A novel CRISPR-CAS system for genome editing
Patent Information
- Application Number
- JP2021533502
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2019-10-10
- Filing Date
- 2019-12-13
- Publication Date
- 2024-12-11
- Estimated Expiration
- 2039-12-13
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Abstract
Description
Technical field
[0001] Cross-reference to related applications This application is based on U.S. Provisional Application No. 62 / 779989, filed December 14, 2018; U.S. Provisional Application No. 62 / 794,427, filed January 18, 2019; U.S. Provisional Application No. 62 / 819409 filed May 24, 2019; U.S. Provisional Application No. 62 / 852788 filed May 24, 2019; , all of which are incorporated herein by reference in their entirety.
[0002] Reference to Sequence Listing Submitted Electronically A definitive copy of the Sequence Listing is available at EFS as a Sequence Listing in ASCII format with a file name of RTS21920B_SequenceListing_ST25.txt, created on December 9, 2019, having a size of 714,386 bytes and filed herewith concurrently. - Submitted electronically via the web. The Sequence Listing contained in this ASCII format document is incorporated herein by reference in its entirety.
[0003] FIELD OF THE DISCLOSURE The present disclosure relates to the field of molecular biology and, in particular, to compositions of novel RNA-guided Cas endonuclease systems and compositions and methods for editing or modifying the genome of cells. [Background technology]
[0004] Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and / or modify specific endogenous chromosomal sequences. Site-specific integration techniques using site-specific recombination systems, like other recombination techniques, have been used to generate targeted insertions of genes of interest in a variety of organisms. Genome editing techniques such as designer zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), or homing meganucleases are available for generating targeted genome perturbations, but these systems lack specificity. There is a tendency to use engineered nucleases that are inexpensive and need to be redesigned for each target site and are therefore expensive and time consuming to produce.
[0005] Archaeal or bacterial adaptation termed CRISPR (Clustered Regularly Arranged Short Palindromic Repeats) containing various domains of effector proteins encompassing diverse activities (recognition, binding and optional cleavage of DNA) Newer techniques using the immune system have been identified.
[0006] Despite the identification and characterization of several of these systems, the identification of novel effectors and systems to effect editing of endogenous and previously introduced heterologous polynucleotides and eukaryotes, especially There remains a need to demonstrate activity in animals and plants.
[0007] Described herein are novel Cas endonucleases, "Cas-alpha," exemplary proteins, and methods and compositions for their use. [Outline of the Invention] [Means for solving the problem]
[0008] Disclosed herein are novel Cas endonuclease compositions and methods of use thereof. These endonucleases of the novel Cas-alpha class have been demonstrated in three different kingdoms of prokaryotes (E. coli) and eukaryotes: the plant, animal and fungal kingdoms. As such, double-stranded DNA can be targeted and guided by a guide polynucleotide for PAM-dependent cleavage.
[0009] In one aspect, it comprises at least one zinc finger-like domain, at least one bridge-helix-like domain, a three-split RuvC domain (comprising noncontiguous RuvC-I, RuvC-II, and RuvC-III domains), A synthetic composition comprising a CRISPR-Cas endonuclease, optionally comprising a heterologous polynucleotide is provided.
[0010] In any aspect, any of the compositions and methods provide at least one component optimized for expression in eukaryotic cells, particularly plant, fungal, or animal cells.
[0011] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding a CRISPR-Cas effector protein from an organism selected from the group consisting of: Acidobacillus sulfuroxidans, and a heterologous polynucleotide. (Acidibacillus sulfuroxidans), Alicyclobacillus acidoterrestris, Aneurinibacillus danicus, Archaea, Bacillus, Bacillus cereus, Bacillus cereus Bacillus megaterium, Bacillus pseudomycoides, Bacillus sp., Bacillus thuringiensis, Bacillus toyonensis, Bacillus wiedmannii ), Bacteroides plebeius, Bos taurus, Brevibacillus centrosporus, Candidatus Aureabacteria bacterium, Candidatus Levybacteria bacterium , Candidatus Micrarchaeota archaeon, Cellulosilyticum ruminicola, Clostridioides difficile, Clostridium botulinum, Clostridium fallax , Clostridium hiranonis, Clostridiumi humii, Clostridium novyi, Clostridium paraputrificum, Clostridium pasteurianum, Clostridium perfringens, Clostridium sp., Clostridium tetani, Clostridium ventriculi, Desulfovibrio fructosivorans, Dorea longicatena, Eubacterium siraeum, Flavobacterium thermophilum ), Gallus gallus, Hepatitis delta virus, Homo sapiens, Human betaherpesvirus 5, Hydrogenivirga sp., Mus musculus, Parageobacillus thermoglucosidasius, Peptoclostridium sp., Phascolarctobacterium sp., Prevotella copri, Ruminiclostridium hungatei, Ruminococcus albus, Ruminococcus sp., Saccharomyces cerevisiae, Simian virus 40, Solanum tuberosum, Sulfrihydrogenibium Azollense (Sulfurihydrogenibi) um azorense), Syntrophomonas palmitatica, Tobacco etch virus, and Zea mays.
[0012] In one aspect, a synthetic composition comprising a eukaryotic cell, a heterologous CRISPR-Cas effector, wherein said heterologous CRISPR-Cas effector protein is less than 800, 790-800, less than 790, 780-790, less than 780, 770- 780, less than 770, 760-770, less than 760, 750-760, less than 750, 740-750, less than 740, 730-740, less than 730, 720-730, less than 720, 710-720, less than 710, 700-710 or less than 700 amino acids, e.g., 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 less than 350 amino acids A synthetic composition is provided comprising:
[0013] In one aspect, a synthetic composition comprising a CRISPR-Cas endonuclease, said CRISPR-Cas endonuclease having, relative to the amino acid position numbers of SEQ ID NO: 17, at least one of the following when aligned to SEQ ID NO: 17: , at least two, at least three, at least four, at least five, at least six, or seven: glycine (G) at position 337, glycine (G) at position 341, Glutamic acid at position 430 (E), leucine at position 432 (L), cysteine at position 487 (C), cysteine at position 490 (C), cysteine at position 507 (C), and / or cysteine at position 512 (C) Or histidine (H).
[0014] In one aspect there is provided a synthetic composition comprising a CRISPR-Cas endonuclease, said CRISPR-Cas endonuclease comprising 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.
[0015] In one aspect, a synthetic composition comprising a CRISPR-Cas endonuclease, said CRISPR-Cas endonuclease comprising one or more zinc finger motifs is provided.
[0016] In one aspect, the 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, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400 sequences selected from the group consisting of 365, 366, 367, 368, 369, 370, and 371; at least 400 or more than 400 contiguous amino acids and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, at least 65%, 65% ~70%, at least 70%, 70%~75%, at least 75%, 75%~80%, at least 80%, 80%~85%, at least 85%, 85%~90%, at least 90%, 90% ~95%, at least 95%, 95%~96%, at least 96%, 96%~97%, at least 97%, 97%~98%, at least 98%, 98%~99%, at least 99%, 99% Synthetic compositions comprising CRISPR-Cas effector proteins sharing ~100% or 100% sequence identity are provided.
[0017] In one aspect, the 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, at least 250, 250-500, at least 500, 500-600, at least 600, 600-700 sequences selected from the group consisting of 365, 366, 367, 368, 369, 370, and 371; at least 700, 700-750, at least 750, 750-800, at least 800, 800-850, at least 850, 850-900, at least 900, 900-950, at least 950, 950 ~1000, at least 1000, or more than 1000 amino acids; 65%~70%, at least 70%, 70%~75%, at least 75%, 75%~80%, at least 80%, 80%~85%, at least 85%, 85%~90%, at least 90%, 90%-95%, at least 95%, 95%-96%, at least 96%, 96%-97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, CRISPR-Cas effectors sharing 99%-100% or 100% sequence identity A synthetic composition is provided that contains a polynucleotide that encodes the protein.
[0018] In one aspect, SEQ ID NOs: 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 at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 RNA sequences selected from the group consisting of 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 contiguous nucleotides and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, at least 65%, 65%-70%, at least 70%, 70%-75%, at least 75%, 75%-80%, at least 80%, 80%-85%, at least 85%, 85%-90%, at least 90%, 90%-95%, at least 95%, 95%-96%, at least 96%, 96%-97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100%, or A synthetic composition is provided comprising a polynucleotide encoding a CRISPR-Cas effector protein capable of hybridizing with a polynucleotide sharing 100% sequence identity.
[0019] Any of the methods or compositions herein can further comprise a heterologous polynucleotide. Heterologous polynucleotides may be selected from the group consisting of: non-coding expression regulatory elements such as promoters, introns, enhancers, or terminators; donor polynucleotides; optional at least one modification compared to the polynucleotide sequence in the cell; guide RNA; guide DNA; guide RNA-DNA hybrid; endonuclease; nuclear localization signal;
[0020] In one aspect, methods are provided for using any of the compositions disclosed herein. In some embodiments, methods are provided for binding a Cas-alpha endonuclease to a polynucleotide target sequence, eg, in the genome of a cell or in vitro. In some embodiments, the Cas-alpha endonuclease forms a complex with a guide polynucleotide, eg guide RNA. In some embodiments, complexes recognize, bind, and optionally generate nicks (single-stranded) or breaks (double-stranded) in polynucleotides at or near the target sequence. In some embodiments, the nick or break is repaired by non-homologous end joining (NHEJ). In some embodiments, nicks or breaks are repaired by homologous recombination repair (HDR) or homologous recombination (HR) using a polynucleotide modified template or donor DNA molecule.
[0021] The novel Cas endonucleases described herein contain a suitable PAM to generate a double-strand break at or adjacent to a target polynucleotide directed by a guide polynucleotide in any prokaryotic or eukaryotic cell. can be generated. In some examples, the cells are plant cells, or animal cells, or fungal cells. In some examples, the plant cell is selected from the group consisting of: corn, soybean, cotton, wheat, canola, rape, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, Alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, and tomato.
[0022] Brief description of drawings and sequence listing The present disclosure can be more fully understood from the following detailed description, which forms a part of this application, and the drawings and sequence listing that accompany this specification. [Brief description of the drawing]
[0023] [Fig. 1-1] Figure 1: Figures 1A-1D show a complete CRISPR-Cas system containing all the components required for acquisition and interference. These co-encode all the proteins required for spacer acquisition and integration (Cas1 and Cas2), as well as a novel protein containing a DNA cleavage domain, Cas-alpha (α), in an operon-like structure flanking the CRISPR array. gene included. In addition, a gene encoding a protein with homology to Cas4 was also encoded at this locus. FIG. 1A shows the locus structure of the Cas-alpha1, Cas-alpha3, and Cas-alpha4 systems. FIG. 1B shows the locus structure of the Cas-alpha2 system. FIG. 1C shows the locus structure of the Cas-alpha6 system. FIG. 1D shows the locus structure of the Cas-alpha 5, 7, 8, 9, 10, and 11 lines. [Fig. 1-2] (As above.) [Fig. 1-3] (As above.) [Fig. 1-4] (As above.)
Figure 2
[0024] The sequence descriptions and sequence listing attached hereto comply with the rules governing the disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.825. The sequence description includes the three-letter code for amino acids as set forth in 37 C.F.R. §§ 1.821 and 1.825, which are incorporated herein by reference.
[0025] SEQ ID NO: 1 is Cas1 encoded by the Cas-alpha 1 locus PRT sequence from Candidatus Micrarchaeota archaeon.
[0026] SEQ ID NO: 2 is Cas1 encoded by the Cas-alpha2 locus PRT sequence from Candidatus Micrarchaeota archaeon.
[0027] SEQ ID NO: 3 is Cas1 encoded by the Cas-alpha3 locus PRT sequence from Candidatus Aureabacteria bacterium.
[0028] SEQ ID NO: 4 is Cas1 encoded by the Cas-alpha4 locus PRT sequence from uncultured archaea.
[0029] SEQ ID NO: 5 is Cas2 encoded by the Cas-alpha 1 locus PRT sequence from Candidatus Micrarchaeota archaeon.
[0030] SEQ ID NO: 6 is Cas2 encoded by the Cas-alpha2 locus PRT sequence from Candidatus Micrarchaeota archaeon.
[0031] SEQ ID NO: 7 is Cas2 encoded by the Cas-alpha3 locus PRT sequence from Candidatus Aureabacteria bacterium.
[0032] SEQ ID NO: 8 is Cas2 encoded by the Cas-alpha4 locus PRT sequence from uncultured archaea.
[0033] SEQ ID NO: 9 is Cas4 encoded by the Cas-alpha 1 locus PRT sequence from Candidatus Micrarchaeota archaeon.
[0034] SEQ ID NO: 10 is Cas4 encoded by the Cas-alpha2 locus PRT sequence from Candidatus Micrarchaeota archaeon.
[0035] SEQ ID NO: 11 is Cas4 encoded by the Cas-alpha3 locus PRT sequence from Candidatus Aureabacteria bacterium.
[0036] SEQ ID NO: 12 is Cas4 encoded by the Cas-alpha4 locus PRT sequence from uncultured archaea.
[0037] SEQ ID NO: 13 is the Cas-alpha 1 endonuclease gene DNA sequence from Candidatus Micrarchaeota archaeon.
[0038] SEQ ID NO: 14 is the Cas-alpha2 endonuclease gene DNA sequence from Candidatus Micrarchaeota archaeon.
[0039] SEQ ID NO: 15 is the Cas-alpha3 endonuclease gene DNA sequence from Candidatus Aureabacteria bacterium.
[0040] SEQ ID NO: 16 is the Cas-alpha4 endonuclease gene DNA sequence from an uncultured archaea.
[0041] SEQ ID NO: 17 is the Cas-alpha 1 endonuclease (Cas14b4) PRT sequence from Candidatus Micrarchaeota archaeon.
[0042] SEQ ID NO: 18 is the Cas-alpha2 endonuclease PRT sequence from Candidatus Micrarchaeota archaeon.
[0043] SEQ ID NO: 19 is the Cas-alpha3 endonuclease PRT sequence from Candidatus Aureabacteria bacterium.
[0044] SEQ ID NO:20 is the Cas-alpha4 endonuclease (Cas14a1) PRT sequence from uncultured archaea.
[0045] SEQ ID NO:21 is the Cas-alpha1 locus DNA sequence from Candidatus Micrarchaeota archaeon.
[0046] SEQ ID NO: 22 is the Cas-alpha2 locus DNA sequence from Candidatus Micrarchaeota archaeon.
[0047] SEQ ID NO:23 is the Cas-alpha3 locus DNA sequence from Candidatus Aureabacteria bacterium.
[0048] SEQ ID NO:24 is the Cas-alpha4 locus DNA sequence from an uncultured archaea.
[0049] SEQ ID NO:25 is the Cas-alpha5 endonuclease gene DNA sequence from Candidatus Micrarchaeota archaeon.
[0050] SEQ ID NO: 26 is the Cas-alpha6 endonuclease gene DNA sequence from uncultured archaea.
[0051] SEQ ID NO: 27 is the Cas-alpha7 endonuclease gene DNA sequence from Parageobacillus thermoglucosidasius.
[0052] SEQ ID NO: 28 is the Cas-alpha 8 endonuclease gene DNA sequence from Acidibacillus sulfuroxidans.
[0053] SEQ ID NO:29 is the Cas-alpha9 endonuclease gene DNA sequence from Ruminococcus sp.
[0054] SEQ ID NO:30 is the Cas-alpha10 endonuclease gene DNA sequence from Syntrophomonas palmitatica.
[0055] SEQ ID NO:31 is the Cas-alpha11 endonuclease gene DNA sequence from Clostridium novyi.
[0056] SEQ ID NO:32 is the Cas-alpha5 endonuclease PRT sequence from Candidatus Micrarchaeota archaeon.
[0057] SEQ ID NO:33 is a Cas-alpha6 endonuclease PRT sequence from uncultured archaea.
[0058] SEQ ID NO:34 is the Cas-alpha7 endonuclease PRT sequence from Parageobacillus thermoglucosidasius.
[0059] SEQ ID NO:35 is the Cas-alpha8 endonuclease PRT sequence from Acidibacillus sulfuroxidans.
[0060] SEQ ID NO:36 is the Cas-alpha9 endonuclease PRT sequence from Ruminococcus sp.
[0061] SEQ ID NO:37 is the Cas-alpha10 endonuclease PRT sequence from Syntrophomonas palmitatica.
[0062] SEQ ID NO:38 is the Cas-alpha11 endonuclease PRT sequence from Clostridium novyi.
[0063] SEQ ID NO:39 is the Cas-alpha5 locus DNA sequence from Candidatus Micrarchaeota archaeon.
[0064] SEQ ID NO: 40 is the Cas-alpha6 locus DNA sequence from an uncultured archaea.
[0065] SEQ ID NO: 41 is the Cas-alpha7 locus DNA sequence from Parageobacillus thermoglucosidasius.
[0066] SEQ ID NO: 42 is the Cas-alpha8 locus DNA sequence from Acidibacillus sulfuroxidans.
[0067] SEQ ID NO: 43 is the Cas-alpha9 locus DNA sequence from Ruminococcus sp.
[0068] SEQ ID NO: 44 is the Cas-alpha 10 locus DNA sequence from Syntrophomonas palmitatica.
[0069] SEQ ID NO: 45 is the Cas-alpha 11 locus DNA sequence from Clostridium novyi.
[0070] SEQ ID NO: 46 is a Cas-alpha1 repeat consensus DNA sequence from Candidatus Micrarchaeota archaeon.
[0071] SEQ ID NO: 47 is a Cas-alpha2 repeat consensus DNA sequence from Candidatus Micrarchaeota archaeon.
[0072] SEQ ID NO:48 is a Cas-alpha3 repeat consensus DNA sequence from Candidatus Aureabacteria bacterium.
[0073] SEQ ID NO:49 is a Cas-alpha4 repeat consensus DNA sequence from uncultured archaea.
[0074] SEQ ID NO:50 is the Cas-alpha5 repeat consensus DNA sequence from Candidatus Micrarchaeota archaeon.
[0075] SEQ ID NO:51 is a Cas-alpha6 repeat consensus DNA sequence from uncultured archaea.
[0076] SEQ ID NO: 52 is a Cas-alpha7 repeat consensus DNA sequence from Parageobacillus thermoglucosidasius.
[0077] SEQ ID NO: 53 is a Cas-alpha 8 repeat consensus DNA sequence from Acidibacillus sulfuroxidans.
[0078] SEQ ID NO:54 is a Cas-alpha9 repeat consensus DNA sequence from Ruminococcus sp.
[0079] SEQ ID NO:55 is a Cas-alpha10 repeat consensus DNA sequence from Syntrophomonas palmitatica.
[0080] SEQ ID NO:56 is the Cas-alpha11 repeat consensus DNA sequence from Clostridium novyi.
[0081] SEQ ID NO: 57 is the RNA sequence of the artifact-derived Cas-alpha1 crRNA (where N represents any nucleotide).
[0082] SEQ ID NO: 58 is the Cas-alpha2 crRNA (where N represents any nucleotide) RNA sequence from an artifact.
[0083] SEQ ID NO: 59 is the Cas-alpha4 crRNA (where N represents any nucleotide) RNA sequence from an artifact.
[0084] SEQ ID NO: 60 is the Cas-alpha1 tracrRNA version 1 RNA sequence from Candidatus Micrarchaeota archaeon.
[0085] SEQ ID NO:61 is the Cas-alpha1 tracrRNA version 2 RNA sequence from Candidatus Micrarchaeota archaeon.
[0086] SEQ ID NO:62 is the Cas-alpha1 tracrRNA version 3 RNA sequence from Candidatus Micrarchaeota archaeon.
[0087] SEQ ID NO:63 is the Cas-alpha1 tracrRNA version 4 RNA sequence from Candidatus Micrarchaeota archaeon.
[0088] SEQ ID NO:64 is the Cas-alpha2 tracrRNA version 1 RNA sequence from Candidatus Micrarchaeota archaeon.
[0089] SEQ ID NO:65 is the Cas-alpha2 tracrRNA version 2 RNA sequence from Candidatus Micrarchaeota archaeon.
[0090] SEQ ID NO:66 is the Cas-alpha2 tracrRNA version 3 RNA sequence from Candidatus Micrarchaeota archaeon.
[0091] SEQ ID NO:67 is the Cas-alpha2 tracrRNA version 4 RNA sequence from Candidatus Micrarchaeota archaeon.
[0092] SEQ ID NO:68 is the Cas-alpha4 tracrRNA version 1 RNA sequence from uncultured archaea.
[0093] SEQ ID NO:69 is an artifact-derived Cas-alpha1 sgRNA version 1 RNA sequence.
[0094] SEQ ID NO:70 is an artifact-derived Cas-alpha1 sgRNA version 2 RNA sequence.
[0095] SEQ ID NO:71 is an artifact-derived Cas-alpha1 sgRNA version 3 RNA sequence.
[0096] SEQ ID NO:72 is an artifact-derived Cas-alpha1 sgRNA version 4 RNA sequence.
[0097] SEQ ID NO:73 is an artifact-derived Cas-alpha2 sgRNA version 1 RNA sequence.
[0098] SEQ ID NO:74 is an artifact-derived Cas-alpha2 sgRNA version 2 RNA sequence.
[0099] SEQ ID NO:75 is an artifact-derived Cas-alpha2 sgRNA version 3 RNA sequence.
[0100] SEQ ID NO:76 is an artifact-derived Cas-alpha2 sgRNA version 4 RNA sequence.
[0101] SEQ ID NO:77 is an artifact-derived Cas-alpha4 sgRNA version 1 RNA sequence.
[0102] SEQ ID NO: 78 is an artifact-derived T2 spacer DNA sequence.
[0103] SEQ ID NO:79 is an artifact-derived complete Cas-alpha1 locus engineered to target a T2 DNA sequence.
[0104] SEQ ID NO:80 is an artifact-derived minimal Cas-alpha1 locus engineered to target a T2 DNA sequence.
[0105] SEQ ID NO:81 is an artifact-derived 10× histidine-tagged PRT sequence.
[0106] SEQ ID NO:82 is an artifact-derived 6x histidine-tagged PRT sequence.
[0107] SEQ ID NO:83 is an artifact-derived maltose binding protein tag PRT sequence.
[0108] SEQ ID NO:84 is the Tobacco etch virus cleavage site PRT sequence from Tobacco etch virus.
[0109] SEQ ID NO:85 is the A1 oligonucleotide DNA sequence derived from the artifact.
[0110] SEQ ID NO:86 is the A2 oligonucleotide DNA sequence derived from the artifact.
[0111] SEQ ID NO:87 is an artifact-derived R0 oligonucleotide DNA sequence.
[0112] SEQ ID NO:88 is the C0 oligonucleotide DNA sequence from an artifact.
[0113] SEQ ID NO:89 is an artifact-derived F1 oligonucleotide DNA sequence.
[0114] SEQ ID NO:90 is an artifact-derived R1 oligonucleotide DNA sequence.
[0115] SEQ ID NO:91 is the bridge amplification portion of the artifact-derived F1 oligonucleotide DNA sequence.
[0116] SEQ ID NO:92 is the bridge amplification portion of the artifact-derived R1 oligonucleotide DNA sequence.
[0117] SEQ ID NO:93 is an artifact-derived F2 oligonucleotide DNA sequence.
[0118] SEQ ID NO:94 is an artifact-derived R2 oligonucleotide DNA sequence.
[0119] SEQ ID NO:95 is an artifact-derived C1 oligonucleotide DNA sequence.
[0120] SEQ ID NO:96 is the sequence resulting from cleavage at position 21 and adapter ligation of the artifact-derived target DNA sequence.
[0121] SEQ ID NO:97 is the adapter portion of the DNA sequence of SEQ ID NO:96 that is derived from an artifact.
[0122] SEQ ID NO:98 is the target portion of the DNA sequence of SEQ ID NO:96, which is derived from an artifact.
[0123] SEQ ID NO:99 is sequence 5' of a PAM DNA sequence derived from an artifact.
[0124] SEQ ID NO: 100 is a fixed double-stranded DNA target DNA sequence derived from an artifact.
[0125] SEQ ID NO: 101 is an artifact-derived T2 target sequence DNA sequence.
