some aspects, the Cas-α ortholog is a polypeptide having at least 80% identity with any one of SEQID NOs: 86-170 or 511-1135. In some aspects, the Cas-α ortholog is a functional variant of any one of SEQID NOs: 86-170 or 511-1135. In some aspects, the Cas-α ortholog is a functional fragment of any one of SEQID NOs: 86-170 or 511-1135. In some aspects, the Cas-α ortholog is a Cas-α polypeptide encoded by a polynucleotide selected from the group consisting of: SEQID NOs: 86-170 or 511-1135. In some aspects, the Cas-α ortholog is a Cas-α polypeptide that recognizes a PAM sequence listed in any one of Tables 4-83. In some aspects, the Cas-α ortholog is a Cas-α polypeptide identified from an organism listed in the sequence listing.

[0724] In some aspects, the Cas-α ortholog is provided as a Cas-α polynucleotide. In some aspects, the Cas-α polynucleotide is selected from the group consisting of: SEQ ID NO: 1-85, or a sequence that is at least 80%, 85%, 90%, 95%, 97%, 99% or 100% identical to any one of SEQ ID NO: 1-85.

[0725] In some aspects, the Cas-α ortholog can be selected from the group consisting of: an unmodified wild-type Cas-α ortholog; a functional Cas-α ortholog variant; a functional Cas-α ortholog fragment; a fusion protein comprising an active or inactivated Cas-α ortholog; a Cas-α ortholog further comprising one or more nuclear localization sequences (NLS) at the C-terminus or at the N-terminus or at both the N and C-termini; a biotinylated Cas-α ortholog; a Cas-α ortholog nickase; a Cas-α ortholog endonuclease; a Cas-α ortholog further comprising a histidine tag; and a mixture of any two or more of the above.

[0726] In some aspects, the Cas-α ortholog is a fusion protein further comprising a nuclease domain, a transcriptional activator domain, a transcriptional repressor domain, an epigenetic modification domain, a cleavage domain, a nuclear localization signal, a cell penetration domain, a translocation domain, a marker, or a transgene that is heterologous to the target polynucleotide sequence or the cell from which the target polynucleotide sequence was obtained or derived.

[0727] In some aspects, it is desirable that multiple Cas-α orthologs. In some aspects, the multiple may include Cas-α orthologs derived from different source organisms or derived from different loci in the same organism. In some aspects, the multiple may include Cas-α orthologs with different binding specificities to target polynucleotides. In some aspects, the multiple may include Cas-α orthologs with different cutting efficiencies. In some aspects, the multiple may include Cas-α orthologs with different PAM specificities. In some aspects, the multiple may include orthologs with different molecular compositions (i.e., polynucleotide Cas-α orthologs and polypeptide Cas-α orthologs).

[0728] The guide polynucleotide can be provided as a single guide RNA (sgRNA), a chimeric molecule comprising a tracrRNA, a chimeric molecule comprising a crRNA, a chimeric RNA-DNA molecule, a DNA molecule, or a polynucleotide comprising one or more chemically modified nucleotides.

[0729] The storage conditions of Cas-α orthologs and / or instructing polynucleotides include parameters of temperature, material state and time. In some aspects, Cas-α orthologs and / or instructing polynucleotides are stored at about -80 degrees Celsius, about -20 degrees Celsius, about 4 degrees Celsius, about 20-25 degrees Celsius or about 37 degrees Celsius. In some aspects, Cas-α orthologs and / or instructing polynucleotides are stored in the form of liquid, frozen liquid or lyophilized powder. In some aspects, Cas-α orthologs and / or instructing polynucleotides are stable for at least one day, at least one week, at least one month, at least one year or even more than one year.

[0730] Any or all possible polynucleotide components of the reaction (e.g., guide polynucleotides, donor polynucleotides, optionally Cas-α polynucleotides) can be provided as part of a vector, construct, linearized or circularized plasmid or as part of a chimeric molecule. Each component can be provided to the reaction mixture alone or together. In some aspects, one or more polynucleotide components are operably linked to a heterologous non-coding regulatory element that regulates its expression.

[0731] The method for modifying a target polynucleotide comprises combining minimal elements into a reaction mixture comprising: a Cas-α ortholog (or a variant, fragment, or other related molecule as described above), a guide polynucleotide (which comprises a sequence that is substantially complementary to or selectively hybridizes with a target polynucleotide sequence of a target polynucleotide), and a target polynucleotide for modification. In some aspects, a Cas-α ortholog is provided as a polypeptide. In some aspects, a Cas-α ortholog is provided as a Cas-α ortholog polynucleotide. In some aspects, the guide polynucleotide is provided as an RNA molecule, a DNA molecule, an RNA: DNA hybrid, or a polynucleotide molecule comprising a chemically modified nucleotide.