[0126] SEQ ID NO: 102 is an artifact-derived Cas-alpha4 T2-1 sgRNA RNA sequence.
[0127] SEQ ID NO: 103 is an artifact-derived Cas-alpha4 T2-2 sgRNA RNA sequence.
[0128] SEQ ID NO: 104 is an artifact-derived Cas-alpha4 T2-1 crRNA RNA sequence.
[0129] SEQ ID NO: 105 is an artifact-derived Cas-alpha4 T2-2 crRNA RNA sequence.
[0130] SEQ ID NO: 106 is the ST-LS1 intron 2 DNA sequence from potato (Solanum tuberosum).
[0131] SEQ ID NO: 107 is the SV40 NLS PRT sequence from Simian virus 40.
[0132] SEQ ID NO: 108 is the Nuc NLS PRT sequence from Mus musculus.
[0133] SEQ ID NO: 109 is the maize UBI promoter DNA sequence from Zea mays.
[0134] SEQ ID NO: 110 is the chicken beta-actin promoter DNA sequence from the red jungle fowl (Gallus gallus).
[0135] SEQ ID NO: 111 is the CMV enhancer DNA sequence from human beta-herpesvirus 5.
[0136] SEQ ID NO: 112 is the maize UBI 5 prime untranslated region DNA sequence from Zea mays.
[0137] SEQ ID NO: 113 is the maize UBI intron 1 DNA sequence from Zea mays.
[0138] SEQ ID NO: 114 is a hybrid intron DNA sequence derived from an artifact.
[0139] SEQ ID NO: 115 is the maize U6 polymerase III promoter DNA sequence from Zea mays.
[0140] SEQ ID NO: 116 is the human U6 polymerase III promoter DNA sequence from Homo sapiens.
[0141] SEQ ID NO: 117 is an artifact-derived Strep II tag PRT sequence.
[0142] SEQ ID NO: 118 is the bGH poly(A) terminator DNA sequence from bovine (Bos taurus).
[0143] SEQ ID NO: 119 is the potato protease inhibitor II (Pin II) terminator DNA sequence from potato (Solanum tuberosum).
[0144] SEQ ID NO: 120 is the Zea mays Wt reference (Liguleless targets 2 and 3) DNA sequence from Zea mays.
[0145] SEQ ID NO: 121 is Mutant 1 (Liguleless target 2 and 3-DNA Exp.) DNA sequence from Zea mays.
[0146] SEQ ID NO: 122 is the Mutant 2 (Liguleless target 2 and 3-DNA Exp.) DNA sequence from Zea mays.
[0147] SEQ ID NO: 123 is Mutant 3 (Liguleless target 2 and 3-DNA Exp.) DNA sequence from Zea mays.
[0148] SEQ ID NO: 124 is the Mutant 4 (Liguleless target 2 and 3-DNA Exp.) DNA sequence from Zea mays.
[0149] SEQ ID NO: 125 is Mutant 5 (Liguleless target 2 and 3-DNA Exp.) DNA sequence from Zea mays.
[0150] SEQ ID NO: 126 is the HEK293 Wt reference (VEGFA target 2) DNA sequence from Homo sapiens.
[0151] SEQ ID NO: 127 is Mutant 1 (VEGFA Target 2-RNP) DNA sequence from Homo sapiens.
[0152] SEQ ID NO: 128 is Mutant 2 (VEGFA Target 2-RNP) DNA sequence from Homo sapiens.
[0153] SEQ ID NO: 129 is the Mutant 3 (VEGFA Target 2-RNP) DNA sequence from Homo sapiens.
[0154] SEQ ID NO: 130 is the Mutant 4 (VEGFA Target 2-RNP) DNA sequence from Homo sapiens.
[0155] SEQ ID NO: 131 is the Mutant 5 (VEGFA target 2-RNP) DNA sequence from Homo sapiens.
[0156] SEQ ID NO: 132 is the HEK293 Wt reference (VEGFA target 3) DNA sequence from Homo sapiens.
[0157] SEQ ID NO: 133 is Mutant 1 (VEGFA target 3-RNP) DNA sequence from Homo sapiens.
[0158] SEQ ID NO: 134 is Mutant 1 (VEGFA target 3-DNA Exp) DNA sequence from Homo sapiens.
[0159] SEQ ID NO: 135 is Mutant 2 (VEGFA target 3-DNA Exp) DNA sequence from Homo sapiens.
[0160] SEQ ID NO: 136 is the ROX3 promoter DNA sequence from Saccharomyces cerevisiae.
[0161] SEQ ID NO: 137 is the GAL promoter DNA sequence from Saccharomyces cerevisiae.
[0162] SEQ ID NO: 138 is an artifact-derived HH ribozyme (where N represents the nucleotide complementary to the 6 nucleotides 3' of the ribozyme) DNA sequence.
[0163] SEQ ID NO: 139 is the HDV ribozyme DNA sequence from Hepatitis delta virus.
[0164] SEQ ID NO: 140 is the SNR52 promoter DNA sequence from Saccharomyces cerevisiae.
[0165] SEQ ID NO: 141 is the SUP4 terminator DNA sequence from Saccharomyces cerevisiae.
[0166] SEQ ID NO: 142 is the sequence DNA sequence shown at the top of Figure 15C from an artifact.
[0167] SEQ ID NO: 143 is the sequence DNA sequence shown at the bottom of Figure 15C from an artifact.
[0168] SEQ ID NO: 144 is the reference DNA sequence shown in Figure 18A from Zea mays.
[0169] SEQ ID NO: 145 is the Mutant 1 DNA sequence from Zea mays.
[0170] SEQ ID NO: 146 is the Mutant 2 DNA sequence from Zea mays.
[0171] SEQ ID NO: 147 is the Mutant 3 DNA sequence from Zea mays.
[0172] SEQ ID NO: 148 is the Mutant 4 DNA sequence from Zea mays.
[0173] SEQ ID NO: 149 is the Mutant 5 DNA sequence from Zea mays.
[0174] SEQ ID NO: 150 is the Mutant 6 DNA sequence from Zea mays.
[0175] SEQ ID NO: 151 is the Mutant 7 DNA sequence from Zea mays.
[0176] SEQ ID NO: 152 is the Mutant 8 DNA sequence from Zea mays.
[0177] SEQ ID NO: 153 is the Mutant 9 DNA sequence from Zea mays.
[0178] SEQ ID NO: 154 is the Mutant 10 DNA sequence from Zea mays.
[0179] SEQ ID NO: 155 is the Mutant 11 DNA sequence from Zea mays.
[0180] SEQ ID NO: 156 is the Mutant 12 DNA sequence from Zea mays.
[0181] SEQ ID NO: 157 is the Mutant 13 DNA sequence from Zea mays.
[0182] SEQ ID NO: 158 is the Mutant 14 DNA sequence from Zea mays.
[0183] SEQ ID NO: 159 is the Mutant 15 DNA sequence from Zea mays.
[0184] SEQ ID NO: 160 is the Mutant 16 DNA sequence from Zea mays.
[0185] SEQ ID NO: 161 is the Mutant 17 DNA sequence from Zea mays.
[0186] SEQ ID NO: 162 is the Mutant 18 DNA sequence from Zea mays.
[0187] SEQ ID NO: 163 is the Mutant 19 DNA sequence from Zea mays.
[0188] SEQ ID NO: 164 is the reference DNA sequence shown in Figure 18B from Zea mays.
[0189] SEQ ID NO: 165 is the Mutant 1 DNA sequence from Zea mays.
[0190] SEQ ID NO: 166 is the Mutant 2 DNA sequence from Zea mays.
[0191] SEQ ID NO: 167 is the Mutant 3 DNA sequence from Zea mays.
[0192] SEQ ID NO: 168 is the Mutant 4 DNA sequence from Zea mays.
[0193] SEQ ID NO: 169 is the Mutant 5 DNA sequence from Zea mays.
[0194] SEQ ID NO: 170 is the reference DNA sequence shown in Figure 19C from Saccharomyces cerevisiae.
[0195] SEQ ID NO: 171 is an artifact-derived repair template DNA sequence.
[0196] SEQ ID NO: 172 is a repair result 1 DNA sequence from Saccharomyces cerevisiae.
[0197] SEQ ID NO: 173 is a repair result 2 DNA sequence from Saccharomyces cerevisiae.
[0198] SEQ ID NO: 174 is a repair result 1 DNA sequence from Saccharomyces cerevisiae.
[0199] SEQ ID NO: 175 is a repair result 1 DNA sequence from Saccharomyces cerevisiae.
[0200] SEQ ID NO: 176 is a repair result 2 DNA sequence from Saccharomyces cerevisiae.
[0201] SEQ ID NO: 177 is an artifact-derived Cas-alpha3 crRNA (where N represents any nucleotide) RNA sequence.
[0202] SEQ ID NO: 178 is an artifact-derived Cas-alpha5 crRNA (where N represents any nucleotide) RNA sequence.
[0203] SEQ ID NO: 179 is an artifact-derived Cas-alpha6 crRNA (where N represents any nucleotide) RNA sequence.
[0204] SEQ ID NO: 180 is an artifact-derived Cas-alpha7 crRNA (where N represents any nucleotide) RNA sequence.
[0205] SEQ ID NO: 181 is an artifact-derived Cas-alpha8 crRNA (where N represents any nucleotide) RNA sequence.
[0206] SEQ ID NO: 182 is an artifact-derived Cas-alpha9 crRNA (where N represents any nucleotide) RNA sequence.
[0207] SEQ ID NO: 183 is an artifact-derived Cas-alpha10 crRNA (where N represents any nucleotide) RNA sequence.
[0208] SEQ ID NO: 184 is an artifact-derived Cas-alpha11 crRNA (where N represents any nucleotide) RNA sequence.
[0209] SEQ ID NO: 185 is the Cas-alpha2 tracrRNA version 5 RNA sequence from Candidatus Micrarchaeota archaeon.
[0210] SEQ ID NO: 186 is the Cas-alpha2 tracrRNA version 6 RNA sequence from Candidatus Micrarchaeota archaeon.
[0211] SEQ ID NO: 187 is the Cas-alpha2 tracrRNA version 7 RNA sequence from Candidatus Micrarchaeota archaeon.
[0212] SEQ ID NO: 188 is a Cas-alpha6 tracrRNA version 1 RNA sequence from uncultured archaea.
[0213] SEQ ID NO: 189 is a Cas-alpha6 tracrRNA version 2 RNA sequence from uncultured archaea.
[0214] SEQ ID NO: 190 is the Cas-alpha6 tracrRNA version 3 RNA sequence from uncultured archaea.
[0215] SEQ ID NO: 191 is a Cas-alpha6 tracrRNA version 4 RNA sequence from uncultured archaea.
[0216] SEQ ID NO: 192 is the Cas-alpha7 tracrRNA version 1 RNA sequence from Parageobacillus thermoglucosidasius.
[0217] SEQ ID NO: 193 is the Cas-alpha7 tracrRNA version 2 RNA sequence from Parageobacillus thermoglucosidasius.
[0218] SEQ ID NO: 194 is the Cas-alpha8 tracrRNA version 1 RNA sequence from Acidibacillus sulfuroxidans.
[0219] SEQ ID NO: 195 is the Cas-alpha8 tracrRNA version 2 RNA sequence from Acidibacillus sulfuroxidans.
[0220] SEQ ID NO: 196 is the Cas-alpha8 tracrRNA version 3 RNA sequence from Acidibacillus sulfuroxidans.
[0221] SEQ ID NO: 197 is the Cas-alpha9 tracrRNA version 1 RNA sequence from Ruminococcus sp.
[0222] SEQ ID NO: 198 is the Cas-alpha9 tracrRNA version 2 RNA sequence from Ruminococcus sp.
[0223] SEQ ID NO: 199 is the Cas-alpha10 tracrRNA version 1 RNA sequence from Syntrophomonas palmitatica.
[0224] SEQ ID NO:200 is the Cas-alpha10 tracrRNA version 2 RNA sequence from Syntrophomonas palmitatica.
[0225] SEQ ID NO:201 is the Cas-alpha10 tracrRNA version 3 RNA sequence from Syntrophomonas palmitatica.
[0226] SEQ ID NO:202 is the Cas-alpha10 tracrRNA version 4 RNA sequence from Syntrophomonas palmitatica.
[0227] SEQ ID NO:203 is the Cas-alpha10 tracrRNA version 5 RNA sequence from Syntrophomonas palmitatica.
[0228] SEQ ID NO:204 is the Cas-alpha11 tracrRNA version 1 RNA sequence from Clostridium novyi.
[0229] SEQ ID NO:205 is the Cas-alpha11 tracrRNA version 2 RNA sequence from Clostridium novyi.
[0230] SEQ ID NO:206 is the Cas-alpha11 tracrRNA version 3 RNA sequence from Clostridium novyi.
[0231] SEQ ID NO:207 is the Cas-alpha11 tracrRNA version 4 RNA sequence from Clostridium novyi.
[0232] SEQ ID NO:208 is an artifact-derived Cas-alpha2 sgRNA version 5 RNA sequence.
[0233] SEQ ID NO:209 is an artifact-derived Cas-alpha2 sgRNA version 6 RNA sequence.
[0234] SEQ ID NO:210 is an artifact-derived Cas-alpha2 sgRNA version 7 RNA sequence.
[0235] SEQ ID NO:211 is an artifact-derived Cas-alpha6 sgRNA version 1 RNA sequence.
[0236] SEQ ID NO:212 is an artifact-derived Cas-alpha6 sgRNA version 2 RNA sequence.
[0237] SEQ ID NO:213 is an artifact-derived Cas-alpha6 sgRNA version 3 RNA sequence.
[0238] SEQ ID NO:214 is an artifact-derived Cas-alpha6 sgRNA version 4 RNA sequence.
[0239] SEQ ID NO:215 is an artifact-derived Cas-alpha7 sgRNA version 1 RNA sequence.
[0240] SEQ ID NO:216 is an artifact-derived Cas-alpha7 sgRNA version 2 RNA sequence.
[0241] SEQ ID NO:217 is an artifact-derived Cas-alpha7 sgRNA version 3 RNA sequence.
[0242] SEQ ID NO:218 is an artifact-derived Cas-alpha8 sgRNA version 1 RNA sequence.
[0243] SEQ ID NO:219 is an artifact-derived Cas-alpha8 sgRNA version 2 RNA sequence.
[0244] SEQ ID NO:220 is an artifact-derived Cas-alpha8 sgRNA version 3 RNA sequence.
[0245] SEQ ID NO:221 is an artifact-derived Cas-alpha8 sgRNA version 4 RNA sequence.
[0246] SEQ ID NO:222 is an artifact-derived Cas-alpha9 sgRNA version 1 RNA sequence.
[0247] SEQ ID NO:223 is an artifact-derived Cas-alpha9 sgRNA version 2 RNA sequence.
[0248] SEQ ID NO:224 is an artifact-derived Cas-alpha9 sgRNA version 3 RNA sequence.
[0249] SEQ ID NO:225 is an artifact-derived Cas-alpha10 sgRNA version 1 RNA sequence.
[0250] SEQ ID NO:226 is an artifact-derived Cas-alpha10 sgRNA version 2 RNA sequence.
[0251] SEQ ID NO:227 is an artifact-derived Cas-alpha10 sgRNA version 3 RNA sequence.
[0252] SEQ ID NO:228 is an artifact-derived Cas-alpha10 sgRNA version 4 RNA sequence.
[0253] SEQ ID NO:229 is an artifact-derived Cas-alpha10 sgRNA version 5 RNA sequence.
[0254] SEQ ID NO:230 is an artifact-derived Cas-alpha11 sgRNA version 1 RNA sequence.
[0255] SEQ ID NO:231 is an artifact-derived Cas-alpha11 sgRNA version 2 RNA sequence.
[0256] SEQ ID NO:232 is an artifact-derived Cas-alpha11 sgRNA version 3 RNA sequence.
[0257] SEQ ID NO:233 is an artifact-derived Cas-alpha11 sgRNA version 4 RNA sequence.
[0258] SEQ ID NO:234 is an artifact-derived Cas-alpha11 sgRNA version 5 RNA sequence.
[0259] SEQ ID NO:235 is an artifact-derived Cas-alpha4 Zea mays codon-optimized gene DNA sequence.
[0260] SEQ ID NO:236 is an artifact-derived Cas-alpha10 Zea mays codon-optimized gene DNA sequence.
[0261] SEQ ID NO: 237 is an artifact-derived Cas-alpha 10 Saccharomyces cerevisiae (Saccharomyces cerevisiae) codon-optimized gene DNA sequence.
[0262] SEQ ID NO:238 is an artifact-derived Cas-alpha4 sgRNA backbone RNA sequence.
[0263] SEQ ID NO:239 is an artifact-derived Cas-alpha10 sgRNA backbone RNA sequence.
[0264] SEQ ID NO:240 is an artifact-derived Cas-alpha4 Liguleless2 sgRNA target sequence RNA sequence.
[0265] SEQ ID NO:241 is an artifact-derived Cas-alpha4 Liguleless3 sgRNA target sequence RNA sequence.
[0266] SEQ ID NO: 242 is an artifact-derived Cas-alpha 10 nptII sgRNA target sequence RNA sequence.
[0267] SEQ ID NO:243 is an artifact-derived Cas-alpha10 ms26 sgRNA target sequence RNA sequence.
[0268] SEQ ID NO:244 is an artifact-derived Cas-alpha10ade2 sgRNA target sequence RNA sequence.
[0269] SEQ ID NO:245 is an artifact-derived Cas-alpha4 VEGFA2 sgRNA target sequence RNA sequence.
[0270] SEQ ID NO:246 is an artifact-derived Cas-alpha4 VEGFA3 sgRNA target sequence RNA sequence.
[0271] SEQ ID NO:247 is an artifact-derived Cas-alpha4 sgRNA targeting Liguleless2 RNA sequence.
[0272] SEQ ID NO:248 is an artifact-derived Cas-alpha4 sgRNA targeting Liguleless3 RNA sequence.
[0273] SEQ ID NO:249 is an artifact-derived Cas-alpha10 sgRNA targeting nptII RNA sequence.
[0274] SEQ ID NO:250 is an artifact-derived Cas-alpha10 sgRNA targeting ms26 RNA sequence.
[0275] SEQ ID NO:251 is an artifact-derived Cas-alpha10 sgRNA targeting ade2 RNA sequence.
[0276] SEQ ID NO:252 is an artifact-derived Cas-alpha4 sgRNA targeting VEGFA2 RNA sequence.
[0277] SEQ ID NO:253 is an artifact-derived Cas-alpha4 sgRNA targeting VEGFA3 RNA sequence.
[0278] SEQ ID NO:254 is the Cas-alpha12 endonuclease PRT sequence from Clostridioides difficile.
[0279] SEQ ID NO:255 is the Cas-alpha13 endonuclease PRT sequence from Clostridium paraputrificum.
[0280] SEQ ID NO:256 is the Cas-alpha14 endonuclease PRT sequence from Clostridium novyi.
[0281] SEQ ID NO:257 is the Cas-alpha15 endonuclease PRT sequence from Ruminococcus albus.
[0282] SEQ ID NO:258 is the Cas-alpha16 endonuclease PRT sequence from Clostridium hiranonis.
[0283] SEQ ID NO:259 is the Cas-alpha 17 endonuclease PRT sequence from Clostridiumihumii.
[0284] SEQ ID NO:260 is the Cas-alpha18 endonuclease PRT sequence from Cellulosilyticum ruminicola.
[0285] SEQ ID NO:261 is the Cas-alpha19 endonuclease PRT sequence from Eubacterium siraeum.
[0286] SEQ ID NO:262 is the Cas-alpha20 endonuclease PRT sequence from Clostridium botulinum.
[0287] SEQ ID NO:263 is the Cas-alpha21 endonuclease PRT sequence from Clostridium botulinum.
[0288] SEQ ID NO: 264 is the Cas-alpha22 endonuclease PRT sequence from Ruminiclostridium hungatei.
[0289] SEQ ID NO:265 is the Cas-alpha23 endonuclease PRT sequence from Desulfovibrio fructosivorans.
[0290] SEQ ID NO:266 is the Cas-alpha24 endonuclease PRT sequence from Bacillus toyonensis.
[0291] SEQ ID NO:267 is the Cas-alpha25 endonuclease PRT sequence from Clostridium paraputrificum.
[0292] SEQ ID NO:268 is the Cas-alpha26 endonuclease PRT sequence from Clostridium ventriculi.
[0293] SEQ ID NO:269 is the Cas-alpha27 endonuclease PRT sequence from Ruminococcus sp.
[0294] SEQ ID NO:270 is the Cas-alpha28 endonuclease PRT sequence from Ruminococcus sp.
[0295] SEQ ID NO:271 is the Cas-alpha29 endonuclease PRT sequence from Peptoclostridium sp.
[0296] SEQ ID NO:272 is the Cas-alpha30 endonuclease PRT sequence from Bacillus sp.
[0297] SEQ ID NO:273 is the Cas-alpha31 endonuclease PRT sequence from Clostridioides difficile.
[0298] SEQ ID NO:274 is the Cas-alpha32 endonuclease PRT sequence from Clostridioides difficile.
[0299] SEQ ID NO:275 is a Cas-alpha33 endonuclease PRT sequence from uncultured archaea.
[0300] SEQ ID NO:276 is a Cas-alpha34 endonuclease PRT sequence from uncultured archaea.
[0301] SEQ ID NO:277 is a Cas-alpha35 endonuclease PRT sequence from uncultured archaea.
[0302] SEQ ID NO:278 is a Cas-alpha36 endonuclease PRT sequence from uncultured archaea.
[0303] SEQ ID NO:279 is a Cas-alpha37 endonuclease PRT sequence from uncultured archaea.
[0304] SEQ ID NO:280 is a Cas-alpha38 endonuclease PRT sequence from uncultured archaea.
[0305] SEQ ID NO:281 is a Cas-alpha39 endonuclease PRT sequence from uncultured archaea.
[0306] SEQ ID NO: 282 is the Cas-alpha 40 endonuclease PRT sequence from a non-cultured archaea.
[0307] SEQ ID NO:283 is a Cas-alpha41 endonuclease PRT sequence from uncultured archaea.
[0308] SEQ ID NO:284 is the Cas-alpha 42 endonuclease PRT sequence from Clostridioides difficile.
[0309] SEQ ID NO: 285 is the Cas-alpha 43 endonuclease PRT sequence from Desulfovibrio fructosivorans.
[0310] SEQ ID NO:286 is the Cas-alpha 44 endonuclease PRT sequence from Clostridium botulinum.
[0311] SEQ ID NO:287 is the Cas-alpha 45 endonuclease PRT sequence from Clostridioides difficile.
[0312] SEQ ID NO:288 is the Cas-alpha46 endonuclease PRT sequence from Clostridioides difficile.
[0313] SEQ ID NO:289 is the Cas-alpha47 endonuclease PRT sequence from Clostridioides difficile.
[0314] SEQ ID NO:290 is the Cas-alpha 48 endonuclease PRT sequence from Clostridioides difficile.
[0315] SEQ ID NO:291 is the Cas-alpha49 endonuclease PRT sequence from Clostridioides difficile.
[0316] SEQ ID NO:292 is the Cas-alpha50 endonuclease PRT sequence from Clostridioides difficile.
[0317] SEQ ID NO:293 is the Cas-alpha51 endonuclease PRT sequence from Clostridioides difficile.
[0318] SEQ ID NO:294 is the Cas-alpha52 endonuclease PRT sequence from Clostridioides difficile.
[0319] SEQ ID NO:295 is the Cas-alpha53 endonuclease PRT sequence from Clostridioides difficile.
[0320] SEQ ID NO:296 is the Cas-alpha54 endonuclease PRT sequence from Clostridioides difficile.
[0321] SEQ ID NO:297 is the Cas-alpha55 endonuclease PRT sequence from Clostridium hiranonis.
[0322] SEQ ID NO:298 is the Cas-alpha56 endonuclease PRT sequence from Clostridioides difficile.
[0323] SEQ ID NO:299 is the Cas-alpha 57 endonuclease PRT sequence from Aneurinibacillus danicus.
[0324] SEQ ID NO:300 is the Cas-alpha 58 endonuclease PRT sequence from Parageobacillus thermoglucosidasius.