[0732] Any storage buffer or reaction mixture can be optimized for stability, efficacy or other parameters. The other components of storage buffer or reaction mixture may include buffer composition, Tris, EDTA, dithiothreitol (DTT), phosphate buffered saline (PBS), sodium chloride, magnesium chloride, HEPES, glycerol, BSA, salt, emulsifier, detergent, chelating agent, redox agent, antibody, nuclease-free water, protease and / or viscosity agent. In some aspects, the storage buffer or reaction mixture also include a buffer solution with at least one of the following components: HEPES, MgCl2, NaCl, EDTA, protease, proteinase K, glycerol, nuclease-free water.

[0733] Incubation conditions will vary according to the desired result. The temperature is preferably at least 10 degrees Celsius, 10 to 15, at least 15, 15 to 17, at least 17, 17 to 20, at least 20, 20 to 22, at least 22, 22 to 25, at least 25, 25 to 27, at least 27, 27 to 30, at least 30, 30 to 32, at least 32, 32 to 35, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40 or even greater than 40 degrees Celsius. The incubation time is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes or even greater than 10 minutes.

[0734] Before, during or after incubation, one or more sequences of one or more polynucleotides in the reaction mixture can be determined by any method known in the art. In one aspect, modification of the target polynucleotide can be determined by comparing one or more sequences of one or more polynucleotides purified from the reaction mixture with the sequence of the target polynucleotide before binding to the Cas-α ortholog.

[0735] The kit may include any one or more of the compositions disclosed herein that can be used for in vitro or in vivo polynucleotide detection, binding and / or modification. The kit comprises a Cas-α ortholog or a polynucleotide Cas-α ortholog encoding such a Cas-α ortholog, and optionally further comprises a buffer component capable of effective storage, and one or more additional compositions, wherein the one or more additional compositions are capable of introducing the Cas-α ortholog or Cas-α ortholog into a heterologous polynucleotide, wherein the Cas-α ortholog or Cas-α ortholog is capable of modifying, adding, deleting or replacing at least one nucleotide of the heterologous polynucleotide. On the other hand, the Cas-α orthologs disclosed herein can be used to enrich one or more polynucleotide target sequences from a mixed pool. On the other hand, the Cas-α orthologs disclosed herein can be fixed on a substrate for in vitro target polynucleotide detection, binding and / or modification.

[0736] For the purpose of storage, purification and / or characterization, the Cas-α endonuclease can be attached, bound or attached to a solid matrix. Examples of solid matrices include, but are not limited to, filters, chromatography resins, assay plates, test tubes, cryogenic vials, etc. The Cas-α endonuclease can be fully purified and stored in a suitable buffer solution, or lyophilized.

[0737] Detection Methods

[0738] Methods for detecting Cas-α:guide polynucleotide complexes bound to target polynucleotides may include any method known in the art, including but not limited to microscopy, chromatography, electrophoresis, immunoprecipitation, filtration, nanopore separation, microarrays, and those described below.

[0739] DNA Electrophoretic Mobility Shift Assay (EMSA): Studies proteins bound to known DNA oligonucleotide probes and assesses the specificity of the interaction. The technique is based on the principle that protein-DNA complexes migrate more slowly than free DNA molecules when subjected to polyacrylamide or agarose gel electrophoresis. Since the rate of DNA migration is retarded upon protein binding, the assay is also called a gel retardation assay. Adding protein-specific antibodies to the binding components results in a larger complex (antibody-protein-DNA) that migrates even slower during electrophoresis, which is called a supershift and can be used to confirm the protein's identity.

[0740] DNA pull-down assay uses a DNA probe labeled with a high-affinity tag (e.g., biotin), which allows recovery or fixed probes. DNA probes can be compounded with the protein of the cell lysate from a similar reaction used in EMSA and then used to purify using agarose or magnetic beads. Then from DNA eluted protein, and detected by Western blotting or identified by mass spectrometry. Alternatively, affinity tag labeled protein can be used, or the antibody separation DNA-protein complex (similar to super-variable assay) for the target protein can be used. In this case, the unknown DNA sequence bound to the protein is detected by Southern blotting or PCR analysis.

[0741] The reporter assay provides real-time in vivo readout of the translation activity of the target promoter.Reporter gene is a fusion of a target promoter DNA sequence and a reporter gene DNA sequence (the reporter gene DNA sequence is customized by the researcher and encoded with a protein that can detect characteristics, such as firefly / Rainilla luciferase or alkaline phosphatase).These genes only produce enzymes when the target promoter is activated.Enzyme then catalyzes substrates to produce light or color changes that can be detected by spectral instruments.The signal from the reporter gene is used as an indirect determinant for the translation of the endogenous protein driven by the same promoter.

[0742] Microplate capture and detection assays use immobilized DNA probes to capture specific protein-DNA interactions and confirm protein identity and relative amounts with target-specific antibodies. Typically, DNA probes are immobilized on the surface of a 96- or 384-well microplate coated with streptavidin. Cell extracts are prepared and added to allow binding proteins to bind to the oligonucleotides. The extracts are then removed and each well is washed several times to remove non-specifically bound proteins. Finally, the proteins are detected using specific antibodies labeled for detection. This method is very sensitive and can detect less than 0.2 pg of target protein / well. This method can also be used for oligonucleotides labeled with other tags (e.g., primary amines that can be fixed on microplates coated with amine-reactive surface chemicals).

[0743] DNA footprinting is one of the most widely used methods to obtain detailed information about individual nucleotides in protein-DNA complexes or even inside living cells. In such experiments, chemicals or enzymes are used to modify or digest DNA molecules. When sequence-specific proteins bind to the DNA, they can protect the binding sites from modification or digestion. This can subsequently be visualized by denaturing gel electrophoresis, where the unprotected DNA is cleaved more or less randomly. It therefore appears as a "ladder" of bands, and the protein-protected sites have no corresponding bands and look like footprints in the banding pattern. By identifying a specific nucleoside at the protein-DNA binding site, a footprint is left there.

[0744] Microscopy techniques include optical, fluorescence, electron and atomic force microscopy (AFM).

[0745] Chromatin immunoprecipitation (ChIP) assays covalently bind proteins to their DNA targets, which can then be unlinked and characterized separately.

[0746] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) exposes the target protein to a random library of oligonucleotides. Those that bind are isolated and amplified by PCR.

[0747] The methods and compositions provided herein include, but are not limited to, the following aspects.

[0748] Aspect 1: A synthetic composition comprising: (a) a guide polynucleotide; (b) a Cas endonuclease comprising a C-terminal trisection RuvC domain, further comprising a bridge helix and at least one zinc finger domain, an alpha helical bundle and multiple beta folds forming a wedge-shaped domain, wherein the Cas endonuclease has a length of less than 650 amino acids; and (c) a target sequence comprising a nucleotide sequence that is complementary to the guide polynucleotide; wherein the guide polynucleotide and the Cas endonuclease form a complex that cuts a double-stranded DNA polynucleotide comprising the target sequence.

[0749] Aspect 2: A synthetic composition comprising: (a) a guide polynucleotide; (b) a Cas endonuclease derived from a taxonomic organism selected from the group consisting of Archaea, Micrarchaeota, Acidibacillus sulfuroxidans, Candidatus Aureabacteria, Candidatus Micrarchaeota, Clostridium novyi, Geobacter thermoglucosidase, Ruminococcus species, and Synechotrophomonas palmi, wherein the Cas endonuclease forms a complex with the guide polynucleotide; and (c) a double-stranded DNA polynucleotide comprising a target sequence bound to the guide polynucleotide; wherein the guide polynucleotide and the Cas endonuclease form a complex that cleaves the double-stranded DNA polynucleotide comprising the target sequence.

[0750] Aspect 3: A synthetic composition as described in Aspect 1 or Aspect 2, wherein the Cas endonuclease further comprises a zinc finger domain near the N-terminus.

[0751] Aspect 4: The synthetic composition of aspect 1 or aspect 2, wherein the double-stranded DNA polynucleotide further comprises a PAM.

[0752] Aspect 5: The synthetic composition of aspect 4, wherein the PAM comprises a plurality of thymine nucleotides.

[0753] Aspect 6: The synthetic composition of aspect 1 or aspect 2, further comprising a heterologous polynucleotide.

[0754] Aspect 7: The synthetic composition of aspect 1 or aspect 2, wherein the guide polynucleotide comprises a 20-nucleotide region complementary to the target sequence.

[0755] Aspect 8: The synthetic composition of aspect 1 or aspect 2, wherein the guide polynucleotide is a duplex molecule comprising tracrRNA and crRNA.

[0756] Aspect 9: The synthetic composition of aspect 1 or aspect 2, wherein the guide polynucleotide is a single guide polynucleotide comprising a Cas endonuclease recognition domain and a variable targeting domain.

[0757] Aspect 10: The synthetic composition of Aspect 6, wherein the heterologous polynucleotide is an expression element.

[0758] Aspect 11: The synthetic composition of Aspect 6, wherein the heterologous polynucleotide is a transgene.

[0759] Aspect 12: The synthetic composition of Aspect 6, wherein the heterologous polynucleotide is a donor DNA molecule.

[0760] Aspect 13: The synthetic composition of aspect 6, wherein the heterologous polynucleotide is a polynucleotide modification template.

[0761] Aspect 14: The synthetic composition of aspect 1 or aspect 2, wherein the CRISPR-Cas endonuclease further comprises a nuclear localization signal.