[0325] SEQ ID NO:301 is the Cas-alpha59 endonuclease PRT sequence from Brevibacillus centrosporus.
[0326] SEQ ID NO:302 is the Cas-alpha60 endonuclease PRT sequence from Clostridium pasteurianum.
[0327] SEQ ID NO:303 is the Cas-alpha61 endonuclease PRT sequence from Eubacterium siraeum.
[0328] SEQ ID NO:304 is the Cas-alpha62 endonuclease PRT sequence from Bacillus toyonensis.
[0329] SEQ ID NO:305 is the Cas-alpha63 endonuclease PRT sequence from Ruminococcus sp.
[0330] SEQ ID NO:306 is the Cas-alpha64 endonuclease PRT sequence from Ruminococcus sp.
[0331] SEQ ID NO:307 is the Cas-alpha65 endonuclease PRT sequence from Clostridium perfringens.
[0332] SEQ ID NO:308 is the Cas-alpha66 endonuclease PRT sequence from Bacillus thuringiensis.
[0333] SEQ ID NO:309 is the Cas-alpha67 endonuclease PRT sequence from Clostridium perfringens.
[0334] SEQ ID NO:310 is the Cas-alpha68 endonuclease PRT sequence from Bacillus cereus.
[0335] SEQ ID NO:311 is the Cas-alpha69 endonuclease PRT sequence from Bacillus toyonensis.
[0336] SEQ ID NO:312 is the Cas-alpha70 endonuclease PRT sequence from Bacillus toyonensis.
[0337] SEQ ID NO:313 is the Cas-alpha71 endonuclease PRT sequence from Bacillus toyonensis.
[0338] SEQ ID NO:314 is the Cas-alpha 72 endonuclease PRT sequence from Alicyclobacillus acidoterrestris.
[0339] SEQ ID NO:315 is the Cas-alpha 73 endonuclease PRT sequence from Clostridium tetani.
[0340] SEQ ID NO:316 is the Cas-alpha74 endonuclease PRT sequence from Candidatus Levybacteria bacterium.
[0341] SEQ ID NO:317 is the Cas-alpha 75 endonuclease PRT sequence from Bacillus cereus.
[0342] SEQ ID NO:318 is the Cas-alpha 76 endonuclease PRT sequence from Bacillus cereus.
[0343] SEQ ID NO:319 is the Cas-alpha 77 endonuclease PRT sequence from Bacillus cereus.
[0344] SEQ ID NO:320 is the Cas-alpha78 endonuclease PRT sequence from Clostridium paraputrificum.
[0345] SEQ ID NO:321 is the Cas-alpha 79 endonuclease PRT sequence from Bacillus cereus.
[0346] SEQ ID NO:322 is the Cas-alpha 80 endonuclease PRT sequence from Bacillus thuringiensis.
[0347] SEQ ID NO:323 is the Cas-alpha 81 endonuclease PRT sequence from Bacillus cereus.
[0348] SEQ ID NO:324 is the Cas-alpha 82 endonuclease PRT sequence from Bacillus toyonensis.
[0349] SEQ ID NO:325 is the Cas-alpha 83 endonuclease PRT sequence from Bacillus cereus.
[0350] SEQ ID NO:326 is the Cas-alpha 84 endonuclease PRT sequence from Bacillus toyonensis.
[0351] SEQ ID NO:327 is the Cas-alpha 85 endonuclease PRT sequence from Bacillus wiedmannii.
[0352] SEQ ID NO:328 is the Cas-alpha 86 endonuclease PRT sequence from Bacillus cereus.
[0353] SEQ ID NO:329 is the Cas-alpha 87 endonuclease PRT sequence from Bacillus cereus.
[0354] SEQ ID NO:330 is the Cas-alpha 88 endonuclease PRT sequence from Bacillus toyonensis.
[0355] SEQ ID NO:331 is the Cas-alpha 89 endonuclease PRT sequence from Bacillus cereus.
[0356] SEQ ID NO:332 is the Cas-alpha90 endonuclease PRT sequence from Bacillus toyonensis.
[0357] SEQ ID NO:333 is the Cas-alpha91 endonuclease PRT sequence from Bacillus thuringiensis.
[0358] SEQ ID NO:334 is the Cas-alpha92 endonuclease PRT sequence from Bacillus cereus.
[0359] SEQ ID NO:335 is the Cas-alpha93 endonuclease PRT sequence from Bacillus cereus.
[0360] SEQ ID NO:336 is the Cas-alpha94 endonuclease PRT sequence from Bacillus cereus.
[0361] SEQ ID NO:337 is the Cas-alpha 95 endonuclease PRT sequence from Bacillus thuringiensis.
[0362] SEQ ID NO:338 is the Cas-alpha96 endonuclease PRT sequence from Bacillus sp.
[0363] SEQ ID NO:339 is the Cas-alpha97 endonuclease PRT sequence from Bacillus cereus.
[0364] SEQ ID NO:340 is the Cas-alpha 98 endonuclease PRT sequence from Bacillus cereus.
[0365] SEQ ID NO:341 is the Cas-alpha99 endonuclease PRT sequence from Bacillus thuringiensis.
[0366] SEQ ID NO:342 is the Cas-alpha 100 endonuclease PRT sequence from Bacillus sp.
[0367] SEQ ID NO:343 is the Cas-alpha 101 endonuclease PRT sequence from Prevotella copri.
[0368] SEQ ID NO:344 is the Cas-alpha 102 endonuclease PRT sequence from Prevotella copri.
[0369] SEQ ID NO:345 is the Cas-alpha 103 endonuclease PRT sequence from Clostridioides difficile.
[0370] SEQ ID NO:346 is the Cas-alpha 104 endonuclease PRT sequence from Clostridioides difficile.
[0371] SEQ ID NO:347 is the Cas-alpha 105 endonuclease PRT sequence from Clostridioides difficile.
[0372] SEQ ID NO:348 is the Cas-alpha 106 endonuclease PRT sequence from Clostridioides difficile.
[0373] SEQ ID NO:349 is the Cas-alpha 107 endonuclease PRT sequence from Clostridioides difficile.
[0374] SEQ ID NO:350 is the Cas-alpha 108 endonuclease PRT sequence from Clostridioides difficile.
[0375] SEQ ID NO:351 is the Cas-alpha 109 endonuclease PRT sequence from Clostridioides difficile.
[0376] SEQ ID NO:352 is the Cas-alpha 110 endonuclease PRT sequence from Flavobacterium thermophilum.
[0377] SEQ ID NO:353 is the Cas-alpha 111 endonuclease PRT sequence from Phascolarctobacterium sp.
[0378] SEQ ID NO:354 is the Cas-alpha 112 endonuclease PRT sequence from Bacillus pseudomycoides.
[0379] SEQ ID NO:355 is the Cas-alpha 113 endonuclease PRT sequence from Bacteroides plebeius.
[0380] SEQ ID NO:356 is the Cas-alpha 114 endonuclease PRT sequence from Clostridium botulinum.
[0381] SEQ ID NO:357 is the Cas-alpha 115 endonuclease PRT sequence from Bacillus pseudomycoides.
[0382] SEQ ID NO:358 is the Cas-alpha 116 endonuclease PRT sequence from Bacillus pseudomycoides.
[0383] SEQ ID NO:359 is the Cas-alpha 117 endonuclease PRT sequence from Clostridium botulinum.
[0384] SEQ ID NO:360 is the Cas-alpha 118 endonuclease PRT sequence from Clostridium botulinum.
[0385] SEQ ID NO:361 is the Cas-alpha 119 endonuclease PRT sequence from Clostridium botulinum.
[0386] SEQ ID NO:362 is the Cas-alpha 120 endonuclease PRT sequence from Hydrogenivirga sp.
[0387] SEQ ID NO:363 is the Cas-alpha 121 endonuclease PRT sequence from Bacillus megaterium.
[0388] SEQ ID NO:364 is the Cas-alpha 122 endonuclease PRT sequence from Clostridium fallax.
[0389] SEQ ID NO:365 is the Cas-alpha123 endonuclease PRT sequence from Bacteroides plebeius.
[0390] SEQ ID NO:366 is the Cas-alpha 124 endonuclease PRT sequence from Bacillus thuringiensis.
[0391] SEQ ID NO:367 is the Cas-alpha 125 endonuclease PRT sequence from Bacillus cereus.
[0392] SEQ ID NO:368 is the Cas-alpha126 endonuclease PRT sequence from Clostridium sp.
[0393] SEQ ID NO:369 is the Cas-alpha127 endonuclease PRT sequence from Bacteroides plebeius.
[0394] SEQ ID NO:370 is the Cas-alpha 128 endonuclease PRT sequence from Dorea longicatena.
[0395] SEQ ID NO:371 is the Cas-alpha 129 endonuclease PRT sequence from Sulfurihydrogenibium azorense.
[0396] Novel CRISPR effector systems and elements comprising such systems, such as, but not limited to, novel guide polynucleotide / endonuclease complexes, guide polynucleotides, guide RNA elements, Cas proteins, and endonucleases, and Compositions and methods are provided for proteins containing endonuclease functionality (domains). Also provided are compositions and methods for direct delivery of endonucleases, cleavage-ready complexes, guide RNAs, and guide RNA / Cas endonuclease complexes. The disclosure further includes compositions and methods for targeted sequence modification, gene editing, and insertion of a polynucleotide of interest into the genome of a cell.
[0397] Terms used in the claims and specification are defined as set forth below unless otherwise specified. As used in this specification and the appended claims, the singular forms "a," "an," and "the" indicate otherwise unless the context clearly indicates otherwise. It must be kept in mind that it includes plural referents unless indicated.
[0398] definition As used herein, "nucleic acid" means a polynucleotide and includes single- or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can also include fragments and modified nucleotides. Thus, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" and "nucleic acid fragment" are used to denote polymers of RNA and / or DNA and / or RNA-DNA that are single- or double-stranded. is used interchangeably with and optionally includes synthetic, non-naturally occurring or modified nucleotide bases. Nucleotides (usually found in the 5'-monophosphate form) are designated by their single-letter designations: "A" for adenosine or deoxyadenosine (for RNA or DNA, respectively); "C" for cytosine or deoxycytosine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), pyrimidine (C) or "Y" for T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.
[0399] The term "genome" when applied to prokaryotic and eukaryotic cells, or biological cells, includes not only the chromosomal DNA found within the nucleus, but also the organellar DNA found within the cellular components of the cell (e.g. mitochondria or plastids). contain.
[0400] "Open reading frame" is abbreviated ORF.
[0401] The term "selectively hybridizes" includes specific nucleic acid target sequences compared to hybridization of nucleic acid sequences to non-target nucleic acid sequences and substantial exclusion of non-target nucleic acids under stringent hybridization conditions. Reference is made to a detectably large degree (eg, at least twice background) of hybridization to . Selectively hybridizing sequences usually have at least about 80% or 90% sequence identity with each other, and up to 100% sequence identity (ie, perfect complementarity).
[0402] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a probe will selectively hybridize to its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of hybridization and / or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow sequence mismatches to be tolerated such that lower similarities are detected (non-homologous probing). Generally, probes are less than about 1000 nucleotides in length, optionally less than 500 nucleotides. Generally, stringent conditions are pH 7.0-8.3 and at least about 30° C. for short probes (eg, 10-50 nucleotides) and at least At a temperature of about 60°C, the salt concentration is about 1.5 M Na ion concentration, typically about 0.01 to 1.0 M Na ion concentration. Stringent conditions may also be created with the addition of destabilizing substances such as formamide. Examples of low stringency conditions include hybridization with a buffer consisting of 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and 1-2X at 50-55°C. Washing in SSC (20×SSC=3.0M NaCl / 0.3M sodium citrate) is included. Examples of moderate stringency conditions include hybridization in 40-45% formamide, 1M NaCl, 1% SDS at 37°C and washing in 0.5-1 x SSC at 55-60°C. Examples of high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37°C and washing in 0.1 x SSC at 60-65°C.
[0403] "Homology" means similar DNA sequences. For example, a "region of homology to a genomic region" found on donor DNA refers to a region of DNA that has sequence similarity to a given "genomic region" of a cell or organism's genome. A homology region can be sufficiently long to promote homologous recombination at the target site to be cleaved. For example, the region of homology may be at least 5-10, 5-15, 5-20, 5-25, 5-25, 5-10, 5-15, 5-25 in length, such that the region of homology has sufficient homology to undergo homologous recombination with the corresponding genomic region. 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, 5~1300, 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 in length. "Sufficient homology" indicates that two polynucleotide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction. This structural similarity includes the full length of each polynucleotide fragment as well as the sequence similarity of the polynucleotides. Sequence similarity is measured in percent sequence identity over the entire length of the sequence and / or sequence identity over a portion of the length of the sequence and conserved regions including localized similarity such as contiguous nucleotides with 100% sequence identity. can be described in terms of sex percentage.
[0404] As used herein, a "genomic region" is a chromosomal segment in the genome of a cell that flanks either side of the target site or that also includes a portion of the target site. This genomic region is 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, 5~1300, 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~ It can contain 3100 bases or more so that this genomic region has sufficient homology to undergo homologous recombination with the corresponding homologous region.
[0405] As used herein, "homologous recombination" (HR) involves the exchange of DNA fragments between two DNA molecules at homologous sites. The frequency of homologous recombination is influenced by many factors. The amount of homologous recombination and the relative proportions of homologous and non-homologous recombination vary between different organisms. In general, the length of the region of homology affects the frequency of homologous recombination events, the longer the region of homology, the higher the frequency. The length of homologous regions required to observe homologous recombination also varies by species. In many cases, at least 5 kb of homology is used, but homologous recombination has been observed with 25-50 bp of homology. For example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. 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. USA 83:5199-203; Liskay et al., (1987) Genetics 115:161-7.
[0406] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to those nucleobases or amino acid residues in two sequences that are identical when aligned for maximum correspondence over a specified window of comparison. means the base.
[0407] The term "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, and the portion of the polynucleotide or polypeptide sequence within the comparison window. may contain additions or deletions (ie, gaps) relative to a reference sequence (containing no additions or deletions) in order to optimally align these two sequences. The percentage is determined by determining the number of positions in both sequences where the same nucleobase or amino acid residue occurs, yielding the number of matched positions, and dividing the number of matched positions by the total number of positions in the comparison window. and multiplying the result by 100 to obtain the percentage sequence identity. Useful examples of percent sequence identity are 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage between 50% and 100%. include but are not limited to. These identities can be determined using any of the programs described herein.
[0408] Sequence alignments and percent identity or similarity calculations can be performed using, but not limited to, the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.) to detect homologous sequences. It can be determined using a variety of designed comparison methods. It will be understood that, within the context of this application, when sequence analysis software is used for analysis, analysis results are based on the "default values" of the mentioned programs, unless otherwise stated. As used herein, "default values" means any set of values or parameters that are initially loaded with the software upon first initialization.
[0409] The "Clustal V method of alignment" is denoted Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191). and corresponds to the alignment method found in the MegAlign™ program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc., Madison, Wis.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. The default parameters for pairwise alignments of protein sequences and calculation of percent identity 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 alignment of the sequences using the Clustal V program, "percent identity" can be obtained by looking up the "sequence distance" table in the same program. The "Clustal W method of alignment" is labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci 8:189-191). and corresponds to the alignment method found in the MegAlign™ v6.1 program of the LASERGENE Bioinformatics Computing Suite (DNASTAR Inc., Madison, Wis.). Default parameters for multiple alignments (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, "percent identity" can be obtained by looking up the "sequence distance" table in the same program. Unless otherwise specified, sequence identity / similarity values given herein refer to values obtained using GAP Version 10 (GCG, Accelrys, San Diego, Calif.) with the following parameters: nucleotides; % sequence identity and similarity using a gap generation penalty weight of 50, gap length extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; amino acid sequence % identity and similarity using a gap generation penalty weight 8, using a gap length extension penalty 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 alignments over two sequences that maximize the number of matches and minimize the number of gaps. GAP considers all possible alignments and gap positions and uses gap creation and gap extension penalties in units of matched bases to produce the alignment with the maximum number of matched bases and minimum gaps. "BLAST" is a search algorithm provided by the National Center for Biotechnology Information (NCBI) used to find regions of similarity between biological sequences. This program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches to identify sequences sufficiently similar to the query sequence such that the similarity is not expected to occur randomly. . BLAST reports the identified sequences and their local alignments to the query sequence. Many levels of sequence identity are useful in identifying polypeptides from other species or that have been naturally or synthetically modified, such polypeptides having the same or similar function or activity. One thing will be well understood by those skilled in the art. Useful examples of percent identity include 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or any percentage from 50% to 100%. but not limited to these. Indeed, any amino acid identity between 50% and 100% can be useful in the description of this disclosure, e.g. %, 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% may be useful.
[0410] Polynucleotide and polypeptide sequences, variants thereof and structural relationships of these sequences are referred to herein by the terms "homology," "homologous," "substantially identical," Can be described by "substantially similar" and "substantially correspond". These are polypeptide sequences or nucleic acids in which changes in one or more amino acid or nucleotide bases do not affect the function of the molecule, such as its ability to mediate expression of a gene or its ability to produce a particular phenotype. points to an array. The terms also refer to modifications of nucleic acid sequences in which the functional properties of the resulting nucleic acid are not substantially altered as compared to the intended unmodified nucleic acid. These modifications include deletions, substitutions and / or insertions of one or more nucleotides in the nucleic acid fragment. Encompassed by substantially similar nucleic acid sequences are sequences exemplified herein (under moderate stringency conditions, e.g., 0.5 x SSC, 0.1% SDS, 60°C), or and are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions are adjusted to convert fragments with moderate similarity, such as homologous sequences from distantly related organisms, to fragments with high similarity, such as genes duplicating functional enzymes from closely related organisms. can be screened. Post-hybridization washes determine stringency conditions.
[0411] A "centimorgan" (cM) or "map unit" is the distance between two polynucleotide sequences, binding genes, markers, target sites, loci, or any pair thereof, where 1% is recombinant. A centimorgan is therefore equivalent to a distance equal to the average recombination frequency of 1% between two linked genes, markers, target sites, loci or any pair thereof.
[0412] An "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide or protein, or biologically active portion thereof, refers to the polynucleotide as it is normally found in its naturally occurring environment. or substantially or essentially free of components that co-occur with or interact with proteins. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of other cellular material or culture medium when chemically synthesized. contains substantially no chemical precursors or other chemicals. Optimally, an "isolated" polynucleotide (optimally, a protein-encoding sequence) will be defined in the genomic DNA of the organism from which it is derived, sequences that naturally flank it ( ie, sequences located at the 5' and 3' ends of the polynucleotide). For example, in various embodiments, the isolated polynucleotide contains in the genomic DNA of the cell from which the polynucleotide is derived the nucleotide sequence that naturally flanks the polynucleotide is about 5 kb, 4 kb, It can be less than 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb. Isolated polynucleotides can be purified from the cells in which they naturally occur. Isolated polynucleotides can be obtained using conventional nucleic acid purification methods known to those of skill in the art. The term also includes recombinant and chemically synthesized polynucleotides.
[0413] The term "fragment" refers to a contiguous set of nucleotides or amino acids. In one embodiment, the fragment comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 Contiguous nucleotides. In one embodiment, the fragment comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 Contiguous amino acids. A fragment may or may not exhibit the function of sequences sharing some percent identity over the entire length of said fragment.
[0414] The terms "functionally equivalent fragment" and "functionally equivalent fragment" are used interchangeably herein. These terms refer to isolated nucleic acid fragments or polypeptide portions or subsequences that exhibit the same activity or function as the longer sequence from which they are derived. In one example, the fragment retains the ability to alter gene expression or produce a particular phenotype, whether or not the fragment encodes an active protein. For example, fragments can be used to design genes that give rise to desired phenotypes in modified plants. Genes are designed to be used repressively by binding the nucleic acid fragment, whether or not it encodes an active enzyme, to the plant promoter sequence in sense or antisense orientation. be able to.
[0415] A "gene" includes, but is not limited to, a functional molecule, such as a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. Included are nucleic acid fragments that express "Native gene" refers to a gene as found in its natural endogenous location with its own regulatory sequences.
[0416] The term "endogenous" refers to sequences or other molecules that are naturally occurring in a cell or organism. In one aspect, an endogenous polynucleotide is normally found in the genome of the cell; ie, it is not heterologous.
[0417] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. If all alleles present at a given genetic locus on a chromosome are the same, the plant is homozygous at that locus. If the alleles present at a given genetic locus on a chromosome differ, the plant is heterozygous at that locus.
[0418] "Coding sequence" means a polynucleotide sequence that encodes a specific amino acid sequence. A "regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequences), within a coding sequence, or downstream (3' non-coding sequences) of a coding sequence, which regulates transcription, RNA processing, and associated coding sequences. or affect stability or translation. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5' untranslated sequences, 3' untranslated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites and stem-loop structures. .
[0419] A "mutant gene" is a gene that has been altered by human intervention. Such a "mutant gene" has a sequence that differs from that of the corresponding non-mutant gene by the addition, deletion or substitution of at least one nucleotide. In certain embodiments of the disclosure, the mutated gene comprises modifications resulting from the guide polynucleotide / Cas endonuclease system disclosed herein. A mutant plant is a plant containing a mutant gene.
[0420] As used herein, a "targeted mutation" is a mutation within a target gene using any method known to those of skill in the art, including methods involving the inducible Cas endonuclease system disclosed herein. Mutations in genes (called target genes), including native genes, are made by altering the target sequence.
[0421] The terms "knockout", "genetic knockout" and "genetic knockout" are used interchangeably herein. Knockout refers to a cellular DNA sequence that has been partially or completely disabled by targeting with a Cas protein, e.g. promoter).
[0422] The terms "knock-in", "gene knock-in", "gene insertion" and "genetic knock-in" are used interchangeably herein. Knock-in refers to the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with Cas proteins (eg by homologous recombination (HR), also using a suitable donor DNA polynucleotide). Examples of knock-ins are the specific insertion of heterologous amino acid coding sequences in the coding regions of genes or the specific insertion of transcriptional regulatory elements at genetic loci.
[0423] A "domain" means a continuous stretch of nucleotides (which can be RNA, DNA and / or combined RNA-DNA sequences) or a continuous stretch of amino acids.
[0424] The term "conserved domain" or "motif" refers to a series of amino acids that are conserved at specific positions in aligned sequences of a set of polynucleotides or evolutionarily related proteins. Amino acids at other positions can vary between homologous proteins, while amino acids that are highly conserved at a particular position represent amino acids that are essential for protein structure, stability or activity. They are identified by the high degree of conservation of the aligned sequences of that protein homolog family, and thus are identifiers or "signatures" that determine whether a protein with a newly determined sequence belongs to a previously identified protein family. ” can be used as
[0425] A "codon modified gene" or "codon preference gene" or "codon optimized gene" is a gene with codon usage designed to mimic the preferred codon usage of a host cell.
[0426] An "optimized" polynucleotide is a sequence that has been optimized to improve expression in a particular heterologous host cell.
[0427] A "plant-optimized nucleotide sequence" is a nucleotide sequence optimized for improved expression in plants, in particular for increased expression in plants. Plant-optimized nucleotide sequences include codon-optimized genes. A plant-optimized nucleotide sequence is obtained by modifying a nucleotide sequence that encodes a protein, such as the Cas endonuclease disclosed herein, using one or more plant-preferred codons for improved expression, e.g. Can be synthesized. See, eg, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage.
[0428] A promoter is a region of DNA involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. An "enhancer" is a DNA sequence capable of stimulating promoter activity, and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. good. Promoters may be derived in their entirety from a native gene, be composed of different elements derived from different promoters found in nature, and / or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct gene expression in different tissue or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that some variations of DNA fragments may have identical promoter activity, since in most cases the exact boundaries of regulatory sequences have not been completely defined.
[0429] Promoters that cause the most gene expression in most cell types are commonly referred to as "constitutive promoters." The term "inducible promoter" is used in response to the presence of an endogenous or exogenous stimulus, e.g. by a chemical compound (chemical inducer), or in response to environmental, hormonal, chemical and / or developmental signals. A promoter that selectively expresses a sequence or functional RNA is meant. Inducible or regulated promoters include, for example, light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounds, or ethanol, abscisic acid (ABA), jasmonate, salicylic acid or safeners such as Promoters that are induced or regulated by chemical substances are included.
[0430] A "translation leader sequence" refers to a polynucleotide sequence located between the promoter and coding sequences of a gene. A translation leader sequence is present upstream of the translation initiation sequence of the mRNA. A translation leader sequence may influence the processing of the primary transcript into mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been reported (eg, Turner and Foster, (1995) Mol Biotechnol 3:225-236).
[0431] A "3' non-coding sequence", "transcription terminator" or "termination sequence" refers to a DNA sequence located downstream of a coding sequence that includes a polyadenylation recognition sequence and regulatory signals capable of affecting mRNA processing or gene sequences. contains other sequences that code for Polyadenylation signals are usually characterized by effecting the addition of a polyadenylate system to the 3' end of the pre-mRNA. The use of different 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.
[0432] "RNA transcript" means the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfectly complementary copy of the DNA sequence, it is called the primary transcript or pre-mRA. When the RNA transcript is the RNA sequence resulting from post-transcriptional processing of the primary transcript pre-RNA, it is referred to as mature RNA or mRNA. "Messenger RNA" or "mRNA" refers to the RNA that is without introns and that can be translated into protein by the cell. "cDNA" refers to DNA that is complementary to and synthesized from an mRNA template using reverse transcriptase. The cDNA may be single-stranded or converted to double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to an RNA transcript that includes the mRNA, which can be translated into protein either intracellularly 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 that blocks expression of a target gene (see, e.g., U.S. Pat. No. 5,107,065). want to be). Complementarity of the antisense RNA can be with any portion of a particular gene transcript, i.e., 5' non-coding sequences, 3' non-coding sequences, introns or coding sequences. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated but affects intracellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts and are intended to define a message antisense RNA.
[0433] The term "genome" refers to the entire set of genetic material (genes and non-coding sequences) present in each cell, or virus or organelle of an organism; and / or the set inherited as a (haploid) unit from one parent refers to the chromosome of
[0434] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence if the expression of the coding sequence can be controlled (ie, the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. In another example, the complementary RNA region can be operably linked, directly or indirectly, to the 5' end of the target mRNA, or the 3' end of the target mRNA, or within the target mRNA, or the first The complementary region of is the 5' end of the target mRNA and its complement is the 3' end of the target mRNA.
[0435] Generally, "host" refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced. As used herein, a "host cell" is an in vivo or in vitro eukaryotic, prokaryotic cell (e.g., bacterial or archaeal cell) cultured as a unicellular entity into which a heterologous polynucleotide or polypeptide has been introduced. cells), or cells derived from multicellular organisms (eg, cell lines). In some embodiments, the cells are selected from the group consisting of: archaeal cells, bacterial cells, eukaryotic cells, eukaryotic unicellular organisms, somatic cells, germ cells, stem cells, plant cells, algal cells, animal cells, invertebrate cells, vertebrate cells, fish cells, frog cells, avian cells , insect cells, mammalian cells, pig cells, bovine cells, goat cells, sheep cells, rodent cells, rat cells, mouse cells, non-human primate cells, and human cells. In some examples the cell is in vitro. In some examples, the cell is in vivo.
[0436] The term "recombinant" refers to the artificial combination of two otherwise separate sequence segments, eg, by chemical synthesis or by manipulation of isolated segments of nucleic acids by genetic engineering techniques.
[0437] The terms “plasmid,” “vector,” and “cassette” refer to additional chromosomal elements, linear or circular, often carrying genes that are not part of the central metabolism of the cell, usually in the form of double-stranded DNA. Such elements may be self-replicating sequences, genomic integration sequences, phage or nucleotide sequences from any source, single- or double-stranded DNA or RNA in linear or circular form, Within, many nucleotide sequences are linked or recombined into unique constructs that can introduce a polynucleotide of interest into a cell. A "transformation cassette" refers to a specific vector that contains a gene and has elements in addition to the gene that facilitate transformation of a particular host cell. An "expression cassette" refers to a specific vector that contains a gene and, in addition to the gene, has elements that allow the host to express the gene.
[0438] The terms "recombinant DNA molecule", "recombinant DNA construct", "expression construct", "construct" and "recombinant construct" are used interchangeably herein. A recombinant DNA construct includes an artificial combination of nucleic acid segments, eg, regulatory and coding sequences, that are not necessarily found together in nature. For example, a recombinant DNA construct may contain regulatory sequences and coding sequences derived from different sources, or may contain regulatory sequences and coding sequences derived from the same source but arranged in a different way that is found in nature. . Such constructs may be used alone or may be used with vectors. When a vector is used, the choice of vector depends on the method used to introduce the vector into the host cell, as is well known to those skilled in the art. For example, plasmid vectors can be used. The skilled artisan is familiar with the genetic elements that must be present on the vector in order to successfully transform, select for and propagate the host cell. The skilled technician also knows that different independent transformation events lead to different expression levels and patterns (Jones et al., (1985) EMBO J 4:2411-2418; De Almeida et al., (1989) Mol Gen. Genetics 218:78-86), therefore, usually multiple events are screened to obtain strains exhibiting desired expression levels and patterns. Such screening may include Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting of protein expression, enzymatic or activity analysis, and / or Standard molecular biological, biochemical and other analytical methods, including phenotypic analysis, can be performed.
[0439] The term "heterologous" refers to a difference between the original environment, location, or composition of a particular polynucleotide or polypeptide sequence and its current environment, location, or composition. As non-limiting examples, polynucleotide sequences from taxonomically derived differences (e.g., Zea mays) may be derived from the rice (Oryza sativa) plant genome, or from Zea mays. Heterologous if inserted into a different subspecies or cultivar; (polynucleotide sequences obtained, isolated, modified, and reintroduced into maize plants). As used herein, "heterologous" in relation to a sequence means a sequence originating from a different species, subspecies, foreign species, or, if from the same species, the composition and / or genomic locus. may refer to sequences that have been substantially altered from their natural form by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide may be from a species different from the species from which the polynucleotide was derived, or from the same / similar species if one or both Either the morphology and / or the genomic locus has been substantially altered, or the promoter is not the native promoter of the polynucleotide to which it is operably linked. Alternatively, one or more of the regulatory regions and / or polynucleotides set forth herein may be entirely synthetic. In another example, a target polynucleotide for cleavage by a Cas endonuclease can be from a different organism than the Cas endonuclease. In another example, the Cas endonuclease and guide RNA can be introduced into the target polynucleotide along with additional polynucleotides that act as templates or donors for insertion into the target polynucleotide. Here the additional polynucleotide is heterologous to the target polynucleotide and / or the Cas endonuclease.
[0440] The term "expression," as used herein, refers to the production of precursor or mature forms of functional end products (eg, mRNA, guide RNA or protein).
[0441] A "mature" protein refers to a post-translationally processed polypeptide (ie, from which any pre- or pro-peptides present in the primary translation product have been removed).
[0442] A "precursor" protein refers to the primary product of translation of mRNA (ie, with pre- and pro-peptides still present). Pre- and pro-peptides can be, but are not limited to, subcellular localization signals.
[0443] "CRISPR" (Clustered Regularly Arranged Short Palindromic Repeat) loci are specific loci that encode components of the DNA cleavage system used, for example, 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 be composed of CRISPR arrays containing short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-related) genes.
[0444] As used herein, an "effector" or "effector protein" is a protein that encompasses activities that include recognizing, binding, and / or cleaving or nicking a polynucleotide target. The effector or effector protein can also be an endonuclease. The "effector complex" of the CRISPR system includes the Cas proteins involved in crRNA and target recognition and binding. Some of the component Cas proteins may further comprise domains involved in target polynucleotide cleavage.
[0445] The term "Cas protein" refers to the protein encoded by the Cas (CRISPR-associated) gene. Cas proteins include proteins encoded by genes of the cas locus and also include adaptation and interference molecules. Interfering molecules of the bacterial adaptive immune complex include endonucleases. The Cas endonucleases described herein contain one or more nuclease domains. Cas endonucleases include the novel Cas-alpha protein, Cas9 protein, Cpf1 (Cas12) protein, C2c1 protein, C2c2 protein, C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or combinations or complexes thereof, but not limited to. Cas proteins, when complexed with suitable polynucleotide moieties, are capable of recognizing, binding, and optionally nicking or cleaving all or part of a specific polynucleotide target sequence ("Cas end nuclease” or “Cas effector protein”. Cas-alpha endonucleases of the present disclosure include those with one or more RuvC nuclease domains. The Cas protein is a functional fragment or functional variant of a native Cas protein, or at least 50, 50-100, at least 100, 100-150, at least 150, 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 500, or more than 500 contiguous amino acids and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, at least 65%, 65%-70%, at least 70%, 70%-75%, at least 75%, 75%-80%, at least 80%, 80%-85%, at least 85%, 85%-90%, at least 90%, at least 90%-95%, at least 95%, 95%-96%, at least 96%, 96%-97% , at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100%, or 100% sequence identity, and at least a portion of the native sequence Further defined as a protein that retains activity.
[0446] "Functional fragment", "functionally equivalent fragment" and "functionally equivalent fragment" of Cas endonuclease are used interchangeably herein to recognize and bind target sites and Refers to a portion or subsequence of a Cas endonuclease sequence of the present disclosure that retains the ability to selectively unwind, nick or cleave (introduce single- or double-stranded breaks within the target site). A portion or partial sequence of this Cas endonuclease may be a complete or partial (functional) peptide of any one of its domains, such as, but not limited to, the entire functional portion of the Cas3 HD domain, the Cas3 helicase domain , including but not limited to Cas5, Cas5d, Cas7 and Cas8b1.
[0447] The terms "functional variant", "functionally equivalent variant" and "functionally equivalent variant" of the Cas endonuclease or Cas effector protein comprising Cas-alpha described herein are Used interchangeably herein, Cas effectors disclosed herein that retain the ability to recognize, bind, optionally untie, nick, or cleave all or part of a target sequence Refers to protein variants.
[0448] A Cas endonuclease may also include a multifunctional Cas endonuclease. The terms "multifunctional Cas endonuclease" and "multifunctional Cas endonuclease polypeptide" are used interchangeably herein and refer to a Cas endonuclease function (at least one protein capable of acting as a Cas endonuclease). and at least one other function, such as, but not limited to, the ability to form a complex (including at least a second protein domain capable of forming a complex with another protein). includes reference to a single polypeptide having In one embodiment, the multifunctional Cas endonuclease is directed to domains typical of Cas endonucleases (internal, upstream (5′), downstream (3′), or both internal 5′ and 3′, or both any combination), comprising at least one additional protein domain.
[0449] The terms "cascade" and "cascade complex" are used interchangeably herein and refer to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP). including. Cascades are PNPs that rely on polynucleotides for complex assembly and stability and target nucleic acid sequence specification. The cascade functions as a surveillance complex that finds and optionally binds a target nucleic acid complementary to the variable targeting domain of the guide polynucleotide.
[0450] The terms "cleavage-ready cascade", "cr cascade", "cleavage-ready cascade complex", "cr cascade complex", "cleavage-ready cascade system", "CRC" and "cr cascade system" are used herein Used interchangeably in the literature, includes reference to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP). Here, one of the cascade proteins is a Cas endonuclease capable of recognizing, binding, optionally unwinding, nicking or cleaving all or part of a target sequence.
[0451] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. A 7-methylguanylate residue is located at the 5' end of messenger RNA (mRNA) in eukaryotic cells. RNA polymerase II (Pol II) transcribes mRNA in eukaryotic cells. Messenger RNA capping generally occurs as follows: the most terminal 5' phosphate group of an mRNA transcript is removed by RNA terminal phosphatase, leaving two terminal phosphate groups. Guanosine monophosphate (GMP) is added to the terminal phosphate groups of transcripts by guanylyltransferases, leaving 5'-5' triphosphate-linked guanines at the transcript ends. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyltransferase.
[0452] The terminology "5'-uncapped" is used herein to mean an RNA having, for example, a 5'-hydroxyl group instead of a 5'-cap. Such RNAs can be referred to, for example, as "uncapped RNAs." Since 5'-capped RNA undergoes nuclear export, non-capped RNA can accumulate more fully in the nucleus after transcription. One or more of the RNA components herein are uncapped.
[0453] As used herein, the term "guide polynucleotide" is capable of forming a complex with a Cas endonuclease, e.g., a Cas endonuclease described herein, which recognizes a DNA target site. refers to a polynucleotide sequence that allows for, optionally binding, and optionally cleaving. A guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (a combined RNA-DNA sequence).
[0454] The terms "functional fragment", "functionally equivalent fragment" and "functionally equivalent fragment" of guide RNA, crRNA or tracrRNA are used interchangeably herein and refer to guide RNA, crRNA or tracrRNA, respectively. Refers to a portion or partial sequence of a guide RNA, crRNA or tracrRNA of the present disclosure that retains the ability to function as crRNA or tracrRNA, respectively.
[0455] "Functional variant", "functionally equivalent variant" and "functionally equivalent variant" of guide RNA, crRNA or tracrRNA (respectively) are used interchangeably herein and refer to guide RNA, crRNA or variants of the guide RNA, crRNA, or tracrRNA of the present disclosure that retain the ability to function as tracrRNA, respectively.
[0456] The terms "single guide RNA" and "sgRNA" are used interchangeably herein to describe a variable targeting domain (linked to a tracr mate sequence that hybridizes to the tracrRNA) fused to a tracrRNA (trans-activating CRISPR RNA). It relates to a synthetic fusion of two RNA molecules, containing crRNA (CRISPR RNA). A single guide RNA may comprise a crRNA or crRNA fragment of a type II CRISPR / Cas system capable of forming a complex with a type II Cas endonuclease and a tracrRNA or tracrRNA fragment, said guide RNA / Cas endonuclease complex comprising , can direct a Cas endonuclease to a DNA target site, where the Cas endonuclease recognizes, optionally binds, and optionally nicks or cleaves (single or double stranded) the DNA target site. cutting).
[0457] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and are a nucleotide sequence capable of hybridizing to (complementary to) one strand (nucleotide sequence) of a double-stranded DNA target site. including. The degree of complementarity between the first nucleotide sequence domain (VT domain) and the target sequence is 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 12-30 nucleotides. The variable targeting domain can consist of DNA sequences, RNA sequences, modified DNA sequences, modified RNA sequences, or any combination thereof.
[0458] The terms "Cas endonuclease recognition domain" or "CER domain" (of a guide polynucleotide) are used interchangeably herein and include nucleotide sequences that interact with Cas endonuclease polypeptides. The CER domain contains the (trans-acting) tracr nucleotide mate sequence followed by the tracr nucleotide sequence. A CER domain may be a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., US Patent Application Publication No. 2015 / 0059010A1, published Feb. 26, 2015), or any combination thereof. Any combination may be used.
[0459] As used herein, the terms "guide polynucleotide / Cas endonuclease complex", "guide polynucleotide / Cas endonuclease system", "guide polynucleotide / Cas complex", "guide polynucleotide / Cas system" , “inducible Cas system,” “polynucleotide-guided endonuclease,” and “PGEN” are used interchangeably herein, comprising at least one guide polynucleotide capable of forming a complex and at least one Cas means an endonuclease, wherein said guide polynucleotide / Cas endonuclease complex is capable of directing the Cas endonuclease to a DNA target site, the Cas endonuclease recognizing and binding to the DNA target site; and optionally allow nicking or cleavage (introducing single- or double-strand breaks). The guide polynucleotide / Cas endonuclease complexes herein are based on known CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13) and suitable polynucleotide components.
[0460] 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 mean at least one RNA component capable of forming a complex and at least one Cas endonuclease. wherein said guide RNA / Cas endonuclease complex is capable of guiding a Cas endonuclease to a DNA target site, wherein the Cas endonuclease recognizes and binds to the DNA target site and optionally nicks or Allows cleavage (introduces single-strand or double-strand breaks).
[0461] 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 are capable of being recognized, bound, and optionally nicked or cleaved by the guide polynucleotide / Cas endonuclease complex, but are not limited to , a nucleotide sequence on a chromosome, episome, locus, or any other DNA molecule in the genome of a cell (eg, chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA). The target site may be an endogenous site in the cell's genome, or the target site may be heterologous to the cell and thus not naturally occurring in the cell's genome, or the target site may be naturally occurring in the cell's genome. It can be found in genomic locations that are heterologous to where it occurs. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein and are either endogenous to or occur in the genome of the cell and refers to a target sequence that is present at the endogenous or developmental location of that target sequence in the genome of "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 artificial target sequences are identical in sequence to an endogenous or natural target sequence in the genome of the cell, but are located at different locations (i.e., non-endogenous or non-occurring locations) in the genome of the cell. obtain.
[0462] A "protospacer adjacent motif" (PAM) herein is a short nucleotide sequence that flanks a target sequence (protospacer) that is recognized (targeted) by the guide polynucleotide / Cas endonuclease system described herein. point to Without the PAM sequence following the target DNA sequence, the target DNA sequence cannot be correctly recognized. The PAM sequence and length herein may vary depending on the Cas protein or Cas protein complex used. PAM sequences can be of any length, but are generally 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 , 19 or 20 nucleotides long.
[0463] "Altered target site", "altered target sequence", "modified target site", "modified target sequence" are used interchangeably herein and include at least one alteration as compared to the unaltered target sequence. Refers to the target sequences disclosed herein, including. Such "modifications" 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 modification, or any combination of (v) (i) to (iv).
[0464] "Modified nucleotide" or "edited nucleotide" refers to a nucleotide sequence of interest that contains at least one alteration as compared to its unmodified nucleotide sequence. Such "modifications" 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 modification, or any combination of (v) (i) to (iv).
[0465] The terms "modifying a target site" and "altering a target site" are used interchangeably herein and refer to a method for generating a modified target site.
[0466] As used herein, "donor DNA" is a DNA construct containing a polynucleotide of interest to be inserted into the Cas endonuclease target site.
[0467] The term "polynucleotide modified template" includes polynucleotides that contain at least one nucleotide modification compared to the nucleotide sequence to be edited. A nucleotide modification can be a substitution, addition or deletion of at least one nucleotide. Optionally, the polynucleotide modification template may further comprise a homologous nucleotide sequence flanking the at least one nucleotide modification, the flanking homologous nucleotide sequence providing sufficient homology to the desired nucleotide sequence to be edited. .
[0468] The term "plant-optimized Cas endonuclease" herein refers to Cas proteins, including multifunctional Cas proteins, encoded by nucleotide sequences optimized for expression in plant cells or plants.
[0469] "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 refers to a nucleotide sequence encoding a Cas protein optimized for expression in plant cells or plants, or a variant or functional fragment thereof. A plant comprising a plant-optimized Cas endonuclease includes a plant comprising a nucleotide sequence encoding a Cas sequence and / or a plant 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.
[0470] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds and their progeny. The plants are monocotyledonous or dicotyledonous. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. "Plant element" is intended to refer to a whole plant or plant component, which may include, but is not limited to, differentiated and / or undifferentiated tissues, such as plant tissues, parts, and cell types. . In one embodiment, the plant element is one of: whole plant, seedling, meristematic tissue, ground tissue, vascular tissue, epidermal tissue, seed, leaf, root, shoot, stem, flower, fruit, stolone, bulb. , tubers, corms, keiki, shoots, buds, tumor tissue, and various forms of cells and cultures (eg, single cells, protoplasts, embryos, callus tissue). It should be noted that protoplasts are not technically "intact" plant cells as found in nature (with all their components), as protoplasts lack cell walls. Refers to a plant tissue or a group of tissues that constitute a morphologically and functionally independent part of a plant.As used herein, "plant element" is synonymous with "part" of a plant, and may include separate tissues and / or organs, and may be used interchangeably with the term "tissue" throughout.Similarly, "plant reproductive element" generally refers to the sexual Any part of a plant that can initiate other plants by reproduction or asexual reproduction, including but not limited to seeds, seedlings, roots, shoots, cuttings, scions, grafts, stolons, bulbs, tubers, It is intended to refer to a corm, cake, or bud.Plant elements can be in plants, or plant organs, tissue cultures, or cell cultures.
[0471] "Progeny" includes subsequent generations of the plant.
[0472] As used herein, the term "plant part" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant masses, and embryos, pollen, intact in plants or plant parts. , ovules, seeds, leaves, flowers, branches, fruits, grains, panicles, cobs, pods, stalks, roots, root tips, anthers, etc., and parts thereof themselves. Grain is intended to mean mature seed produced by the grower for purposes other than seed cultivation or propagation. Progeny, variants and mutants of regenerated plants are also included within the scope of the invention, provided that a portion thereof contains the introduced polynucleotide.
[0473] The term "monocotyledonous" or "monocot" refers to the subclass of angiosperms, also known as the class "monocotyledoneae," whose seeds are generally composed of one Contains only early leaves or cotyledons. The term includes references to whole plants, plant elements, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells, and their progeny.
[0474] The term "dicotyledonous" or "dicot" refers to the subclass of angiosperms, also known as the "dicotyledoneae," whose seeds are generally two. contains only early leaves or cotyledons of The term includes references to whole plants, plant elements, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells, and their progeny.
[0475] As used herein, a "male sterile plant" is a plant that does not produce viable or otherwise fertile male gametes. As used herein, a "female sterile plant" is a plant that does not produce viable or otherwise fertile female gametes. It is recognized that male and female sterile plants can be female fertile and male twisted, respectively. In addition, male fertile (other than female twisted) plants can produce viable progeny when crossed with female fertile plants, and female fertile (other than male twisted) plants can produce male It has been found that when crossed with fertile plants they can produce viable progeny.
[0476] As used herein, the term "non-conventional yeast" refers to any yeast that is neither a Saccharomyces yeast species (e.g., S. cerevisiae) nor a Schizosaccharomyces yeast species. . (See "Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols", K. Wolf, K. D. Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003).
[0477] In the context of the present disclosure, the term "crossed" or "cross" or "crossing" refers to the fusion of gametes by pollination to produce progeny (i.e., cells, seeds or plants). means. The term refers to sexual crossing (pollination of one plant by another plant) and selfing (self-pollination, i.e., whether pollen and ovules (endoplasmic reticulum and megaspores) are of the same plant or genetically of the same plant).
[0478] The term "introgression" means the transfer of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a particular locus can be transmitted to at least one progeny plant by sexual crossing between two parental plants, wherein at least one parental A plant carries the desired allele in its genome. Alternatively, for example, allele transfer can be performed, for example, by recombination between two donor genomes in fused protoplasts, where at least one donor protoplast has the desired allele in its genome. ing. The desired allele can be, for example, a transgene, a modified (mutated or edited) natural allele or a selected allele of a marker or QTL.
[0479] The term "isogenic strain" is a comparative term, a reference organism that is genetically identical but treated differently. In one example, two genetically identical maize plant embryos, one undergoing a treatment (such as the introduction of a CRISPR-Cas effector endonuclease), and two different groups of controls, the other without such treatment. can be divided into Thus, phenotypic differences between the two groups may be due to treatment alone and not to any inherent peculiarities of the plant's endogenous genetic makeup.
[0480] "Introducing" means 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 enters into the cells of the organism or into the cells themselves. is intended.
[0481] A "polynucleotide of interest" includes a nucleotide sequence that encodes a protein or polypeptide that improves crop desirability, ie, traits of agronomic benefit. Target polynucleotides include agroeconomically important traits, herbicide resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, virus resistance, fertility or sterility, Polynucleotides encoding traits of interest for crop traits, commercial products, phenotypic markers, or other traits of agricultural or commercial importance include, but are not limited to. Polynucleotides of interest may also be utilized in sense or antisense orientations. Additionally, two or more polynucleotides of interest can be utilized or "stacked" together to provide additional benefits.
[0482] A "complex trait locus" includes a genomic locus having multiple transgenes that are genetically linked together.
[0483] The compositions and methods herein can provide plants with improved "agronomic traits" or "agronomically important traits" or "agronomically beneficial traits", wherein such traits include , which may include, but are not limited to: disease resistance, drought tolerance, heat tolerance, cold tolerance compared to plants of isogenic lines not modified by the methods or compositions described herein , salt tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water efficiency, improved nitrogen utilization, improved nitrogen fixation, pest tolerance, herbivore tolerance, pathogen tolerance, improved yield, enhanced health, improved vitality , improved growth, improved photosynthesis, fortification, altered protein content, altered oil content, increased biomass, increased root length, improved root structure, altered metabolites, altered proteome, increased seed weight increase, modified seed carbohydrate composition, modified seed oil composition, modified seed protein composition, modified seed nutritional composition.