[0762] Aspect 15: A synthetic composition as described in Aspect 1 or Aspect 2, wherein the CRISPR-Cas endonuclease is Cas-α, or a functional fragment thereof.

[0763] Aspect 16: A synthetic composition as described in aspect 1 or aspect 2, wherein the CRISPR-Cas endonuclease is a catalytically inactive Cas-α.

[0764] Aspect 17: A synthetic composition as described in aspect 1 or aspect 2, wherein the CRISPR-Cas endonuclease is a fusion protein comprising a functional fragment of Cas-α.

[0765] Aspect 18: The synthetic composition of Aspect 17, wherein the fusion protein further comprises another nuclease domain.

[0766] Aspect 19: The synthetic composition of aspect 1 or aspect 2, further comprising at least one additional polypeptide.

[0767] Aspect 20: The synthetic composition of Aspect 19, wherein the additional polypeptide is selected from the group consisting of: Cas1, Cas2 and Cas4.

[0768] Aspect 21: The synthetic composition of aspect 1 or aspect 2, further comprising a cell.

[0769] Aspect 22: The synthetic composition of Aspect 21, wherein the cell is a eukaryotic cell.

[0770] Aspect 23: The synthetic composition of Aspect 21, wherein the cell is a plant cell.

[0771] Aspect 24: The synthetic composition of Aspect 23, wherein the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

[0772] Aspect 25: The synthetic composition of Aspect 23, wherein the plant cell is from an organism selected from the group consisting of: maize, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turf grass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower and tomato.

[0773] Aspect 26: The synthetic composition of aspect 21, further comprising a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the genome of the cell

[0774] Aspect 27: A polynucleotide encoding the synthetic composition as described in aspect 1 or aspect 2.

[0775] Aspect 28: The polynucleotide of aspect 27, further comprising at least one additional polynucleotide.

[0776] Aspect 29: The polynucleotide of aspect 28, wherein the at least one additional polynucleotide is an expression element.

[0777] Aspect 30: The polynucleotide of aspect 28, wherein the at least one additional polynucleotide is a gene.

[0778] Aspect 31: The synthetic composition of Aspect 30, wherein the gene is selected from the group consisting of: cas1, cas2 and cas4.

[0779] Aspect 32: The polynucleotide of aspect 28, wherein at least one polynucleotide is contained in a recombinant construct.

[0780] Aspect 33: The synthetic composition of Aspect 1 or Aspect 2, wherein at least one component is attached to a solid substrate.

[0781] Aspect 34: A synthetic composition comprising a target double-stranded DNA polynucleotide, a guide polynucleotide complementary to a sequence in the double-stranded DNA polynucleotide, and a Cas endonuclease having at least 80% identity with a sequence selected from the group consisting of: SEQ ID NO: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37 and 38, or a functional fragment or variant thereof.

[0782] Aspect 35: A synthetic composition comprising a target double-stranded DNA polynucleotide, a polynucleotide encoding a guide polynucleotide complementary to a sequence in the double-stranded DNA polynucleotide, and a cas endonuclease gene having at least 80% identity with a sequence selected from the group consisting of: SEQ ID NO: 13, 14, 15, 16, 25, 26, 27, 28, 29, 30 and 31, or a functional fragment or variant thereof.

[0783] Aspect 36: A method of introducing a site-specific modification at a target sequence in the genome of a cell, the method comprising: introducing a composition synthesized from any of aspects 1-35 into the cell.

[0784] Aspect 37: A method for producing an organism with a modified genome, the method comprising: (a) introducing a heterologous composition into at least one cell of the organism, the heterologous composition comprising: i. a Cas-α endonuclease or a Cas-α polynucleotide encoding the Cas-α endonuclease, ii. a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, wherein the guide polynucleotide and the Cas-α endonuclease are capable of forming a complex that can recognize, bind, and optionally nick or cut the target sequence, iii. and a polynucleotide modification template comprising at least one region complementary to a PAM sequence adjacent to a DNA target sequence recognized by the Cas-α complex, wherein the at least one region corresponding to the PAM sequence comprises at least one nucleotide mismatch; (b) incubating the cells, (c) producing a complete organism from the cells, and (d) verifying at least one nucleotide modification in the genome of at least one cell of the organism compared to the target sequence in the genome of the cell before the introduction of the heterologous composition of (a).

[0785] Aspect 38: The method of aspect 36 or 37, wherein the cell is a eukaryotic cell.

[0786] Aspect 39: The method of aspect 38, wherein the eukaryotic cell is derived from or obtained from an animal or a plant.

[0787] Aspect 40: The method of aspect 39, wherein the plant is a monocot or a dicot.

[0788] Aspect 41: The method of aspect 39, wherein the plant is selected from the group consisting of: maize, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turf grass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower and tomato.

[0789] Aspect 42: The method of aspect 36 or 37, further comprising introducing a heterologous polynucleotide.