[0484] An "agronomic trait potential" is a plant that exhibits a phenotype, preferably an improved agronomic trait, at some point in its life cycle, or that transmits said phenotype to other relevant plant elements of the same plant. It is intended to mean the capabilities of an element.
[0485] As used herein, the terms "reduced", "less", "slower" and "increased", "faster", "enhanced", "greater" Refers to a reduction or increase in the properties of a modified plant element or resulting plant compared to an unmodified plant element or resulting plant. For example, the reduction in property is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5%-10%, at least 10%, 10%-20%, at least 15%, at least 20%. , 20%-30%, at least 25%, at least 30%, 30%-40%, at least 35%, at least 40%, 40%-50%, at least 45%, at least 50%, 50%-60%, at least about 60%, 60%-70%, 70%-80%, at least 75%, at least about 80%, 80%-90%, at least about 90%, 90%-100%, at least 100%, 100%-200 %, at least 200%, at least about 300%, at least about 400%) or more than an untreated control, and the increase in property is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5%-10%, at least 10%, 10%-20%, at least 15%, at least 20%, 20%-30%, at least 25%, at least 30%, 30%-40%, at least 35 %, at least 40%, 40%-50%, at least 45%, at least 50%, 50%-60%, at least about 60%, 60%-70%, 70%-80%, at least 75%, at least about 80 %, 80%-90%, at least about 90%, 90%-100%, at least 100%, 100%-200%, at least 200%, at least about 300%, at least about 400% or more, untreated control can be higher.
[0486] As used herein, the term "before" refers to the upstream or 5' occurrence of one sequence, with respect to sequence position, of another.
[0487] The abbreviations have the following meanings: "sec" means seconds, "min" means minutes, "h" means hours, "d" means days, "µL" means means microliter, "mL" means milliliter, "L" means liter, "μM" means "micromolar", "mM" means "millimolar", " "M" means "mole", "mmol" means millimoles, "μmole" or "umole" means micromole, "g" means grams, "μg" or "ug" means microgram, "ng" means nanogram, "U" means unit, "bp" means base pair, and "kb" means kilobase.
[0488] Classification of CRISPR-Cas systems CRISPR-Cas systems have been classified according to component sequence and structural analysis. Includes class 1 systems with multi-subunit effector complexes (including types I, III, and IV) and class 2 systems with single protein effectors (including types II, V, and VI). , multiple CRISPR / Cas systems have been described (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015 Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6):e60; and Koonin et al. 2017, Curr Opinion Microbiology 37:67-78).
[0489] A CRISPR-Cas system comprises at least a CRISPR RNA (crRNA) molecule and at least one CRISPR-associated (Cas) protein to form a crRNA ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus comprises an array of identical repeats interspersed with DNA targeting spacers that encode crRNA components and operon-like units of cas genes that encode Cas protein components. The resulting ribonucleoprotein complex recognizes polynucleotides in a sequence-specific manner (Jore et al., Nature Structural & Molecular Biology 18, 529-536 (2011)). crRNAs sequence-specifically bind effectors (proteins or complexes) to double-stranded DNA sequences by base-pairing with complementary DNA strands while displacing the non-complementary strands, forming a so-called R-loop. It functions as a guide RNA that allows (Jore et al., 2011. Nature Structural & Molecular Biology 18, 529-536).
[0490] RNA transcripts (pre-crRNA) of CRISPR loci are cleaved specifically at repeat sequences by CRISPR-associated (Cas) endoribonucleases 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 by species.
[0491] Different cas genes encoding proteins with different domains exist in different CRISPR systems. The cas operon contains genes encoding one or more effector endonucleases and other Cas proteins. Protein subunits include Makarova et al.2011, Nat Rev Microbiol.2011 9(6):467-477; Makarova et al.2015, Nature Reviews Microbiology Vol.13:1-15; and Koonin et al.2017, Current Opinion Microbiology 37:67-78). Types of domains include those involved in expression (pre-crRNA processing, e.g., Cas 6 or RNase III), those involved in buffering (effector modules for crRNA and target binding, and domains for target cleavage). ), those involved in adaptation (spacer insertion, eg Cas1 or Cas2), and those involved in assistance (regulatory or helper or unknown function). Some domains can serve more than one purpose, among which Cas9, for example, contains a domain for endonuclease function and a domain for targeted cleavage.
[0492] Cas endonucleases are induced by single CRISPR RNAs (crRNAs) through direct RNA-DNA base pairing to recognize DNA target sites adjacent to protospacer adjacent motifs (PAMs) (Jore, M.M. et al. al., 2011, Nat. Struct. Mol. Biol. 18:529-536, Westra, E.R. et al., 2012, Molecular Cell 46:595-605, and Sinkunas, T. et al., 2013, EMBO J. 32:385-394).
[0493] Class I CRISPR-Cas system Class I CRISPR-Cas systems include types I, III, and IV. A unique feature of Class I systems is the presence of effector endonuclease complexes instead of single proteins. The cascade complex contains an RNA recognition motif (RRM) and a nucleic acid-binding domain that is the corefold of the diverse RAMP (repeat-related mysterious protein) protein superfamily (Makarova et al. 2013, Biochem Soc Trans 41, 1392- 1400; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). RAMP protein subunits contain Cas5 and Cas7 (which comprise the backbone of the crRNA-effector complex, where the Cas5 subunit binds to the 5' handle of crRNA and interacts with the large subunit) and are often , contains Cas6, which is loosely associated with effector complexes and generally functions as a repeat-specific RNase in pre-crRNA processing (Charpentier et al., FEMS Microbiol Rev 2015,39:428-441; Niewoehner et al., RNA 2016, 22:318-329).
[0494] The type I CRISPR-Cas system contains a complex of effector proteins called the cascade (CRISPR-associated complexes for antiviral defense), including at least Cas5 and Cas7. Effector complexes work together with a single CRISPR RNA (crRNA) and Cas3 to defend against invading viral DNA (Brouns, S.J.J. et al. Science 321:960-964; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). The type I CRISPR-Cas locus contains a metal-dependent nuclease with a single-stranded DNA (ssDNA)-stimulated superfamily 2 helicase that has demonstrated the ability to unwind double-stranded DNA (dsDNA) and RNA-DNA duplexes. contains the signature gene cas3 (or variant cas3′ or cas3″) encoding (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). recruits into DNA complexes to cleave and degrade DNA targets (Westra, E.R. 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 may be the active endonuclease involved in crRNA processing, while Cas5 and Cas7 are non-active endonucleases. Although functioning as catalytic RNA-binding proteins, in type I-C systems crRNA processing can be catalyzed by Cas5 (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). (Makarova et al. 2011, Nat Rev Microbiol. 2011 9(6):467-477; Koonin et al. 2017, Curr Opinion Microbiology 37:67-78), at least the protein subunits Cas7 and Cas5. and a modified type I CRISPR-related complex (cascade) for adaptive antiviral defense comprising Cas6, one of these subunits synthetically fused to the Cas3 endonuclease or the modified restriction endonuclease FokI (International Publication No. 2013 / 098244 pamphlet published on July 4, 2013).
[0495] The type III CRISPR-Cas system, which contains multiple cas7 genes, targets ssRNA or ssDNA and functions as either an RNase and a target RNA-activating DNA nuclease (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) DNases that couple target RNA binding / cleavage with ssDNA degradation. Upon infection with foreign DNA, CRISPR RNA (crRNA)-induced binding of the Csm or Cmr complex to new transcripts recruits Cas10 DNase to the actively transcribed phage DNA, resulting in transcription of the transcript. and phage DNA are both degraded, but host DNA is not. The Cas10 HD domain is responsible for ssDNase activity and the Csm3 / Cmr4 subunits are responsible for the endoribonuclease activity of the Csm / Cmr complex. The 3' flanking sequence of target RNA is critical for the ssDNase activity of Csm / Cmr: base pairing with the 5' handle of crRNA protects host DNA from degradation.
[0496] Type IV systems contain typical type I cas5 and cas7 domains in addition to cas8-like domains, but may lack the CRISPR arrays characteristic of most other CRISPR-Cas systems.
[0497] Class II CRISPR-Cas system Class II CRISPR-Cas systems include types II, V, and VI. A unique feature of class II systems is the presence of a single Cas effector protein instead of effector complexes. Type II and V Cas proteins contain a RuvC endonuclease domain that adopts the RNase H-fold.
[0498] Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to direct the Cas endonuclease to a DNA target. crRNA base-pairs with a spacer region complementary to one strand of a double-stranded DNA target and tracrRNA (trans-activating CRISPR RNA) to form an RNA duplex that allows the Cas endonuclease to cleave the DNA target and regions that leave blunt ends. Spacers are obtained by a poorly understood process involving the Cas1 and Cas2 proteins. Type II CRISPR / Cas loci generally contain the cas1 and cas2 genes in addition to the cas9 gene (Chylinski et al., 2013, RNA Biology 10:726-737; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type II CRISR-Cas loci can encode tracrRNAs that are partially complementary to repeats in each CRISPR array and can include other proteins such as Csn1 and Csn2. The presence of cas9 in the vicinity of cas1 and cas2 genes is characteristic of type II loci (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).
[0499] The type V CRISPR / Cas system contains a single Cas endonuclease, including Cpf1 (Cas12) (Koonin et al., Curr Opinion Microbiology 37:67-78, 2017), which, unlike Cas9, is capable of targeted cleavage. It is an active RNA-guided endonuclease that does not necessarily require additional transactivating CRISPR (tracr) RNA for its activation.
[0500] The type VI CRISPR-Cas system has two HEPN (higher eukaryotic and prokaryotic nucleotide-binding) domains, but no HNH domain, nor a RuvC domain, and contains a cas13 gene that is independent of tracrRNA activity. Most of the HEPN domains contain conserved motifs that constitute metal-independent endo-RNase active sites (Anantharam et al., Biol Direct 8:15, 2013). Because of this feature, the Type VI system is thought to act on RNA targets instead of DNA targets common to other CRISPR-Cas systems.
[0501] Novel CRISPR-Cas system A novel CRISPR-Cas system, its components, and methods of using said components are disclosed herein. This system contains a novel Cas effector protein, Cas-alpha.
[0502] The novel CRISPR-Cas system components described herein are derived from or modified from one or more subunits from different Cas systems, two or more different bacterial or archaeal prokaryotes It may contain subunits and / or synthetic or engineered components.
[0503] Described herein are newly identified CRISPR-Cas systems that include novel sequences of cas genes. Further novel cas genes and proteins are described.
[0504] One of the several features of the novel Cas-alpha system is the locus structure as shown in Figures 1A-1D. In some embodiments, the Cas-alpha genomic locus comprises the cas1 gene, the cas2 gene, the cas4 gene, and the cas-alpha gene encoding the effector protein Cas-alpha. A CRISPR array containing repeats of nucleotide sequences can be found before or after the gene encoding the Cas-alpha endonuclease. In some embodiments, the cas-alpha locus comprises a cas-alpha gene encoding an effector protein and a CRISPR array comprising repeats, but any one or more of the cas1, cas2, and / or cas4 genes. may not contain
[0505] CRISPR-Cas system components Cas-protein A large number of proteins are involved in the 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, and others. can be coded to
[0506] Two proteins, Cas1 and Cas2, are conserved among many CRISPR systems (described, for example, in Koonin et al., Curr Opinion Microbiology 37:67-78, 2017). Cas1 is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments. In some systems, Cas1 forms a stable complex with Cas2, which is essential for spacer acquisition and insertion for the CRISPR system (Nunez et al., Nature Str Mol Biol 21:528-534, 2014 ).
[0507] Many other proteins, including Cas4, which may have similarities to the RecB nuclease, have been identified in various systems and are thought to play a role in capturing new viral DNA sequences for incorporation into CRISPR arrays. (Zhang et al., PLOS One 7(10):e47232, 2012).
[0508] Some proteins may encompass multiple functions. For example, the class 2 type II system signature protein Cas9 has been demonstrated to be involved in pre-crRNA processing, target binding, and target cleavage.
[0509] The novel Cas-alpha proteins disclosed herein include effector proteins (endonucleases) and adaptation proteins. Cas endonucleases have been identified from several bacterial and archaeal sources, including those shown in Figures 7A-7K.
[0510] Cas endonuclease and effector Endonucleases are enzymes that cleave the phosphodiester bonds of polynucleotide chains and include restriction endonucleases that cleave DNA at specific sites without damaging the bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-related) effector endonucleases.
[0511] The Cas endonuclease, either as a single effector protein or in an effector complex with other components, unwinds the DNA duplex at the target sequence and the polynucleotide ( Optionally, at least one DNA strand is cleaved, mediated by recognition of the target sequence by, but not limited to, crRNA or guide RNA. Such recognition and cutting of a target sequence by a Cas endonuclease generally depends on whether the correct protospacer adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. occurs. Alternatively, the Cas endonucleases herein may lack DNA cleaving or nicking activity when complexed with suitable RNA components, but still be able to specifically bind to DNA target sequences. (See also US Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and US Patent Application No. 2015 / 0059010, published February 26, 2015).
[0512] Cas endonucleases can occur as individual effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).
[0513] Cas endonucleases that have been described include, for example, Cas3 (characteristic of class 1 type I systems), Cas9 (characteristic of class 2 type II systems) and Cas12 (Cpf1) (characteristic of class 2 type V systems). but not limited to these.
[0514] Cas3 (and its variants Cas3' and Cas3'') functions as a single-stranded DNA nuclease (HD domain) and an ATP-dependent helicase. Functional activity can be obtained by abolishing ATPase-dependent helicase activity (either by deletion, knockout of the Cas3-helicase domain, or by mutagenesis of key residues, or by mutagenesis of key residues, as previously described). (Sinkunas, T, et al., 2013, EMBO J.32:385-394) by assembling the reaction in the absence of ATP as in Cleavage readiness cascade involving the modified Cas3 endonuclease. can be converted to a nickase (because the HD domain is still functional).Abolition of HD endonuclease activity includes, but is not limited to, mutagenesis of key residues in the HD domain. A cleavage-ready cascade comprising a modified Cas3 endonuclease can be converted to a helicase, which can be accomplished by known methods, and abrogation of both Cas helicase and Cas3 HD endonuclease activity, including, but not limited to: , which can be achieved by methods known in the art, such as mutagenesis of key residues in both the helicase and HD domains, to bind the cleavage-ready cascade containing the modified Cas3 endonuclease to the target sequence. It can be converted into a binding protein.
[0515] "Cas9" (previously called Cas5, Csn1, or Csx12) forms a complex with cr and tracr nucleotides or with a single guide polynucleotide to specifically target all or part of a DNA target sequence. Cas endonuclease that recognizes and cleaves Cas9 recognizes 3'GC-rich PAM sequences in target dsDNA. The Cas9 protein contains a RuvC nuclease with HNH (H-N-H) nucleases flanking the RuvC-II domain. The RuvC nuclease and the HNH nuclease can each cleave a single DNA strand at the target sequence (coordinated activity of both domains results in DNA double-strand breaks, whereas activity of one domain results in nicking). ). In general, the RuvC domain comprises subdomains I, II, and III, with domain I located near the N-terminus of Cas9 and subdomains II and III located in the middle of the protein and flanking the HNH domain. (Hsu et al, Cell 157:1262-1278). The Cas9 endonuclease is typically derived from the type II CRISPR system, which includes a DNA cleavage system that utilizes the Cas9 endonuclease complexed with at least one polynucleotide component. For example, Cas9 can be complexed with CRISPR RNA (crRNA) and transactivating CRISPR RNA (tracrRNA). In another example, Cas9 can be complexed with a single guide RNA (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).
[0516] Cas12 (previously called Cpf1, and variants c2c1, c2c3, CasX, and CasY) contained the RuvC nuclease domain and generated a 5' overhang on the dsDNA target. Some variants do not require tracrRNA, unlike Cas9 functionality. Cas12 and its variants recognize 5'AT-rich PAM sequences on target dsDNA. An insertion domain called Nuc of the Cas12a protein has been demonstrated to be involved in targeted strand cleavage (Yamano et al., Cell 2016,165:949-962). Further mutation studies in other Cas12 proteins demonstrated that the Nuc domain contributes to induction and target binding, together with the RuvC domain responsible for cleavage (Swarts et al., Mol Cell 2017, 66:221-233 e224).
[0517] Cas endonucleases and effector proteins can be used for targeted genome editing (by single and multiple double-strand breaks and nicks) and targeted genome regulation (by attaching epigenetic effector domains to Cas proteins or sgRNAs). can be done. Cas endonucleases can also be engineered to function as RNA-guided recombinases and via RNA tethers can serve as scaffolds for the assembly of multiple protein and nucleic acid complexes (Mali et al., 2013, Nature Methods Vol. 10: 957-963).
[0518] Cas-alpha endonuclease Cas-alpha endonucleases are defined as functional RNA-guided PAM-dependent dsDNA-cleaving proteins of less than 800 amino acids, divided into three subdomains and further containing a bridging helix and one or more zinc finger motifs at the C-terminus. a RuvC catalytic domain; 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.
[0519] The Cas-alpha endonuclease comprises, when aligned to SEQ ID NO: 17, relative to amino acid position numbers of SEQ ID NO: 17, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or 7:337 Glycine (G) at position 341, Glycine (G) at position 430, 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 Cysteine (C), and / or cysteine (C) or histidine (H) at position 512. The Cas-alpha endonuclease is The following motifs: GxxxG, ExL, Cx n C, Cx n (C or H) (where n=1 or more amino acids).
[0520] The RuvC domain has been demonstrated in the literature to contain endonuclease functionality. Cas-alpha endonucleases can be isolated or identified from genetic loci comprising arrays containing cas-alpha genes and multiple repeats that encode effector proteins. In some embodiments, the cas-alpha locus can further comprise part or all of the cas1 gene, the cas2 gene, and / or the cas4 gene.
[0521] A zinc finger motif is a domain that coordinates one or more zinc ions, usually through cysteine and histidine side chains, to stabilize their fold. Zinc fingers are named for the pattern of cysteine and histidine residues coordinating the zinc ion (e.g., C4 means the zinc ion is coordinated to four cysteine residues, C3H is the zinc meaning that the ion is coordinated to three cysteine residues and one histidine residue).
[0522] Cas-alpha proteins contain one or more zinc finger (ZFN) coordination motifs that can form a zinc binding domain. Zinc finger-like motifs can aid in the separation of target and non-target strands and loading of guide RNA into DNA targets. A Cas-alpha protein containing one or more zinc finger motifs can provide additional stability to the ribonucleoprotein complex on the target polynucleotide. Cas-alpha proteins contain a C4 or C3H zinc binding domain.
[0523] Several Cas-alpha proteins and polynucleotides are shown in Figures 7A-7K, and key structural motifs of the endonuclease proteins are shown in Figures 8A-8K, respectively.
[0524] Cas-alpha endonuclease is an RNA-guided endonuclease that can bind and cleave double-stranded DNA targets that contain (1) a sequence with homology to the nucleotide sequence of the guide RNA and (2) a PAM sequence. be. In some embodiments, the PAM is T-rich. In some embodiments, the PAM is C-rich.
[0525] A Cas-alpha endonuclease functions as a double-strand break inducer and can also be a nickase, or a single-strand break inducer. In some embodiments, a catalytically inactive Cas-alpha endonuclease can be used to target or direct to a target DNA sequence, but does not induce cleavage. In some embodiments, a catalytically inactive Cas-alpha protein can be used with a functional endonuclease that cleaves the target sequence. In some embodiments, a catalytically inactive Cas-alpha protein can be combined with a base-editing molecule such as a deaminase. In some aspects, the deaminase can be cytidine deaminase. In some aspects, the deaminase can be an adenine deaminase. In some embodiments, the deaminase can be ADAR-2.
[0526] The Cas-alpha endonuclease further includes SEQ ID NOs: 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, at least 50, 50-100, at least 100 of any of 363, 364, 365, 366, 367, 368, 369, 370 and 371, or a functional fragment or functional variant thereof retaining at least a portion of the activity , 100-150, at least 150, 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 500, or more than 500 contiguous amino acids and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, at least 65%, 65%-70%, at least 70%, 70%-75%, at least 75%, 75%-80%, at least 80%, 80%-85%, at least 85%, 85%-90%, at least 90%, at least 90%-95%, at least 95%, 95%-96%, at least 96%, 96%-97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100%, or RNA-induced with 100% sequence identity Defined as derived double-stranded DNA-cleaving protein. A "functional fragment" of a Cas-alpha endonuclease is the ability to recognize or bind or nick a single strand of a double-stranded polynucleotide, or the ability to cleave both strands of a double-stranded polynucleotide; Or holds any combination of them.
[0527] The Cas-alpha endonuclease is at least 50, 50-100, at least 100, 100-150, at least 150 of any of SEQ ID NO: 13, 14, 15, 16, 25, 26, 27, 28, 29, 30 or 31 , 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 500, 500-550, at least 600, 600-650, at least 650, 650-700, at least 700, 700-750, at least 750, 750-800, at least 800, 800-850, at least 850, 850-900, at least 900, 900-950, at least 950, 950 ~1000, at least 1000, or more than 1000 contiguous nucleotides; 65%~70%, at least 70%, 70%~75%, at least 75%, 75%~80%, at least 80%, 80%~85%, at least 85%, 85%~90%, at least 90%, 90%-95%, at least 95%, 95%-96%, at least 96%, 96%-97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, A polynucleotide having 99% to 100% or 100% sequence identity, or 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, 33 1, 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 may be encoded by a polynucleotide encoding any one.
[0528] Cas endonucleases, effector proteins, or functional fragments thereof for use in the disclosed methods may be from natural sources or modified such that a genetically modified host cell expresses a nucleic acid sequence encoding the protein. It can be isolated from any recombinant source. Alternatively, the Cas protein can be produced using cell-free protein expression systems or produced synthetically. Effector Cas nucleases may be isolated and introduced into heterologous cells, or may be modified from their native form to exhibit a different type or magnitude of activity than their natural source. Such modifications include, but are not limited to fragments, variants, substitutions, deletions, and insertions.
[0529] Fragments and variants of Cas endonuclease and Cas effector proteins can be obtained by methods such as site-directed mutagenesis and synthetic construction. Methods of measuring endonuclease activity are well known in the art, e.g. WO 2016 / 186953 and WO 2016 / 186946 published November 24, 2016, but are not limited thereto.
[0530] Cas endonucleases can include modified forms of Cas polypeptides. Modified forms of Cas polypeptides can include amino acid changes (eg, deletions, insertions or substitutions) that reduce the naturally occurring nuclease activity of the Cas protein. For example, in some instances, a modified form of a Cas protein is less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the corresponding wild-type Cas polypeptide. (U.S. Patent Application Publication No. 2014 / 0068797, published March 6, 2014). In some instances, modified forms of Cas polypeptides have substantially no nuclease activity and are referred to as catalytically "inactivated Cas" or "inactivated cas(dCas)." Inactivated Cas / Deactivated Cas contains deactivated Cas endonuclease (dCas). Catalytically inactive Cas effector proteins can be fused to heterologous sequences to induce or modify activity.
[0531] The Cas endonuclease may be part of a fusion protein comprising one or more heterologous protein domains (eg, one, two, three or more domains in addition to the Cas protein). Such fusion proteins may comprise any additional protein sequences and optionally a linker sequence between any two domains, eg Cas and the first heterologous domain. Examples of protein domains that can be fused to the Cas proteins herein include 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], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP ], HcRed, DsRed, Cyan Fluorescent Protein [CFP], Yellow Fluorescent Protein [YFP], Blue Fluorescent Protein [BFP]), and domains with one or more of the following activities: methyl demethylase activity, demethylase activity, transcription activation activity (eg VP16 or VP64), transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Cas proteins also bind 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. It can be fused with proteins.