[0790] Aspect 43: The method of aspect 42, wherein the heterologous polynucleotide is a donor DNA molecule.

[0791] Aspect 44: The method of aspect 42, wherein the heterologous polynucleotide is a polynucleotide modification template comprising a sequence that is at least 50% identical to a sequence in the cell.

[0792] Aspect 45: A progeny of an organism obtained by the method of aspect 37, wherein said progeny retains said at least one nucleotide modification in at least one cell.

[0793] Aspect 46: A method for modifying the genomic sequence of a target cell, the method comprising providing a Cas endonuclease comprising an amino acid sequence having at least 95% to 100% identity with one of SEQ ID NOs: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37 and 38 and a guide polynucleotide targeting the genomic sequence of the target cell; and introducing a double-stranded break into the genomic sequence of the target cell, thereby modifying the genomic sequence of the target cell.

[0794] Although the present invention has been clearly shown and described with reference to preferred embodiments and various alternative embodiments, it will be appreciated by those skilled in the art that various changes in form and detail may be made thereto without departing from the spirit and scope of the present invention. For example, although the following specific examples may use specific target sites or target organisms to illustrate the methods and embodiments described herein, the principles in these examples may be applied to any target site or target organism. Therefore, it should be understood that the scope of the present invention is encompassed by the embodiments of the present invention described herein and in the specification, rather than by the specific examples exemplified below. For all purposes, all cited patents, applications, and publications mentioned in this application are incorporated herein by reference in their entirety, to the extent that they are each individually and specifically incorporated by reference.

[0795] Examples

[0796] Below are examples of specific embodiments of some aspects of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. With regard to the numerals (e.g., amounts, temperatures, etc.) used, efforts have been made to ensure their accuracy, but some experimental errors and deviations should still be allowed.

[0797] Example 1: Identification and characterization of novel Cas-α-like CRISPR-Cas systems

[0798] In this example, a method for identifying novel class 2 CRISPR (clustered regularly interspaced short palindromic repeats)-Cas ( C The invention relates to a method for RISPR-associated) loci using the identification of operon-like genetic structures and protein structure analysis.

[0799] First, CRISPR arrays were detected in microbial sequences using PILER-CR (Edgar, R. (2007) BMC Bioinformatics, 8: 18) and MinCED (Bland, C. et al. (2007) BMC Bioinformatics, 8: 209) software programs. Next, known CRISPR-Cas systems were removed from the dataset by searching for homology to known CRISPR-associated (Cas) proteins encoded near the CRISPR array (20 kb 5′ and 20 kb 3′ if possible) using a set of position-specific scoring matrices (PSSMs) containing all known Cas protein families (as described in Makarova, K. et al. (2015) Nature Reviews Microbiology, 13: 722-736). To help completely remove known Class 2 CRISPR-Cas systems, multiple sequence alignments of protein sequences from a collection of orthologs from each family of Class 2 CRISPR-Cas endonucleases (e.g., Cas9, Cpf1 (Cas12a), C2c1 (Cas12b), C2c2 (Cas13), C2c3 (Cas12c)) were performed using MUSCLE (Edgar R. (2004) Nucleic Acids Res. 32: 1792-1797). These alignments were inspected, organized, and used to build a profile hidden Markov model (HM) using HMMER (Eddy, SR (1998) Bioinformatics 14: 755-763; Eddy, SR (2011) PLoS Comp. Biol. 7: e1002195). The resulting HMM model was then used to further identify and remove known Class 2 CRISPR-Cas systems from the data set. Next, the remaining CRISPR loci were evaluated for the presence of genes encoding proteins (Cas1 and Cas2) that are considered important for spacer insertion and adaptation using a PSSM-specific search as described above (Makarova, K. et al. (2015) Nature Reviews Microbiology, 13: 722-736). CRISPR loci containing cas1 and cas2 genes were then selected and further examined to determine the proximity, order, and directionality of the undefined genes encoded in the loci relative to the cas1 and cas2 genes and the CRISPR array.Only those CRISPR loci that formed an operon-like structure, in which large (≥1500bp open reading frame) undefined genes were close to the cas1 and cas2 genes and in the same transcriptional orientation, were selected for further analysis. Next, the sequence and structural features of the proteins encoded in the undefined genes were analyzed to indicate Class 2 endonucleases capable of cutting DNA. First, based on the similarity between the candidate sequences and known proteins, various bioinformatics tools were used to reveal their conserved functional features, from pairwise comparisons, to family tree searches, to structural threading, to manual structural inspection. In general, homologous sequences of new candidate proteins are first collected by performing a PSI-BLAST (Altschul, SF et al. (1997) Nucleic Acids Res. [Nucleic Acids Research] 25: 3389-3402) search against the National Center for Biotechnology Information (NCBI) non-redundant (NR) protein set, with a cutoff e value of 0.01. After reducing redundancy at an identity level of approximately 90%, groups of homologous sequences with different member inclusion thresholds (e.g., >60%, 40%, or 20% identity) were aligned using the multiple sequence alignment tools MSAPRobs (Liu, Y. et al. (2010) Bioinformatics. 26: 1958-1964) and Clustalw to reveal conserved motifs. The most conserved homologous sequences were searched for sequence family profiles by HMMER (Eddy, SR (1998) Bioinformatics. 14: 755-763) against a number of domain databases including Pfam, Superfamily, and SCOP (Murzin, AG et al. (1995) J. Mol. Biol. 247: 536-540) and self-made profiles based on internal building block structures. In addition, the resulting candidate homologous sequence alignments were also used to generate candidate protein profiles with predicted secondary structures. The candidate profiles were further used for profile-profile searches against pdb70_hhm and Pfam_hhm profile databases by HHSEARCH (Soding, J. et al. (2006) Nucleic Acids Res. 34: W374-378). In the next step, all detected sequence-structure relationships and conserved motifs were inserted into 3D structure templates using MODELLER or manually mapped to known structural references on DiscoveryStudio (BIOVIA) and Pymol (Schrodinger). Finally, to verify and confirm the potential biological relevance as a Class 2 endonuclease, catalytic or most conserved residues and key structural integrity were manually inspected and evaluated based on the biochemical function of the protein.After the key features indicative of class 2 endonucleases were identified structurally (e.g., one or more DNA cleavage domains), other proteins encoded within the locus (5 kb 5' and 5 kb 3' (where possible) from the end of the newly defined CRISPR-Cas system) were next examined for homology to known protein families using InterProScan software (EMBL-EBI, UK) and compared to the NCBI NR protein set using the BLAST program (Altschul, SF et al. (1990) J. Mol. Biol. 215: 403-410). Genes encoding proteins with similarity (at least 30% identity) to known proteins were annotated in the CRISPR-Cas locus.