[0532] Catalytically active and / or inactive Cas endonucleases can be fused to heterologous sequences (US 2014 / 0068797, published Mar. 6, 2014). Suitable fusion partners include, but are not limited to, indirectly increasing transcription by acting directly on the target DNA or polypeptides associated with the target DNA (e.g., histones or other DNA binding proteins). Included are polypeptides that provide activity. Further suitable fusion partners include, but are not limited to, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating Polypeptides that provide activity, SUMOylation activity, de-SUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity or demyristoylation activity are included. Further suitable fusion partners include, but are not limited to, polypeptides that directly cause increased transcription of the target nucleic acid (e.g., transcriptional activators or fragments thereof, transcriptional activators, small molecule / drug-responsive transcriptional regulators, etc.). (proteins or fragments thereof) that induce A partially active or catalytically inactive Cas-alpha endonuclease can also be fused to another protein or domain, such as the Clo51 nuclease or the FokI nuclease, to generate a double-strand break ( Guilinger et al. Nature biotechnology, volume 32, number 6, June 2014).
[0533] Catalytically active or inactive Cas proteins, such as the Cas-alpha protein described herein, also include molecules that direct editing of single or multiple bases in a polynucleotide sequence, e.g. can be fused with a site-specific deaminase that can change, for example, C G to T A or 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.”Nature 533(7603)(2016):420-4. (nickases) or inactivated (catalytically inactive) Cas-alpha endonucleases and deaminases (e.g., but not limited to, cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.) Base editing repair inhibitors and glycosylase inhibitors (eg, uracil glycosylase inhibitors (which prevent removal of uracil)) are contemplated as other components of the base editing system in some embodiments.
[0534] Cas endonucleases described herein can be obtained by methods known in the art, e.g. It can be expressed and purified.
[0535] So far, it recognizes a specific PAM sequence (International Publication No. 2016 / 186953 published on November 24, 2016, International Publication No. 2016 / 186946 published on November 24, 2016, and Zetsche B et al.2015. Cell 163, 1013), many Cas endonucleases have been reported that can cleave target DNA at specific positions. Of course, one of ordinary skill in the art can base these methods on the methods and embodiments described herein using the novel inducible Cas system, as these methods can use any inducible endonuclease system. It is understood that adaptations are possible.
[0536] A Cas effector protein may contain a heterologous nuclear localization sequence (NLS). The heterologous NLS amino acid sequences herein can have sufficient strength, for example, to drive the accumulation of detectable amounts of Cas protein in the nucleus of the yeast cells herein. NLSs are short sequences (e.g., 2-20 residues) of one (single segment) or multiple (e.g., two segmented segments) of basic, positively charged residues (e.g., lysine and / or arginine). It may be included and may be positioned anywhere in the Cas amino acid sequence so long as it is exposed on the protein surface. The NLS may, for example, be operably linked to the N-terminus or C-terminus of the Cas proteins herein. For example, two or more NLS sequences can be linked to a Cas protein, eg, to the N-terminus and C-terminus of the Cas protein. The Cas endonuclease gene has an SV40 nuclear targeting signal upstream of the Cas codon region and a bisecting VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region. can be operatively connected to the Non-limiting examples of suitable NLS sequences herein include those disclosed in US Pat. Nos. 6,660,830 and 7,309,576.
[0537] guide polynucleotide Guide polynucleotides permit target recognition, target binding, and optionally target cleavage by the Cas endonuclease, and can be single or double molecules. A guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (a combined RNA-DNA sequence). Optionally, the guide polynucleotide has at least one nucleotide, phosphodiester bond or linkage modification such as, but not limited to, locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2 '-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, phosphorothioate linkage, linkage to cholesterol molecule, linkage to polyethylene glycol molecule, linkage to spacer 18 (hexaethylene glycol chain) molecule, or ring can include a 5' to 3' covalent linkage that results in a ligation. Guide polynucleotides that contain only ribonucleic acid are also referred to as "guide RNA" or "gRNA" (U.S. Patent Application Publication Nos. 2015 / 0082478, published March 19, 2015 and published February 26, 2015). Published U.S. Patent Application Publication No. 2015 / 0059010). Guide polynucleotides may be genetically engineered or synthetically produced.
[0538] Guide polynucleotides include chimeric non-naturally occurring guide RNAs that contain regions that are not found together in nature (ie, they are heterologous to each other). For example, a first nucleotide sequence domain (called a variable targeting domain or VT domain) capable of hybridizing to a nucleotide sequence in target DNA linked to a second nucleotide sequence capable of recognizing the Cas endonuclease A chimeric non-natural guide RNA comprising a first nucleotide sequence and a second nucleotide sequence that are not found joined together in nature.
[0539] A guide polynucleotide can be a double molecule (also called a double-stranded guide polynucleotide) comprising cr nucleotide sequences (such as crRNA) and tracr nucleotide sequences (such as tracrRNA). Optionally, there is a linker polynucleotide that joins crRNA and tracrRNA to form a single guide, eg, sgRNA.
[0540] The cr nucleotides are a first nucleotide sequence domain (called the variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in the target DNA, and a second that is part of the Cas endonuclease recognition (CER) domain. (also called the tracr mate sequence). The tracr mate sequences can hybridize to tracr nucleotides along regions of complementarity and together form a Cas endonuclease recognition domain, or CER domain. CER domains are capable of interacting with Cas endonuclease polypeptides. The cr and tracr nucleotides of the double-stranded guide polynucleotide can be RNA, DNA, and / or combined RNA-DNA sequences. In some embodiments, the cr nucleotide molecule of the double-stranded guide polynucleotide is "crDNA" (when composed of a continuous stretch of DNA nucleotides) or "crNNA" (when composed of a continuous stretch of RNA nucleotides). ), or “crDNA-RNA” (when composed of a combination of DNA and RNA nucleotides). The cr nucleotides can include fragments of crRNAs naturally occurring in Bacteria and Archaea. The sizes of naturally occurring crRNA fragments in Bacteria and Archaea that may be present in the cr nucleotides disclosed herein are 2, 3, 4, 5, 6 1, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides can be, but are not limited to. In some embodiments, the crRNA molecule is selected from the group consisting of SEQ ID NOs:57, 58 and 59.
[0541] In some embodiments, the tracr nucleotide sequence is "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 that induces the RNA / Cas9 endonuclease complex is double stranded RNA comprising double stranded crRNA-tracrRNA. The tracrRNA (trans-activating CRISPR RNA) contains, in the 5′ to 3′ direction, (i) a sequence that anneals to the repeat region of CRISPR type II crRNA, and (ii) a stem-loop containing portion (Deltcheva et al., Nature 471:602-607). A double-stranded guide polynucleotide can form a complex with a Cas endonuclease, and said guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) binds the Cas endonuclease to the genome. can be directed to a target site and the Cas endonuclease recognizes the target site, binds to the target site, and optionally nicks or cleaves (introduces a single- or double-strand break) the target site enable the (U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015 and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015).
[0542] In some embodiments, the tracrRNA molecule is selected from the group consisting of SEQ ID NOs:60-68.
[0543] In one aspect, the guide polynucleotide is a guide polynucleotide capable of forming a PGEN as described herein, said guide polynucleotide comprising a first nucleotide sequence domain complementary to the nucleotide sequence of the target DNA. and a second nucleotide sequence domain that interacts with said Cas endonuclease polypeptide.
[0544] 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 in the group consisting of DNA sequences, RNA sequences, and combinations thereof A guide polynucleotide selected from
[0545] In one aspect, the guide polynucleotide is a guide polynucleotide as described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain comprise a stability-enhancing RNA backbone modification, a stability-enhancing A guide polynucleotide selected from the group consisting of DNA backbone modifications and combinations thereof (Kanasty et al., 2013, Common RNA-backbone modifications, Nature Materials 12:976-977; published March 19, 2015). See U.S. Patent Application Publication No. 2015 / 0082478, published February 26, 2015, U.S. Patent Application Publication No. 2015 / 0059010).
[0546] The guide RNA comprises a duplex molecule comprising a chimeric non-natural crRNA linked to at least one tracrRNA. Chimeric non-natural crRNAs include crRNAs that contain regions that are not found together in nature (ie, they are heterologous to each other). For example, it contains a first nucleotide sequence domain (called the variable targeting domain or VT domain) capable of hybridizing to a nucleotide sequence in the target DNA linked to a second nucleotide sequence (also called the tracrmate sequence). In crRNA, the first and second sequences are not found naturally joined together.
[0547] A guide polynucleotide can also be a single molecule (also called a single guide polynucleotide) comprising a cr nucleotide sequence linked to a tracr nucleotide sequence. A single guide polynucleotide contains a first nucleotide sequence domain (called the variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in the target DNA, and a Cas endonuclease recognition domain that interacts with the Cas endonuclease polypeptide. Includes domains (CER domains). In some embodiments, the sgRNA molecule is selected from the group consisting of SEQ ID NOs:69-77.
[0548] The VT domain and / or CER domain of the single guide polynucleotide can comprise RNA sequences, DNA sequences or combined RNA-DNA sequences. A single guide polynucleotide composed of sequences derived from cr and tracr nucleotides can be called a "single guide RNA" (if it is composed of a continuous stretch of RNA nucleotides) or a "single guide DNA" (a continuous stretch of DNA nucleotides). ), or “single guide RNA-DNA” (if it is composed of a combination of RNA and DNA nucleotides). A single guide polynucleotide can form a complex with a Cas endonuclease, and said guide polynucleotide / Cas endonuclease complex (also called a guide polynucleotide / Cas endonuclease system) directs the Cas endonuclease to the genomic target site. Cas endonuclease recognizes its target site, binds to the target site, and optionally nicks or cleaves (introduces a single- or double-strand break) the target site. to enable. (U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015 and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015).
[0549] Chimeric non-natural single guide RNAs (sgRNAs) include sgRNAs containing regions that are not found together in nature (ie, they are heterologous to each other). For example, a first nucleotide sequence domain (also called a tracrmate sequence) capable of hybridizing to a nucleotide sequence in the target DNA, linked to a second nucleotide sequence (also called a tracrmate sequence) that is not found linked together in nature. sgRNAs containing variable targeting domains or called VT domains).
[0550] The nucleotide sequence linking the cr and tracr nucleotides of the single-stranded guide polynucleotide can comprise an RNA sequence, a DNA sequence, or a combined RNA-DNA sequence. In one embodiment, the nucleotide sequence (also referred to as a "loop") connecting the cr and tracr nucleotides of the single guide polynucleotide is 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, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86 , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the cr and tracr nucleotides of the single guide polynucleotide may comprise a tetraloop sequence, such as, but not limited to, a GAAA tetraloop sequence.
[0551] The guide polynucleotide can be chemically synthesized (such as, but not limited to, Hendel et al. 2015, Nature Biotechnology 33, 985-989), generated in vitro the guide polynucleotide, and / or generated the guide RNA. It can be produced by methods known in the art, including but not limited to self-splicing (such as Xie et al., (2015), PNAS 112:3570-3575).
[0552] Protospacer Adjacent Motif (PAM) A "protospacer adjacent motif" (PAM) herein refers to a short nucleotide sequence that flanks a target sequence (protospacer) that is recognized (targeted) by the guide polynucleotide / Cas endonuclease system. A Cas endonuclease cannot correctly recognize a target DNA sequence without a PAM sequence following the target DNA sequence. The PAM sequence and length herein may vary depending on the Cas protein or Cas protein complex used. PAM sequences can be of any length, but are generally 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18 , 19 or 20 nucleotides long.
[0553] "Randomized PAM" and "randomized protospacer flanking motif" are used interchangeably herein to indicate the target sequence (protospacer) recognized (targeted) by the guide polynucleotide / Cas endonuclease system. A random DNA sequence that flanks a Randomized PAM sequences can be of any length, but are generally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19 or 20 nucleotides long. Randomized nucleotides include either nucleotides A, C, G or T.
[0554] Guide polynucleotide / Cas endonuclease complex The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding, and optionally nicking, unwinding, or cleaving all or part of a target sequence.
[0555] A guide polynucleotide / Cas endonuclease complex capable of cleaving both strands of a DNA target sequence is typically a Cas protein that has all of the endonuclease domains in a functional state (e.g., the wild-type endonuclease domain, or variants thereof that retain the activity of part or all of each endonuclease domain). Thus, a wild-type Cas protein (e.g., a Cas protein disclosed herein), or a variant thereof that retains the activity of part or all of each endonuclease domain of the Cas protein, can be used in both the DNA target sequence and the is a suitable example of a Cas protein capable of cleaving the chain of .
[0556] A guide polynucleotide / Cas endonuclease complex capable of cleaving a single strand of a DNA target sequence can be characterized herein as having nickase activity (eg, partial cleavage capability). A Cas nickase usually contains a monofunctional endonuclease domain that allows Cas to cleave (ie, nick) only a single strand of a DNA target sequence. For example, a Cas9 nickase can include (i) a mutated, dysfunctional RuvC domain, and (ii) a functional HNH domain (eg, a wild-type HNH domain). As another example, a Cas9 nickase can include (i) a functional RuvC domain (eg, 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 Patent Application Publication No. 2014 / 0189896, published Jul. 3, 2014. A pair of Cas nickases can be used to increase the specificity of DNA targeting. Generally, this provides two Cas nickases that target DNA sequences on opposite strands within that region and nick them in close proximity for the desired targeting by combining RNA components with different guide sequences. can be implemented by Such breaks near each DNA strand cause double-strand breaks (i.e., DSBs with single-stranded overhangs), which subsequently lead to non-homologous end-joining NHEJ (mutation incomplete repair). ), or as a substrate for homologous recombination HR. Each nick in these embodiments is, for example, at least about 5, 5-10, at least 10, 10-15, at least 15, 15-20, at least 20, 20-30, at least 30, 30-40, at least 40, 40-50, at least 50, 50-60, at least 60, 60-70, at least 70, 70-80, at least 80, 80-90, at least 90, 90-100, or 100 or more (or 5-100 Any integer) of bases can be separated from each other. One or two Cas nickase proteins herein can be used in a Cas nickase pair. For example, a Cas9 nickase that has a mutated RuvC domain but a functional HNH domain (ie, Cas9 HNH+ / RuvC−) can be used (eg, Streptococcus pyogenes Cas9 HNH+ / RuvC−). Each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) is brought close together (maximum 100 base pairs apart) can be directed to specific DNA sites.
[0557] The guide polynucleotide / Cas endonuclease complex in certain embodiments can bind to a DNA target site sequence, but does not cleave any strand at the target site sequence. Such complexes may contain a Cas protein whose entire nuclease domain is mutated and dysfunctional. For example, a Cas9 protein that can bind to a DNA target site sequence but does not cleave any strand at the target site sequence can contain both a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. A Cas protein herein that binds to, but does not cleave, a target DNA sequence can be used to modulate gene expression, e.g., where the Cas protein is a transcription factor (or part thereof) ( For example, it may be fused with a repressor or activator, such as any of those disclosed herein.
[0558] In one aspect, the guide polynucleotide / Cas endonuclease complex (PGEN) described herein is a PGEN, wherein said Cas endonuclease optionally comprises at least one protein subunit or a functional PGEN covalently or non-covalently linked or assembled into fragments.
[0559] In one embodiment of the 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 said Cas endonuclease polypeptide comprises at least one protein subunit or functional fragment thereof, said guide polynucleotide is a chimeric non-natural guide polynucleotide, said guide polynucleotide / Cas endonuclease complex comprises a target sequence can be recognized, bound, and optionally nicked, unwound, or cleaved.
[0560] The Cas effector protein can be the Cas-alpha effector protein disclosed herein.
[0561] In one embodiment of the present disclosure, the guide polynucleotide / Cas effector complex is a guide polynucleotide / Cas effector protein complex (PGEN) comprising at least one guide polynucleotide and a Cas-alpha effector protein, wherein said guide polynucleotide The nucleotide / Cas effector protein complex can recognize, bind, and optionally nick, unwind, or cleave all or part of the target sequence.
[0562] A PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein further comprises one copy or multiple copies of at least one protein subunit or functional fragment thereof. In some embodiments, said protein subunit is selected from the group consisting of Cas1 protein subunit, Cas2 protein subunit, Cas4 protein subunit, and any combination thereof. A PGEN can be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein further comprises at least two different protein subunits selected from the group consisting of Casl, Cas2 and Cas4.
[0563] The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein is selected from the group consisting of Cas1, Cas2, and optionally one further Cas protein, including Cas4, at least three It further comprises three different protein subunits, or functional fragments thereof.
[0564] In one aspect, the guide polynucleotide / Cas endonuclease complex (PGEN) described herein is a PGEN, wherein said Cas endonuclease is covalently bound to at least one protein subunit or functional fragment thereof or PGEN that is non-covalently linked. PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein polypeptide comprises one further Cas protein subunit comprising a Cas1 protein subunit, a Cas2 protein subunit, optionally a Cas4 protein. covalently or non-covalently linked or assembled to one or more copies of at least one protein subunit, or functional fragment thereof, selected from the group consisting of units, and any combination thereof It is The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein is at least two selected from the group consisting of Casl, Cas2, and optionally one further Cas protein, including Cas4. Covalently or non-covalently linked or assembled into two different protein subunits. The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein is from the group consisting of Casl, Cas2, and optionally one additional Cas protein, including Cas4, and combinations thereof Covalently or non-covalently linked to at least three different protein subunits of choice.
[0565] Any component of the guide polynucleotide / Cas effector protein complex, the guide polynucleotide / Cas effector protein complex itself, and the polynucleotide-modified template and / or donor DNA may be treated with heterologous cells by any method known in the art. Or it can be introduced into an organism.
[0566] Recombinant constructs for cell transformation A disclosed guide polynucleotide, a Cas endonuclease, a polynucleotide modification template, donor DNA, a guide polynucleotide / Cas endonuclease system disclosed herein, and optionally one or more polynucleotides of interest. A combination of any one of them, including further, can be introduced into the cell. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast and plant cells, as well as plants and plants produced by the methods described herein. seeds.
[0567] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989) describes it in more detail. Transformation methods are well known to those of skill in the art and are described below.
[0568] Vectors and constructs include circular plasmids and linear polynucleotides, which include a polynucleotide of interest and, optionally, other components, including linkers, adapters, regulatory elements or analytical elements. In some examples, recognition sites and / or target sites can be contained within introns, coding sequences, 5'UTRs, 3'UTRs and / or regulatory regions.
[0569] Components for the expression and utilization of novel CRISPR-Cas systems in prokaryotic and eukaryotic cells The present invention further provides guide RNAs / organisms capable of recognizing, binding, and optionally nicking, unwinding, or cleaving all or part of a target sequence in prokaryotic or eukaryotic cells / organisms. An expression construct is provided for expressing the Cas system.
[0570] In one embodiment, the expression construct of the present disclosure comprises a promoter operably linked to a nucleotide sequence encoding a Cas gene (or an optimized plant containing a Cas endonuclease gene as described herein) , and a promoter operably linked to the guide RNA of the disclosure. Promoters are capable of driving expression of operably linked nucleotide sequences in prokaryotic or eukaryotic cells / organisms.
[0571] Nucleotide sequence modifications of the guide polynucleotide, the VT domain, and / or the CER domain include 5′ caps, 3′ polyadenylation tails, riboswitch sequences, stability control sequences, sequences that form dsRNA duplexes, modifications or sequences that target intracellular locations, modifications or sequences that result in tracking, modifications or sequences that generate binding sites for proteins, locked nucleic acids (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2'-fluoro A nucleotide, 2'-fluoro U nucleotide; 2'-O-methyl RNA nucleotide, phosphorothioate linkage, linked to cholesterol molecule, linked to polyethylene glycol molecule, linked to spacer 18 molecule, 5' to 3 covalently linked to ', or any combination thereof, but is not limited thereto. These modifications may result in at least one additional advantageous characteristic, which may be alteration or modulation of stability, intracellular targeting, tracking, fluorescent labeling, It is selected from the group of binding sites, altered binding affinity for complementary target sequences, altered resistance to cellular degradation, and increased cell permeability.
[0572] A method of expressing RNA components such as gRNAs in eukaryotic cells for Cas9-mediated DNA targeting is to use RNA polymerase III (Pol III) promoters, which contain precisely defined modifications. allows transcription of RNAs with missing 5' and 3' ends (DiCarlo et al., Nucleic Acids Res. 41:4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3: e161). This strategy has been successfully applied to cells of several different species, including maize and soybean (US2015 / 0082478, published March 19, 2015). A method for expressing RNA components without a 5' cap has been described (WO 2016 / 025131, published February 18, 2016).
[0573] A variety of methods and compositions can be used to obtain a cell or organism having a polynucleotide of interest inserted into a target site for Cas endonuclease. Such methods may employ homologous recombination (HR) to integrate the polynucleotide of interest at the target site. In one method described herein, a polynucleotide of interest is introduced into a biological cell by a donor DNA construct.
[0574] The donor DNA construct further comprises first and second regions of homology that flank the polynucleotide of interest. The first and second regions of homology of the donor DNA are homologous to the first and second genomic regions respectively present in or adjacent to the target site of the genome of the cell or organism.
[0575] Donor DNA can be ligated to a guide polynucleotide. Ligated donor DNA may allow co-localization of target and donor DNA useful for genome editing, gene insertion and target genome regulation, and endogenous HR machinery may be greatly reduced in function. It may be useful for targeting postmitotic cells that are thought to be living (Mali et al., 2013, Nature Methods Vol. 10:957-963).
[0576] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and can range from about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400bp, 250-500bp, 300-600bp, 350-750bp, 400-800bp, 450-900bp, 500-1,000bp, 600-1,250bp, 700-1,500bp, 800-1,750bp, 900-2,000bp, 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 full length and / or with unit integer values within the range up to the full length of the target site or including the entire area. These ranges include all integers within this range, e.g. 13, 14, 15, 16, 17, 18, 19 and 20bp are included. The amount of homology can also be described in percent sequence identity over the fully aligned length of 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%~99%, 99%, 99%~100% , or percent sequence identity of 100%. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or regional percent sequence identity, e.g. Homology can be described as a 75-150 bp region with at least 80% sequence identity to the region of the target locus. Sufficient homology can also be explained by the predictive ability of two polynucleotides to hybridize specifically under conditions of high stringency, see, for example, 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). See Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).
[0577] 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 "homologous region" of the donor DNA and the "genomic region" of the organism's genome is at least 50%, 55%, 60%, such that the sequences undergo homologous recombination. , 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.
[0578] A region of homology on the donor DNA can have homology to any sequence that flanks the target site. In some instances, the region of homology shares considerable sequence homology to genomic sequences immediately adjacent to the target site, but this region of homology may be further 5' or 3' to the target site. It will be appreciated that regions can be designed to have sufficient homology. A homologous region can also have homology to fragments of the target site in addition to downstream genomic regions.
[0579] In one embodiment, the first region of homology further comprises a first fragment of the target site and the second region of homology comprises a second fragment of the target site, wherein the first fragment and the second fragment are different .
[0580] Polynucleotide of interest Polynucleotides of interest, which are described in detail herein, include polynucleotides that reflect the interests of commercial markets and commercial markets related to crop development. Target crops and markets will change, and new crops and technologies will also emerge as developing countries expand their global markets. Moreover, as our understanding of agronomic traits and characteristics such as yield and hybrid vigor increases, the selection of genes for genetic modification will change accordingly.
[0581] General categories of polynucleotides of interest include, for example, genes involved in information, such as zinc fingers, genes involved in transduction, such as kinases, and genes involved in housekeeping, such as heat shock proteins. More specific polynucleotides of interest include, but are not limited to, crop yield, grain quality, grain nutrient content, starch and carbohydrate quality and quantity, and grain size, sucrose load, protein quality and Genes involved in traits of agronomic interest, such as those affecting abundance, nitrogen fixation and / or utilization, fatty acid and oil composition, abiotic stresses (drought, nitrogen, temperature, salinity, toxic metals or trace elements, genes encoding proteins that confer resistance to toxins such as pesticides and herbicides); genes that encode proteins that confer resistance to organism-related disease progression, etc., but are not limited to these.
[0582] Agronomically important traits such as oil, starch and protein content can be genetically modified in addition to using conventional breeding methods. Modifications include increasing oleic acid, saturated and unsaturated oil content, increasing lysine and sulfur levels, providing essential amino acids, and also modifying starch. Modifications of the holidothionin protein are described in US Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.
[0583] A polynucleotide sequence of interest may encode a protein involved in the induction of disease or pest resistance. "Disease resistance" or "pest resistance" means that plants avoid harmful symptoms that are the result of plant-pathogen interactions. A pest resistance gene may encode resistance to pests that inhibit high yields, such as lice, armyworm, European corn borer. Disease and insect resistance genes such as lysozyme or cecropins to protect antibacterial properties, or proteins such as defensins, glucanases or chitinases to protect antifungal properties, or Bacillus thuringiensis endotoxins to control nematodes or insects. , protease inhibitors, collagenases, lectins or glucosidases are all examples of useful gene products. Genes encoding disease resistance traits include detoxification genes such as against fumonisins (U.S. Pat. No. 5,792,931), avirulent (avr) genes 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).
[0584] Insect resistance genes can encode resistance to pests that inhibit high yields, such as lice, cutworms, European corn borer. Such genes include, for example, the Bacillus thuringiensis toxic protein gene (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109).
[0585] As a protein obtained by expression of a "herbicide tolerance protein" or a "nucleic acid molecule encoding herbicide tolerance", the ability to tolerate higher concentrations of herbicides than cells that do not express the protein, or the ability to express the protein Examples include proteins that confer on cells the ability to tolerate a particular concentration of herbicide for a longer period of time than cells that do not. Herbicide tolerance traits are herbicides that act to inhibit the action of acetolactate synthase (ALS, also called acetohydroxyacid synthase, AHAS), particularly sulfonylurea (UK: sulphonylurea). )-type herbicides, genes encoding tolerance to herbicides that act to inhibit the action of glutamine synthetase, such as phosphinothricin or basta (e.g., the bar gene), tolerance to glyphosate into plants by genes encoding (e.g., EPSP synthase and GAT genes), genes encoding resistance to HPPD inhibitors (e.g., HPPD genes), or other such genes known in the art. can be introduced. For example, U.S. Pat. and US Pat. No. 9,187,762. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene mutant encodes resistance to the herbicide chlorsulfuron.
[0586] Additionally, the polynucleotide of interest can also include an antisense sequence complementary to at least a portion of the messenger RNA (mRNA) for the targeted gene sequence of interest. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. An antisense sequence may be modified so long as the sequence hybridizes to the corresponding mRNA and prevents its expression. Antisense constructs with 70%, 80% or 85% sequence identity to the corresponding antisense sequences can be used in this manner. In addition, antisense nucleotide moieties can be used to interfere with target gene expression. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides or more can be used.
[0587] Additionally, the polynucleotide of interest can also be used in a sense orientation to suppress expression of endogenous genes in the plant. Methods for suppressing gene expression in plants by means of polynucleotides in the sense orientation are known in the art. The method generally involves transforming the plant with a DNA construct comprising a promoter driving expression in the plant, operably linked to at least a portion of the nucleotide sequence corresponding to the transcription of the endogenous gene. is included. Typically, such nucleotide sequences will exhibit substantial sequence identity, generally greater than about 65% sequence identity, greater than about 85% sequence identity, or greater than about 95% sequence identity, to the transcribed sequence of the endogenous gene. Greater than % sequence identity. See US Pat. Nos. 5,283,184 and 5,034,323.
[0588] A polynucleotide of interest may also be a phenotypic marker. Phenotypic markers are screening or selectable markers, including visual and selectable markers, whether positively or negatively selectable. Any phenotypic marker can be used. In particular, selectable or screening markers often identify, or select against, or against a molecule or cell containing a molecule or cell under certain conditions. contains a DNA segment that enables These markers can encode activities such as, but not limited to, the production of RNA, peptides or proteins, or provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions such as be able to.
[0589] Examples of selectable markers include, but are not limited to, DNA segments containing restriction enzyme sites; spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT)). DNA segments encoding products that provide resistance to otherwise toxic compounds, including antibiotics; DNA segments encoding products that are otherwise missing in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding readily identifiable products (e.g., phenotypic markers such as β-galactosidase, GUS; green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP); ) fluorescent proteins, such as fluorescent proteins, and cell surface proteins); generation of novel primer sites for PCR (e.g. alignment of two previously unaligned DNA sequences), restriction endonucleases or other DNA modifications. Included are the inclusion of DNA sequences that have been unaffected or subjected to the action of enzymes, chemicals, etc., as well as the inclusion of DNA sequences that are required for specific modifications (eg, methylation) that allow their identification.
[0590] Additional selectable markers include genes that confer resistance to herbicidal compounds such as sulfonylureas, glufosinate-ammonium, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). For example, acetolactate synthase (ALS) resistant to sulfonylureas, imidazolinones, triazolopyrimidinesulfonamides, pyrimidinyl salicylates and sulfonylaminocarbonyltriazolinones (Shaner and Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69 -110); glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate (EPSPS) (Saroha et al., 1998, J. Plant Biochemistry & Biotechnology Vol 7:65-72);
[0591] Polynucleotides of interest include genes that can be stacked or used in combination with other traits, such as, but not limited to, herbicide tolerance or any other trait described herein. Polynucleotides and / or traits of interest are described in U.S. Patent Application Publication No. 2013 / 0263324, published Oct. 3, 2013 and International Publication No. WO 2013 / 112686, published Aug. 1, 2013. As described, multiple trait loci can be stacked together.
[0592] A polypeptide of interest includes any protein or polypeptide encoded by a polynucleotide of interest described herein.
[0593] Further provided is a method of identifying at least one plant cell that contains in its genome a polynucleotide of interest integrated at a target site. Various methods can be used to identify plant cells with insertions at or near the target site of the genome. Such methods can be considered direct analysis of the target sequence to detect alterations in the target sequence and include, but are not limited to, PCR, sequencing, nuclease digestion, Southern blotting and Any combination of these is included. See, for example, US Patent Application Publication No. 2009 / 0133152, published May 21, 2009. The method also includes recovering plants from plant cells containing the polynucleotide of interest integrated into the genome. Plants may be sterile or fertile. Any polynucleotide of interest can be provided, integrated into a target site in the genome of a plant, and expressed in the plant.
[0594] Sequence optimization for expression in plants Methods for synthesizing plant preference genes are available in the art. See, eg, US 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. These modifications include, for example, removal of one or more sequences encoding spurious polyadenylation signals, removal of one or more exon-intron splice site signals, removal of one or more transposon-like repeats, and Removal of such other well-characterized sequences that may be detrimental to gene expression is included. The GC content of the sequences can be adjusted to the average level for a given plant host (calculated with reference to known genes expressed in that host plant cell). If possible, modify the sequence to avoid predicted hairpin secondary structures in one or more mRNAs. Accordingly, the "plant-optimized nucleotide sequences" of this disclosure include one or more modifications of such sequences.
[0595] expression element Any polynucleotide encoding a Cas protein or other CRISPR system component disclosed herein can be operably linked to heterologous expression elements to facilitate transcription or regulation in a host cell. Such expression elements include, but are not limited to, promoters, leaders, introns, terminators. An expression element may be "minimal", which means a short sequence derived from a natural source that still functions as an expression regulator or modifier. Alternatively, an expression element may be "optimized," which means that its polynucleotide sequence has been altered from its natural state so as to function with more desirable characteristics in a particular host cell. (For example, but not limited to, a bacterial promoter may be "maize optimized" to improve its expression in maize plants). Alternatively, the expression element may be "synthetic," meaning that the expression element is designed in silico and synthesized for use in the host cell. Synthetic expression elements may be wholly or partially synthetic (including fragments of naturally occurring polynucleotide sequences).
[0596] It has been shown that certain promoters are able to induce RNA synthesis at a higher rate than others. These are called "strong promoters". Certain other promoters have been shown to induce synthesis of RNA at high levels only in certain cell or tissue types, with promoters preferentially inducing RNA synthesis in certain tissues and If they induce RNA synthesis at low levels, they are often called "tissue-specific promoters" or "tissue-preferred promoters."
[0597] A plant promoter includes promoters capable of initiating transcription in plant cells. For a description 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. .
[0598] 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 (US Pat. No. 5,659,026);
[0599] Tissue-preferred promoters can be used to target enhanced expression within specific plant tissues. Tissue-preferred promoters include, for example, International Publication No. 2013 / 103367, published July 11, 2013, Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Hansen et al., (1997) Mol Gen Genet 254:337-43; Russell et al., (1997) Transgenic Res 6:157-68; Rinehart et al., (1996) Plant Physiol 112:1331-41; Van Camp et al., (1996) Plant Physiol 112:525-35; Canevascini et al., (1996) Plant Physiol 112:513-524; Lam, (1994) Results Probl Cell Differ 20:181-96; and Guevara-Garcia et al., (1993) Plant J 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. USA 90:9586-90; Simpson et al., (1958) EMBO J 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 synthetase 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 regulatory elements in the green bean GRP 1.8 gene); Sanger et al., (1990) Plant Mol Biol. 14:433-43 (root-specific promoter of A. tumefaciens mannopine synthase (MAS)); Bogusz et al., (1990) Plant Cell 2:633-41 (Parasponia andersonii). and root-specific promoters isolated from Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A. rhizogenes rolC and rolD root-induced genes); 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 the rolB promoter (Capana et al., (1994) Plant Mol Biol 25:681-91; the phaseolin gene (Murai et al., (1983) Science 23:476-82; Sengopta-Gopalen et al.); USA 82:3320-4), U.S. Pat. See also US Pat. Nos. 5,401,836, 5,110,732 and 5,023,179.
[0600] 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 Cim1 (cytokinin-inducible message); cZ19B1 (maize 19 kDa zein); and milps (myoinositol-1-phosphate synthase); Examples include, but are not limited to, those disclosed in 2000 / 011177 and US Pat. No. 6,225,529. Seed-preferred promoters for dicotyledonous plants include, but are not limited to, bean-β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. Seed-preferred promoters for monocotyledonous plants include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, schranken 1, schranken 2, globulin 1, oleosin and nuc1. See also WO 2000 / 012733, published Mar. 9, 2000. This document discloses seed-preferred promoters from the END1 and END2 genes.
[0601] Chemically inducible (regulated) promoters can be used to regulate gene expression in prokaryotic and eukaryotic cells or organisms by application of exogenous chemical regulatory agents. The promoter may be a chemically inducible promoter, where application of a chemical induces gene expression, or a chemically repressible promoter, where 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 preemergence herbicides (GST-II-27, WO 1993 / 001294 published Jan. 21, 1993), and the tobacco PR-1a promoter that is activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Other chemically regulated promoters include steroid-responsive promoters (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 and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,814,618); 5,789,156).
[0602] Pathogen-inducible promoters that are induced after pathogen infection include, but are not limited to, those that regulate the expression of PR proteins, SA proteins, beta-1,3-glucanase, chitinase, and the like.
[0603] Stress-inducible promoters include the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91). Those skilled in the art have protocols for simulating stress conditions such as drought, osmotic stress, salt stress and temperature stress, and assessing stress tolerance of plants subjected to simulated or naturally occurring stress conditions. Familiarity with the protocol for
[0604] Another example of an inducible promoter useful for plant cells is the ZmCAS1 promoter described in US Patent Application Publication No. 2013 / 0312137, published Nov. 21, 2013.
[0605] A wide variety of new promoters useful in plant cells are constantly being discovered, many examples can be found in Okamuro and Goldberg, eds. (1989) The Biochemistry of Plants, Vol. 115, Stumpf and Conn, eds (New York, NY). : Academic Press), pp.1-82.
[0606] Genome modification by novel CRISPR-Cas system components As described herein, guided Cas endonucleases can recognize and bind DNA target sequences and introduce single-strand (nicks) or double-strand breaks. When a single-strand or double-strand break is introduced into DNA, the cell's DNA repair machinery is activated to repair the break. Error-prone DNA repair machinery can create mutations at double-strand break sites. The most common repair mechanism for joining broken ends together is the non-homologous end joining (NHEJ) pathway (Bleuyard et al., (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically maintained by repair, although deletions, insertions or other rearrangements (such as chromosomal translocations) are possible (Siebert and Puchta, 2002, Plant Cell 14:1121). -31; Pacher et al., 2007, Genetics 175:21-9).
[0607] DNA double-strand breaks appear to be effective factors for stimulating the homologous recombination pathway (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). A 2- to 9-fold increase in homologous recombination between artificially constructed homologous DNA repeats in plants has been observed using DNA cutting agents (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).
[0608] Homologous recombination repair (HDR) is a mechanism that repairs intracellular double- and single-stranded DNA breaks. Homologous recombination repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber.2010 Annu.Rev.Biochem.79:181-211). The most common form of HDR is called homologous recombination (HR) with the longest sequence homology requirement between donor and acceptor DNA. Other forms of HDR include single-strand annealing (SSA) and break-induced replication, which require shorter sequence homology compared to HR. Homologous recombination repair for nicking (single-strand breaks) can occur by a different mechanism than HDR for double-strand breaks (Davis and Maizels. PNAS (0027-8424), 111(10), p. E924-E932).
[0609] For example, modification of the genomes of prokaryotic and eukaryotic living or biological cells 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 231:186-93) and insects (Dray and Gloor, 1997, Genetics 147:689-99). Homologous recombination has also been achieved in other organisms. For example, at least 150-200 bp of homology was required for homologous recombination in the protozoan parasite Leishmania (Papadopoulou and Dumas, (1997) Nucleic Acids Res 25:4278-86). . In the filamentous fungus Aspergillus nidulans, gene replacement has been achieved with as little as 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 cell lines (ES) 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.).
[0610] gene targeting The guide polynucleotide / Cas system described herein can be used for gene targeting.
[0611] In general, DNA targeting can be carried out by cleaving one or both strands at specific polynucleotide sequences in cells in which Cas proteins are associated with suitable polynucleotide moieties. When a single-strand or double-strand break is induced in DNA, the cell's DNA repair machinery is activated and repairs the break by non-homologous end joining (NHEJ) or homologous recombination repair (HDR) processes to target It can lead to modification of the site.
[0612] The length of the target site DNA sequence may vary, e.g. , 28, 29, 30, or 30 or more nucleotides. The target site may be palindromic, in other words it is still possible for a sequence on one strand to read the same in opposite directions on the complementary strand. The nicks / cleavage sites can be within the target sequence, or the nicks / cleavage sites can be outside the target sequence. In another variation, cleavage can occur at nucleotide positions directly opposite each other, resulting in blunt-end cleavage, and in other cases, the nicks are offset to create single-stranded overhangs, also called "sticky ends." (which can be a 5' overhang or a 3' overhang). Active variants of genomic target sites can also be used. Such active variants are at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% active against a given target site. %, 97%, 98%, 99% or more sequence identity, active variants retain biological activity and can therefore be recognized and cleaved by a Cas endonuclease.
[0613] Assays for measuring single- or double-strand cleavage of target sites by endonucleases are known in the art and generally involve the overall activity and specificity of an agent to a DNA substrate containing a recognition site. to measure.
[0614] The targeting method herein can be performed such that, for example, more than one DNA target site is targeted in the method. Such methods may optionally be characterized as multiplex methods. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sites may be targeted simultaneously. Multiplex methods are generally provided herein with a plurality of different RNA components, each of which is designed to direct the guide polynucleotide / Cas endonuclease complex to a unique DNA target site. targeting method.
[0615] gene editing The process of editing a genome sequence that combines a DSB and a modified template generally involves a DSB inducer capable of recognizing a target sequence in a chromosomal sequence and introducing a DSB into the genomic sequence, or a nucleic acid encoding the DSB inducer, and a target to be edited. introducing into the host cell at least one polynucleotide-modified template that contains at least one nucleotide modification compared to the nucleotide sequence of . A polynucleotide modification template can further comprise a nucleotide sequence flanking at least one nucleotide modification. The flanking sequences are substantially homologous to chromosomal regions flanking the DSB. Genome editing using DSB inducers, such as Cas-gRNA complexes, is described, for example, in U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, published February 26, 2015. WO 2015 / 026886, WO 2016 / 007347 published 14 January 2016, and WO 2016 / 025131 published 18 February 2016 It is
[0616] Several uses of guide RNA / Cas endonuclease systems have been described (e.g., US Patent Application Publication No. 2015 / 0082478A1, published March 19, 2015, published February 26, 2015). See International Publication No. WO 2015 / 026886, published Feb. 26, 2015, and U.S. Patent Application Publication No. 2015 / 0059010, published Feb. 26, 2015), which includes nucleotide sequences of interest (such as regulatory elements ), insertion of a polynucleotide of interest, gene knockout, gene knockin, splicing site modification and / or introduction of an alternative splicing site, modification of a nucleotide sequence encoding a target protein, amino acid and / or protein fusion, and gene silencing by expressing inverted repeats in the gene of interest, but are not limited to these.
[0617] Proteins can be modified in a variety of ways, including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of proteins can be prepared by mutations within the DNA. Methods of mutagenesis and nucleotide sequence alteration include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. See Patent 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 a protein is found, for example, in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). . Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferred. Conservative deletions, insertions, and amino acid substitutions are not expected to cause drastic changes in protein properties, and the effects of substitutions, deletions, insertions, or combinations thereof can be assessed in routine screening assays. can. Assays for double-strand break-inducing activity are known and generally measure the overall activity and specificity of an agent on a DNA substrate containing a target site.
[0618] Described herein are methods of genome editing using Cas endonucleases and complexes comprising Cas endonucleases and guide polynucleotides. Following characterization of the guide RNA and PAM sequences, endonucleases and related CRISPR RNA (crRNA) components can be utilized to modify chromosomal DNA in other organisms, including plants. In order to facilitate optimal expression and nuclear localization (for eukaryotic cells), the gene containing complex was quantified as described in WO 2016 / 186953 published Nov. 24, 2016. and then delivered to cells as a DNA expression cassette by methods known in the art. The components necessary to contain the active complex are also available as RNA with or without modifications that protect the RNA from degradation, i.e. as capped or uncapped mRNA (Zhang, Y. et al., 2016 , Nat. Commun. 7:12617), or as a Cas protein guide polynucleotide complex (WO 2017 / 070032, published April 27, 2017), or any combination thereof. Furthermore, one or more parts of the complex and the crRNA can be expressed from a DNA construct, while the other components can be expressed as RNA with or without modifications that protect the RNA from degradation, i.e. capped or as uncapped mRNA (Zhang et al.2016Nat.Commun.7:12617), or as a Cas protein guide polynucleotide complex (WO2017 / 070032 published April 27, 2017), or can be delivered as any combination of To produce crRNA in vivo, a tRNA-derived element is used to generate the crRNA transcript, e.g., as described in WO 2017 / 105991, published Jun. 22, 2017. Endogenous RNAse can also be recruited to cleave into mature forms that can direct the complex to the DNA target site. Nickase complexes can be used individually or in concert to generate single or multiple DNA nicks on one or both of the DNA strands. Furthermore, the cleavage activity of the Cas endonuclease can be inactivated by modifying key catalytic residues in its cleavage domain (Sinkunas, T et al, 2013, EMBO J. 32:385-394). , resulting in an RNA-guided helicase that can be used to enhance homologous recombination repair, induce transcriptional activation, or remodel DNA local structures. Furthermore, both the Cas cleavage domain and the helicase domain activity are knocked out, resulting in other DNA cleavages, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA reassembly, DNA deamination, DNA unwinding, DNA assembly. It can be used in combination with recombination enhancement, DNA integration, DNA inversion, and DNA repair agents.
[0619] Transcriptional directions of the CRISPR-Cas system tracrRNA (if present) and other components of the CRISPR-Cas system (e.g., variable targeting domains, crRNA repeats, loops, anti-repeats) were published on November 24, 2016. It can be estimated as described in WO 2016 / 186946 and WO 2016 / 186953 published on November 24, 2016.
[0620] Once the appropriate guide RNA requirements are established, as described herein, the PAM preferences for each new system disclosed herein can be examined. If the cleavage complex results in degradation of the randomized PAM library, either by mutagenesis of key residues or by assembling the reaction in the absence of ATP as previously described (Sinkunas, T. et al. al., 2013, EMBO J.32:385-394), the complex can be converted to a nickase by abolishing the ATPase-dependent helicase activity. Two regions of PAM randomization separated by two protospacer targets can be utilized to generate double-stranded DNA breaks that are captured and sequenced to support cleavage by each complex. You can look up the PAM array for
[0621] In one embodiment, the invention describes a method of modifying a target site in the genome of a cell. The method comprises introducing into a cell at least one PGEN described herein, and identifying at least one cell having an alteration in said target, wherein said alteration at said target site comprises (i) at least one substitution of one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, chemical modification of at least one nucleotide, and (v) any combination of (i) to (iv) selected from the group consisting of
[0622] The nucleotide to be edited may be located inside or outside the target site recognized and cleaved by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification at a target site recognized and cleaved by a Cas endonuclease. In another embodiment, at least 1, 2, 3, 4, 5, 6, 7, 8, 9 are located between at least one nucleotide to be edited and the genomic target site. , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 1, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides.
[0623] Knockouts may be generated by indels (insertions or deletions of nucleotide bases in the target DNA sequence by NHEJ), or by specific removal of sequences that reduce or completely abolish the function of sequences at or near the target site.
[0624] Guide polynucleotide / Cas endonuclease-directed targeted mutations can occur within nucleotide sequences located within or outside the genomic target site recognized and cleaved by the Cas endonuclease.
[0625] A method of editing a nucleotide sequence in the genome of a cell can be a method that does not use exogenous selectable markers by restoring the function of non-functional gene products.
[0626] In one embodiment, the invention describes a method for modifying a target site in the genome of a cell. The method comprises introducing into a cell at least one PGEN as described herein and at least one donor DNA, wherein said donor DNA comprises a polynucleotide of interest and, optionally, said target site or its Further comprising identifying at least one cell that has integrated said polynucleotide of interest in its vicinity.
[0627] In one aspect, the methods disclosed herein may employ homologous recombination (HR) to provide for integration of the polynucleotide of interest into the target site.
[0628] Various methods and compositions can be used to produce cells or organisms with a polynucleotide of interest inserted into a target site by the activity of the CRISPR-Cas system components described herein. In one method described herein, a polynucleotide of interest is introduced into a biological cell by a donor DNA construct. As used herein, "donor DNA" is a DNA construct containing a polynucleotide of interest to be inserted into the Cas endonuclease target site. The donor DNA construct further comprises first and second regions of homology that flank the polynucleotide of interest. The first and second regions of homology of the donor DNA are homologous to the first and second genomic regions respectively present in or adjacent to the target site of the genome of the cell or organism.
[0629] Donor DNA can be bound to a guide polynucleotide. Ligated donor DNA may allow co-localization of target and donor DNA useful for genome editing, gene insertion and target genome regulation, and endogenous HR machinery may be greatly reduced in function. It may be useful for targeting postmitotic cells that are thought to be living (Mali et al., 2013, Nature Methods Vol. 10:957-963).
[0630] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and can range from about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400bp, 250-500bp, 300-600bp, 350-750bp, 400-800bp, 450-900bp, 500-1,000bp, 600-1,250bp, 700-1,500bp, 800-1,750bp, 900-2,000bp, 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 full length and / or with unit integer values within the range up to the full length of the target site or including the entire area. These ranges include all integers within this range, e.g. 13, 14, 15, 16, 17, 18, 19 and 20bp are included. The amount of homology can also be described in percent sequence identity over the fully aligned length of 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%, 99%, 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 regional percent sequence identity, e.g. Homology can be described as a 75-150 bp region with at least 80% sequence identity to the region of the target locus. Sufficient homology can also be explained by the predictive ability of two polynucleotides to hybridize specifically under conditions of high stringency, see, for example, 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). See Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).
[0631] Episomal DNA molecules can also ligate within double-strand breaks, e.g., incorporate T-DNA into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J 17:6086-95). If the sequences surrounding the double-strand break are modified, for example, by exonuclease activities involved in the maturation of the double-strand break, the gene conversion pathway may be altered, for example, in nondividing somatic cells or sister chromatids after DNA replication. The original structure can be restored if homologous sequences such as are available (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).
[0632] In one embodiment, the disclosure includes a method of editing a nucleotide sequence in the genome of a cell, the method comprising introducing at least one PGEN as described herein, and a polynucleotide modified template ( wherein said polynucleotide modified template comprises at least one nucleotide modification of said nucleotide sequence), optionally further comprising selecting at least one cell comprising the edited nucleotide sequence.