[0800] Four novel Class 2 CRISPR-Cas systems were preliminarily identified from unknown microorganisms (Table 1). Figure 1A and 1B As shown, encoded at each locus is a complete CRISPR-Cas system, including all components required for capture and interference. These include genes that collectively encode the proteins required for capture and integration of spacers (Cas1, Cas2, and optionally Cas4) and a novel protein (containing the DNA cleavage domain Cas-alpha (α), located in an operator-like structure adjacent to the CRISPR array) (Table 1).

[0801] Table 1. Novel Class 2 CRISPR-Cas systems Cas-alpha (α) 1, 2, 3 and 4

[0802]

[0803] Next, the Cas-α endonucleases were compared to the NCBI NR protein set using BLAST and then analyzed with MinCED to find proteins close to the CRISPR array (≤5 kb), resulting in seven additional CRISPR systems (Table 2). The loci discovered for these new proteins are shown in Figure 1C and 1D In addition to a partial cas1 gene, the locus encoding Cas-α6 also contains complete cas2 and cas4 genes ( Figure 1C ), while Cas-α5, 7, 8, 9, 10, and 11 contain only endonuclease genes adjacent to the CRISPR array ( Figure 1D ). The loci of Cas-α18 and 19 are depicted in Fig.21A Its mechanism of action is Fig. 21B shown.

[0804] Table 2a. Cas-alpha (α) endonucleases 5-11

[0805]

[0806]

[0807] Structural analysis of these proteins showed that they are different from previously described Class 2 CRISPR-Cas endonucleases with double-stranded DNA target recognition and cleavage capabilities. First, the size of the endonucleases (422-613 amino acids) is significantly compact compared to other known Class 2 CRISPR-Cas systems. Second, the first half of the protein from the amino (N) terminus is highly variable in sequence composition, as evident from the lack of conservation of even single amino acids (except for the start methionine). Nevertheless, secondary structure prediction (PSIPRED (Jones, JT (1999) J. Mol. Biol. [Journal of Molecular Biology] 292: 195-202)) indicates that the presence of mixed β-strands and α-helices suggests the presence of a wedge-shaped (WED) or oligonucleotide binding domain (OBD) structure and helical bundle in the N-terminal region of all Cas-α proteins. In the carboxyl (C)-terminal half of the protein, the key catalytic residues and structure containing the trisection RuvC domain are conserved ( Figure 2 In addition, all proteins contain a bridge-helix domain and a zinc finger domain inserted between RuvC subdomains I-II and II-III, respectively ( Figure 2 ). It is important to note that additional zinc finger-like motifs were detected in Cas-α-1, 2, 3, 4, and 10 proteins. For Cas-α-1, 2, 3, and 4, the second zinc finger motif is located near the N-terminus (e.g., amino acid positions 70-96 and 63-111 in Cas-α-1 and 2, respectively) ( Fig. 8A -D) For Cas-alpha-10, two additional zinc finger motifs were found in the C-terminal half of the protein ( Figure 8J Here, one of the additional zinc finger domains is positioned in tandem with the first one between RuvC II and III subdomains (Cas-α-10 amino acid positions 376-422), and the third one is located after RuvC subdomain III (Cas-α-10 amino acid positions 466-482) ( Figure 8J ). For Cas-α1-11, examples of recovered Cas-α sequences and motifs are shown in Fig. 8A -K. Figure 9 depicts how some Cas-α domains interact with hybrid double-stranded target DNA / guide RNA, using the Cas12b (C2c1) protein backbone (PDB: 5wti) as a reference.

[0808] Sequence analysis of Cas-α1 to 129, using MUSCLE multiple sequence alignment, revealed unique motifs characteristic of Cas-α endonucleases associated with the amino acid position numbers of SEQ ID NO: 17 (Table: glycine (G) at position 337, glycine (G) at position 341, glutamic acid (E) at position 430, leucine (L) at position 432, cysteine ​​(C) at position 487, cysteine ​​(C) at position 490, and / or cysteine ​​(C) at position 507). Cas-α endonucleases contain the following motifs: GxxxG, ExL, Cx n C and Cx n (C, H)(where x n =2-4 residues). Cas-α endonucleases contain one or more zinc finger domains. Table 2b includes some conserved motifs found in Cas-α endonucleases.

[0809] Table 2b. Conserved motifs of Cas-alpha (α) endonuclease

[0810] Motifs are described by the starting amino acid (aa) position of each sequence (ending if >= 6 aas). x = any amino acid (n = any number, where applicable). G = glycine, E = glutamic acid, L = leucine, C = cysteine, H = histidine.

[0811]

[0812]

[0813]

[0814]

[0815] Example 2: Cas-α guide RNA approach

[0816] In this example, methods are described for identifying one or more guide RNAs that support double-stranded DNA target recognition and cleavage by a novel set of Class 2 CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) endonucleases, Cas-α.

[0817] One method relies on computational prediction to determine one or more sRNAs required to form a functional complex with the Cas-α endonuclease. In short, the CRISPR array can be used to generate one or more CRISPR RNAs (one or more crRNAs), taking into account the possible transcription directions of the CRISPR array and the various configurations of the repeat sequences and spacers that may be preferred by the endonuclease (e.g., repeat sequences: spacers, spacers: repeat sequences, or repeat sequences: spacers: repeat sequences). In addition, one or more trans-encoded CRISPR-associated RNAs (one or more tracrRNAs) can be identified by computation in the locus as described in Karvelis, T. et al. (2015) Genome Biology. [Genome Biology] 16: 253. In short, BLAST or manual alignment can be used between the CRISPR repeat consensus sequence and the locus sequence. The homologous region (separated from the CRISPR array) can then be examined by analyzing the possible transcription directions of the inferred one or more tracrRNAs for the secondary structures and possible termination signals present in the RNA versions of the sense and antisense genomic DNA sequences surrounding the anti-repeated sequences. One or more tracrRNAs can then be double-stranded or engineered with various crRNA predictions to form one or more chimeric non-natural single guide RNAs (one or more sgRNAs). One or more crRNAs, one or more tracrRNAs, and one or more sgRNAs can be synthesized (IDT equivalent) or T7 transcribed with TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) or equivalent for further experiments.

[0818] Another approach relies on sequencing of small RNAs (sRNA-seq) produced by the novel Class 2 CRISPR-Cas loci. This can be performed similarly to the method described in Zetsche, B. et al. (2015) Cell. [Cell] 163: 1-13. Briefly, the CRISPR-Cas loci are placed on E. coli plasmid DNA, followed by centrifugation to obtain cultures containing the plasmid-borne CRISPR-Cas genome, total RNA is extracted using the TRIzol Max Bacterial Isolation Kit (Thermo Fisher Scientific), small RNA is isolated using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific), and libraries are prepared for sequencing using the TruSeq Small RNA Library Preparation Kit (Illumina). Expression of the locus can be enhanced using known E. coli promoters. After sequencing on a MiSeq instrument (Innomina) or equivalent, the resulting sequence data is mapped (Bowtie 2 software (Langmead, B. et al. (2012) Nat. Methods. 9:357-359) or equivalent) or returned to the genome to determine the transcription and maturation patterns of one or more sRNAs encoded in the genome.

[0819] A...

Claims

1. A synthetic composition comprising: (a) a Cas endonuclease as shown in the amino acid sequence of SEQ ID NO: 37; (b) a target double-stranded DNA polynucleotide, wherein the target double-stranded DNA polynucleotide is heterologous to the source of the Cas endonuclease, and (c) a guide polynucleotide comprising a variable targeting domain, wherein the variable targeting domain comprises a region complementary to the target double-stranded DNA polynucleotide; wherein the Cas endonuclease recognizes a PAM sequence on the target double-stranded DNA polynucleotide, and wherein the guide polynucleotide and the Cas endonuclease form a complex that binds to the target double-stranded DNA polynucleotide.

2. The synthetic composition of claim 1, wherein the Cas endonuclease is provided as a polynucleotide encoding the Cas endonuclease.

3. The synthetic composition of claim 1, wherein the Cas endonuclease cleaves the double-stranded DNA polynucleotide.

4. The synthetic composition of claim 1, further comprising a heterologous polynucleotide.

5. The synthetic composition of claim 4, wherein the heterologous polynucleotide is an expression element.

6. The synthetic composition of claim 5, wherein the heterologous polynucleotide is a transgene.

7. The synthetic composition of claim 5, wherein the heterologous polynucleotide is a donor DNA molecule.

8. The synthetic composition of claim 5, wherein the heterologous polynucleotide is a polynucleotide modification template.

9. The synthetic composition of claim 1, wherein the Cas endonuclease is part of a fusion protein.

10. The synthetic composition of claim 1, further comprising a deaminase.

11. The synthetic composition of claim 9, wherein the fusion protein further comprises a heterologous nuclease domain.

12. The synthetic composition of claim 1, further comprising a eukaryotic cell.

13. The synthetic composition of claim 12, wherein the eukaryotic cell is a plant cell, an animal cell, or a fungal cell.

14. The synthetic composition of claim 13, wherein the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

15. The synthetic composition of claim 13, wherein the plant cell is from an organism selected from the group consisting of maize, soybean, cotton, wheat, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turf grass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.

16. The synthetic composition of claim 13, wherein the plant cell is from canola.

17. A polynucleotide encoding the Cas endonuclease of the synthetic composition of claim 1.

18. The polynucleotide of claim 17, further comprising: at least one additional polynucleotide.

19. The polynucleotide of claim 18, wherein the at least one additional polynucleotide is an expression element.

20. The polynucleotide of claim 18, wherein the at least one additional polynucleotide is a gene.

21. The synthetic composition of claim 1, wherein at least one component is attached to a solid substrate.

22. A method for introducing targeted editing in a target polynucleotide, the method comprising providing a heterologous composition comprising: (a) a Cas endonuclease as shown in the amino acid sequence of SEQ ID NO: 37; wherein the Cas endonuclease recognizes a PAM sequence on the target polynucleotide; (b) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a portion of the target polynucleotide, wherein the guide polynucleotide and the Cas-α endonuclease form a complex that can recognize and bind to the target polynucleotide; The methods are not intended for the purpose of treating disease in living animals or humans.

23. The method of claim 22, further comprising introducing at least one nucleotide modification in the genome of at least one cell of the organism compared to the target sequence in the genome of the cell prior to the introduction of the heterologous composition, further comprising incubating the cells and producing a whole organism from the cells, and determining the presence of the at least one nucleotide modification in the genome of at least one cell of the organism compared to the target sequence in the genome of the cell prior to the introduction of the heterologous composition.

24. The method of claim 23, wherein the cell is a eukaryotic cell.

25. The method of claim 24, wherein the eukaryotic cell is derived or obtained from an animal, a fungus or a plant.

26. The method of claim 25, wherein the plant is a monocot or a dicot.

27. The method of claim 25, wherein the plant is selected from the group consisting of maize, soybean, cotton, wheat, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turf grass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.

28. The method of claim 25, wherein the plant is canola.

29. The method of claim 23, further comprising: Introduction of heterologous polynucleotides.

30. The method of claim 29, wherein the heterologous polynucleotide is a donor DNA molecule.

31. The method of claim 29, wherein the heterologous polynucleotide is a polynucleotide modification template comprising a sequence that is at least 50% identical to a sequence in the cell.

32. A method of obtaining progeny of an organism, wherein the progeny retains at least one nucleotide modification in at least one cell, the method comprising providing a heterologous composition comprising: (a) a Cas endonuclease as shown in the amino acid sequence of SEQ ID NO: 37; wherein the Cas endonuclease recognizes a PAM sequence on a target polynucleotide; (b) a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a portion of the target polynucleotide, wherein the guide polynucleotide and the Cas-α endonuclease form a complex that can recognize and bind to the target polynucleotide; introducing at least one nucleotide modification in the genome of at least one cell of the organism as compared to a target sequence in the genome of the cell prior to introduction of the heterologous composition; The cells are incubated and a whole organism is generated from the cells, and the presence of at least one nucleotide modification in the genome of at least one cell of the organism is determined compared to the target sequence in the genome of the cell prior to the introduction of the heterologous composition, wherein the cell is a non-human cell.

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