[0633] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow editing (modification) of the genomic nucleotide sequence of interest. (See also U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and International Publication No. WO 2015 / 026886, published February 26, 2015).
[0634] The polynucleotide of interest and / or trait is, as described in WO 2012 / 129373 published September 27, 2012 and WO 2013 / 112686 published August 1, 2013, Can be stacked together in compound trait loci. The guide polynucleotide / Cas9 endonuclease system described herein provides a system for efficiently generating double-strand breaks, allowing stacking of traits at complex trait loci.
[0635] A guide polynucleotide / Cas system as described herein that mediates gene targeting is used in methods for directing heterologous gene insertion and / or for generating complex trait loci comprising multiple heterologous genes, September 2012 It can be used in a manner similar to that disclosed in WO 2012 / 129373, published May 27, in which double-strand break-induced Instead of using agents, the guide polynucleotide / Cas system disclosed herein is used. Transgenes can be propagated as a single locus by inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or 5 centimorgans (cM) of each other ( See, e.g., U.S. Patent Application Publication No. 2013 / 0263324, published October 3, 2013, or International Publication No. WO 2012 / 129373, published March 14, 2013). After selection of transgene-containing plants, plants containing (at least) one transgene can be crossed to form an F1 containing both transgenes. Of these F1 (F2 or BC1) offspring, 1 / 500 offspring will have two different transgenes recombined on the same chromosome. This composite locus can then be propagated with both introduced traits as a single locus. By repeating this process, any number of desired traits can be accumulated.
[0636] Additional uses of the guide RNA / Cas endonuclease system have been described (e.g., US Patent Application Publication No. 2015 / 0082478, published March 19, 2015, published February 26, 2015). WO 2015 / 026886, U.S. Patent Application Publication No. 2015 / 0059010 published February 26, 2015, WO 2016 / 007347 published January 14, 2016, and PCT Application WO 2016 / 025131, published February 18, 2016), including alterations or substitutions of nucleotide sequences of interest (such as regulatory elements), polynucleotides of interest, Insertions, gene knockouts, gene knockins, splicing site modifications and / or introduction of alternative splicing sites, modification of the nucleotide sequence encoding the protein of interest, amino acid and / or protein fusions, and expression of inverted repeats in the gene of interest Includes, but is not limited to, gene silencing by chance.
[0637] Characteristics resulting from the gene-editing compositions and methods described herein can be evaluated. Chromosomal intervals that correlate with a phenotype or trait of interest can be identified. Various methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are delineated to include markers that will be linked to the gene controlling the trait of interest. In other words, chromosomal intervals are determined such that any marker that lies within that interval (including terminal markers that define the boundaries of the interval) can be used as a marker for a particular trait. In one embodiment, a chromosomal interval includes at least one QTL, and may in fact include more than one QTL. The close proximity of multiple QTLs within the same interval can obscure the association of a particular marker with a particular QTL, as one marker links to more than one QTL. Conversely, for example, if two markers in close proximity co-segregate with a desired phenotypic trait, does each of these markers specify the same QTL or two different QTLs? Please, it is often unclear. The term "quantitative trait locus" or "QTL" refers to a region of DNA associated with differential expression of a quantitative phenotypic trait in at least one genetic background, eg, in at least one breeding population. QTL regions encompass or are closely associated with genes that affect the trait in question. A "QTL allele" can include multiple genes or other genetic elements within a contiguous genomic region or linkage group, such as a haplotype. A QTL allele can refer to haplotypes within a particular window, which is a contiguous genomic region that can be defined and tracked by a set of one or more polymorphic markers. A haplotype can be defined by a unique fingerprint of the allele at each marker position within a particular window.
[0638] Introduction of CRISPR-Cas system components into cells The methods and compositions disclosed herein do not rely on any particular method of introducing sequences into an organism or cell, but simply enter the polynucleotide or polypeptide inside at least one cell of this organism. Only. Introduction includes references to integration of nucleic acids into eukaryotic or prokaryotic cells, where the nucleic acid can be integrated into the genome of the cell, and nucleic acids, proteins or polynucleotide-protein complexes (PGEN, RGEN). includes reference to transient (direct) provision of
[0639] Methods of introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated transfer, transfection, transduction, cell-permeable peptides, mesoporous silica nanoparticles (MSN) ) mediated direct protein delivery, topical application, sexual mating, sexual breeding, and any combination thereof.
[0640] For example, guide polynucleotides (guide RNA, cr nucleotides + tracr nucleotides, guide DNA and / or guide RNA-DNA molecules) are directly (transiently) introduced into cells as single-stranded or double-stranded polynucleotide molecules. obtain. The guide RNA (or crRNA+tracrRNA) is also present in the cell, the guide RNA (or crRNA+ It can be introduced indirectly by introducing a recombinant DNA molecule containing a heterologous nucleic acid segment encoding tracrRNA). A particular promoter can be, but is not limited to, the RNA polymerase III promoter, which allows the transcription of RNAs with rigorously defined, unmodified 5' and 3' ends (Ma et al., 2014). Ther. Nucleic Acids 3:e161; DiCarlo et al., 2013, Nucleic Acids Res. 41:4336-4343; Any promoter capable of transcribing the guide RNA in the cell can be used, including heat shock / heat inducible promoters operably linked to the nucleotide sequence encoding the guide RNA.
[0641] Plant cells differ from animal cells (such as human cells), fungal cells (such as yeast cells) and protoplasts, for example plant cells contain a plant cell wall that can act as a barrier to the delivery of components.
[0642] Cas endonuclease and / or guide RNA and / or ribonucleoprotein complex and / or delivery of polynucleotides encoding any one or more of the foregoing to plant cells by methods known in the art, For example, but not limited to, Rhizobiales-mediated transformation (e.g., Agrobacterium, Ochrobactrum, particle-mediated delivery (particle bombardment), (e.g., protoplast to ), electroporation, cell-permeable peptides, or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery.
[0643] Cas endonucleases, such as the Cas endonucleases described herein, can be expressed by the Cas polypeptide itself (referred to as direct delivery of the Cas endonuclease), the mRNA encoding the Cas protein, and / or the guide polynucleotide / Cas endonuclease complex. It can be introduced into cells by direct introduction using any method known in the art. A Cas endonuclease can also be indirectly introduced into a cell by introducing a recombinant DNA molecule encoding the Cas endonuclease. The endonuclease can be transiently introduced into the cell or integrated into the genome of the host cell using any method known in the art. Cellular uptake of endonucleases and / or inducible polynucleotides can be performed using cell penetrating peptides (CPPs) as described in WO 2016 / 073433 published May 12, 2016. can be promoted. Any promoter capable of expressing the Cas endonuclease in the cell can be used, including a heat shock / heat inducible promoter operably linked to the nucleotide sequence encoding the Cas endonuclease. be
[0644] Direct delivery of polynucleotide-modified templates to plant cells can be achieved by particle-mediated delivery, including but not limited to polyethylene glycol (PEG)-mediated transfection of protoplasts, whisker-mediated transformation, electroporation, particles. Any other direct delivery method such as bombardment, cell-permeable peptides, or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery can be used successfully for delivery of polynucleotide-modified templates in eukaryotic cells such as plant cells. can.
[0645] 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 methods. Donor DNA may be transiently present in the cell or could be introduced by a viral replicon. In the presence of the Cas endonuclease and target site, the donor DNA is inserted into the transformed plant genome.
[0646] Direct delivery of any one of the inducible Cas system components can facilitate enrichment and / or visualization of cells that receive the guide polynucleotide / Cas endonuclease complex components. ). For example, the guide polynucleotide / Cas endonuclease component (and / or the guide polynucleotide / Cas endonuclease complex itself) may be combined with a phenotypic marker, including but not limited to CRC (Bruce et al. 2000 The Plant Cell 12:65- 79) by direct co-delivery together with the mRNA encoding the transcriptional activator, as described in WO2017 / 070032, published April 27, 2017, Function of non-functional gene products can be restored, allowing selection and enrichment of cells without the use of exogenous selectable markers.
[0647] Introduction of the guide RNA / Cas endonuclease complexes described herein (representing the cleavage-ready complexes described herein) into cells may involve the individual components of said complexes either separately or Collectively into cells, either directly (delivered directly as RNA for the guide and protein and protein subunits for the Cas endonuclease, or functional fragments thereof) or as recombinant constructs expressing the components (guide RNA, Cas endonuclease). nucleases, protein subunits, or functional fragments thereof). Introducing a guide RNA / Cas endonuclease complex (RGEN) into a cell involves introducing the guide RNA / Cas endonuclease complex into the cell as a ribonucleotide-protein. Ribonucleotide-proteins can be assembled prior to introduction into cells, as described herein. The components that make up the guide RNA / Cas endonuclease ribonucleotide protein (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) are introduced in vitro or into a cell (see herein can be assembled by any means known in the art prior to being targeted for genomic modification as described).
[0648] Direct delivery of RGEN ribonucleoproteins enables genome editing at targeted sites in the cellular genome in such a way that the complexes are then rapidly disassembled and their existence within the cell is transient. . The transient presence of this RGEN complex can lead to reduced off-targets. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) by plasmid DNA sequences results in sustained expression of RGEN from these plasmids, which can increase off-targets (Cradick, T.J. et al. (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. 31:822-826).
[0649] Direct delivery involves any one component (e.g., at least one guide RNA, at least one Cas protein, and optionally one additional protein) with a delivery matrix comprising microparticles (such as, but not limited to, gold particles, tungsten particles, and silicon carbide whisker particles) (2017 See also WO 2017 / 070032, published April 27). The delivery matrix may comprise any one of the components, eg Cas endonuclease, which is attached to a solid matrix (eg impacting particles).
[0650] 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.
[0651] In one aspect, the guide polynucleotide / Cas endonuclease complex comprises a guide RNA and a Cas endonuclease protein forming a guide RNA / Cas endonuclease complex, and at least one protein subunit of the complex as RNA and protein, respectively. It is a complex that is introduced into cells.
[0652] In one aspect, the guide polynucleotide / Cas endonuclease complex comprises guide RNA and a Cas endonuclease protein that form a guide RNA and Cas endonuclease complex (cleavage-ready complex), and at least one protein subgroup of the complex. A complex where the units are preassembled in vitro and introduced into the cell as a ribonucleotide-protein complex.
[0653] 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), meristematic 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 11). :1126-1134;Shaheen A.and M. Arshad 2011 Properties and Applications of Silicon Carbide(2011),345-358 Editor(s):Gerhardt,Rosario.Publisher:InTech,Rijeka,Croatia.CODEN:69PQBP;ISBN:978 -953-307-201-2), direct gene transfer (Paszkowski et al., (1984) EMBO J 3:2717-22), and ballistic particle acceleration (U.S. Pat. Nos. 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, ed. Gamborg & Phillips (Springer-Verlag, Berlin); McCabe et al., (1988) Biotechnology 6:923-6; Weissinger et al., (1988) Ann Rev Genet 22:421-77; nford 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 96:319-24 (soybean); Datta et al., (1990) Biotechnology 8:736-40 (rice); Klein et al. USA 85:4305-9 (corn); Klein et al., (1988) Biotechnology 6:559-63 (corn); U.S. Patent No. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol 91:440-4 (corn); Fromm et al., (1990) Biotechnology 8:833-9 (corn); 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. -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 84:560-6 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-505 (electroporation); Li et al., (1993) Plant Cell R ep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996) Nat Biotechnol 14:745-50 (Agrobacterium tumefaciens via corn).
[0654] Alternatively, polynucleotides can be introduced into plants or plant cells by contacting the cells or organisms with a virus or viral nucleic acid. Generally, such methods involve incorporating the polynucleotide into a viral DNA or RNA molecule. In some examples, the polypeptide of interest can be first synthesized as part of a viral polyprotein, which can then be proteolytically processed in vivo or in vitro to produce the desired recombinant protein. Methods for introducing polynucleotides, including viral DNA or RNA molecules, into plants and expressing the encoded proteins therein are known, see, for example, US Pat. See US Pat. Nos. 5,866,785, 5,589,367 and 5,316,931.
[0655] Polynucleotides or recombinant DNA constructs can be provided or introduced into prokaryotic and eukaryotic cells or organisms using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, direct introduction of polynucleotide constructs into plants.
[0656] Incorporating nucleic acids and proteins in any manner, including using molecules that facilitate the uptake of any or all components (proteins and / or nucleic acids) of an inducible Cas system, such as cell-permeable peptides and nanocarriers. , can be provided to cells. See also US Patent Application Publication No. 2011 / 0035836, published February 10, 2011, and European Patent Application Publication No. 2821486A1, published January 7, 2015.
[0657] Other methods of introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts can be used, such as plastid transformation, and methods of introducing polynucleotides into tissues from seedlings or mature seeds.
[0658] Stable transformation is intended to mean that a nucleotide construct introduced into an organism is integrated into the organism's genome and can be passed on to its progeny. Transient transformation is intended to mean that either the polynucleotide is introduced into the organism but does not integrate into the organism's genome, or the polypeptide is introduced into the organism. Transient transformation means that the introduced composition is expressed or present only transiently within the organism.
[0659] A variety of methods are available to identify those cells with altered genomes at or near target sites without the use of screening marker phenotypes. Such methods can be considered direct analysis of the target sequence to detect alterations in the target sequence and include, but are not limited to, PCR, sequencing, nuclease digestion, Southern blotting and Any combination of these is included.
[0660] cells and plants Polynucleotides and polypeptides of the present disclosure can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, mammalian, bacterial, fungal, insect, yeast, non-conventional yeast and plant cells, and those produced by the methods described herein. plants and seeds. Monocotyledonous and dicotyledonous plants, as well as any plant containing plant elements, can be used with the compositions and methods described herein.
[0661] Examples of monocotyledonous plants that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum. (Sorghum bicolor, Sorghum vulgare), barnyard (e.g. pearl millet, Pennisetum glaucum), millet (Panicum miliaceum), millet (Setaria italica) Setaria italica), finger millet (Eleusine coracana), wheat (Triticum species, e.g. Triticum aestivum, Triticum monococcum), sugarcane (Saccharum species) species (Saccharum spp.)), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapples (Ananas comosus), bananas (Musa spp.), palms, ornamental plants, turfgrass and other grasses.
[0662] Examples of dicotyledonous plants that can be used include, but are not limited to, soybean (Glycine max), Brassica species (e.g., but not limited to, rapeseed or canola) (Brassica Brassica napus, B. campestris, Brassica rapa, Brassica juncea), Alfalfa (Medicago sativa), Tobacco (Nicotiana tabacum) (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum, Gossypium barbadense), and peanut ( Arachis hypogaea), tomatoes (Solanum lycopersicum), potatoes (Solanum tuberosum).
[0663] Further plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea sp. Coffea spp.), coconut (Cocos nucifera), citrus (Citrus spp.), cocoa (Theobroma cacao), tea tree (Camellia sinensis), banana (Musa species), Avocado (Persea americana), Fig (Ficus casica), Guava (Psidium guajava), Mango (Mangifera indica) )), Olive (Olea europaea), Papaya (Carica papaya), Cashew (Anacardium occidentale), Macadamia (Macadamia integrifolia), Almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamental plants, and conifers.
[0664] Vegetables that can be used include tomatoes (Lycopersicon esculentum), lettuce (e.g. lettuce (Lactuca sativa)), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas. (Lathyrus sp.), and cucumber (C. sativus), cantaloupe (C. cantalupensis), and muskmelon (C. melo), etc. members of the genus Cucumis. Ornamental plants include azaleas (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa sp.), Daffodils (Narcissus sp.), Petunias (Petunia hybrida), Carnations (Dianthus caryophyllus), Poinsettia (Euphorbia pulcherrima) pulcherrima)) and chrysanthemums.
[0665] Conifers that can be used include lodge pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus elliotii). pines such as Pinus contorta, and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); American hemlock (Tsuga canadensis); Picea glauca); redwoods (Sequoia sempervirens); Sakhalin firs, such as European fir (Abies amabilis) and balsam fir (Abies balsamea); (Thuja plicata) and Alaskan Yellow Cedar (Chamaecyparis nootkatensis).
[0666] In certain embodiments of the disclosure, a fertile plant is a plant that produces viable male and female gametes and is a self-fertile plant. Such self-fertile plants are capable of producing progeny plants without the contribution of the gametes of other plants or the genetic material contained therein. Other embodiments of the present disclosure may use plants that are not self-fertile because the plants do not produce viable or otherwise fertile male gametes or female gametes or both.
[0667] The disclosure finds use in breeding plants containing one or more introduced traits.
[0668] A non-limiting example of how two traits can be stacked in the genome at a genetic distance of, for example, 5 cM from each other is described as follows: integration into the first DSB target site within the genome window A first plant containing the first transgenic target site and without the first genomic locus of interest is transgenic to a second transgenic plant containing the genomic locus of interest at a different genomic insertion site within the genomic window. Crossbreed with plants. The second plant does not contain the first transgenic target site. Approximately 5% of the plant progeny from this cross have the first transgenic target site integrated at the first DSB target site and the first genomic locus of interest integrated at a different genomic insertion site within the genomic window. have both Progeny plants with both sites within the defined genomic window were selected from the second transgenic target site integrated into the second DSB target site and / or the second genomic locus of interest within the defined genomic window. and lacking the first transgenic target site and the first genomic locus of interest. After this, progeny are selected that have the first transgenic target site, the first genomic locus of interest, and the second genomic locus of interest integrated at a different genomic insertion site within the genomic window. Using such methods, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, Transgenic target sites integrated into 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more DSB target sites and / or purposes integrated into different sites within the genomic window Transgenic plants can be produced that contain multiple trait loci with the genomic loci. In this way, a variety of complex trait loci can be generated.
[0669] cells and animals Polynucleotides and polypeptides of the disclosure can be introduced into animal cells. Animal cells include, but are not limited to, organisms of the phylum including chordates, arthropods, mollusks, annelids, cnidarians, or echinoderms; or mammals, insects, birds, amphibians, reptiles, Or organisms of classes including fishes. In some embodiments, the animal is human, mouse, C. elegans, rat, fruit fly (Drosophila sp.), zebrafish, chicken, dog, cat, guinea pig, hamster, chicken, chicken , killifish, sea lampreys, blowfish, tree frogs (eg, Xenopus species), monkeys, or chimpanzees. 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. cells, osteocytes, germ cells, somatic cells, stem cells, pluripotent stem cells, induced pluripotent stem cells, progenitor cells, meiotic cells, and mitotic cells. In some embodiments, multiple cells from an organism can be used.
[0670] The disclosed novel Cas9 orthologs can be used to edit the genome of animal cells in a variety of ways. In one aspect, it may be desirable to delete one or more nucleotides. In another aspect, it may be desirable to insert one or more nucleotides. In one aspect, it may be desirable to substitute one or more nucleotides. In another aspect, it may be desirable to modify one or more nucleotides by covalent or non-covalent interaction with another atom or molecule.
[0671] Genome modification by Cas9 orthologs can be used to effect genotypic and / or phenotypic changes in target organisms. Such alterations are preferably associated with improvement of a desired phenotype or physiologically important trait, correction of an intrinsic defect, or expression of some type of expression marker. In some embodiments, the desired phenotype or physiologically important trait is the animal's overall health, fitness or fertility, the animal's ecological fitness, or the relationship between the animal and other organisms in its environment. related to the relationship or interaction of In some embodiments, the desired phenotype or physiologically important trait is selected from the following group: overall health improvement, disease reversal, disease amelioration, disease stabilization, disease prevention, parasites Treatment of infections, treatment of viral infections, treatment of retroviral infections, treatment of bacterial infections, treatment of neurological disorders (e.g., but not limited to multiple sclerosis), correction of endogenous genetic defects ( For example, but not limited to, metabolic disorders, achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Barth syndrome, breast cancer, Charcot-Marie-Tooth disease, colorectal cancer, catless syndrome, Crohn's disease, cellular fibrosis, Durham disease, Down's syndrome, Duchenne syndrome, Duchenne muscular dystrophy, factor V Leiden thrombotic predisposition, familial hypercholesterolemia, familial Mediterranean fever, Fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter's syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland syndrome, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, skin cancer, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner's syndrome, palatiocardiofacial syndrome, WAGR syndrome, and Wilson's disease), treatment of congenital immune disorders (e.g., but not limited to immunoglobulin subclass deficiency), acquired immune disorders (e.g., but not limited to, AIDS and other HIV-related disorders), cancer, and disease, including rare or "orphan" conditions, for which there are no other effective treatment options. treatment.
[0672] Cells genetically engineered using the compositions or methods disclosed herein may be used for purposes such as gene therapy, e.g., to treat disease, or for antiviral, antipathogenic, or anticancer purposes. As therapeutic agents, they can be implanted into subjects for the production of genetically engineered organisms in agriculture or for biological research.
[0673] Detection, binding and modification of polynucleotides in vitro The compositions disclosed herein can further be used as compositions for use in in vitro methods in some embodiments involving isolated polynucleotide sequences. The isolated polynucleotide sequence may contain one or more target sequences for modification. In some aspects, the isolated polynucleotide sequence can be genomic DNA, a PCR product, or a synthesized oligonucleotide.
[0674] Composition Modifications of the target sequence can be in the form of nucleotide insertions, nucleotide deletions, nucleotide substitutions, addition of atomic molecules to existing nucleotides, nucleotide modifications, or binding of heterologous polynucleotides or polypeptides to said target sequence. Insertion of one or more nucleotides can be accomplished by including a donor polynucleotide in the reaction mixture, said donor polynucleotide inserting into the double-stranded break produced by said Cas-alpha ortholog polypeptide. . Insertions may be by non-homologous end joining or by homologous recombination.
[0675] In one aspect, the sequence of the target polynucleotide is known prior to modification and compared to the sequence of the polynucleotide obtained from treatment with the Cas-alpha ortholog. In one aspect, the sequence of the target polynucleotide is unknown prior to modification and treatment with Cas-alpha orthologues is used as part of the method to determine the sequence of said target polynucleotide.
[0676] Polynucleotide modifications with Cas-alpha orthologs can be accomplished by using full-length polypeptides identified from the Cas locus, or fragments, modifications, or variants of polypeptides identified from the Cas locus. In some embodiments, the Cas-alpha ortholog is obtained or derived from an organism listed in Table 1. In some embodiments, the Cas-alpha ortholog is a polypeptide sharing at least 80% identity with any of SEQ ID NOs:86-170 or 511-1135. In some embodiments, said Cas-alpha ortholog is a functional variant of any of SEQ ID NOs:86-170 or 511-1135. In some embodiments, the Cas-alpha ortholog is a functional fragment of any of SEQ ID NOs:86-170 or 511-1135. In some embodiments, the Cas-alpha ortholog is a Cas-alpha polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs:86-170 or 511-1135. In some embodiments, the Cas-alpha ortholog is a Cas-alpha polypeptide that recognizes a PAM sequence listed in any of Tables 4-83. In some embodiments, the Cas-alpha ortholog is a Cas-alpha polypeptide identified from an organism listed in the sequence listing.
[0677] In some aspects, Cas-alpha orthologs are provided as Cas-alpha polynucleotides. In some embodiments, the Cas-alpha polynucleotide is at least 80%, 85%, 90%, 95%, 97%, 99%, Or selected from the group consisting of sequences that share 100%.
[0678] In some embodiments, the Cas-alpha ortholog is an unmodified wild-type Cas-alpha ortholog, a functional Cas-alpha ortholog variant, a functional Cas-alpha ortholog fragment, a fusion protein comprising an active or inactivated Cas-alpha ortholog; Cas-alpha orthologs, biotinylated Cas-alpha orthologs, Cas-alpha ortholog nickases further comprising one or more nuclear localization sequences (NLS) at the C-terminus or N-terminus or at both the N-terminus and C-terminus , a Cas-alpha ortholog endonuclease, a Cas-alpha ortholog further comprising a histidine tag, and mixtures of any two or more thereof.
[0679] In some embodiments, the Cas-alpha ortholog is a nuclease domain, transcriptional activator domain, transcriptional repressor domain, epigenetic modification domain, cleavage domain, nuclear localization signal, cell permeation domain, translocation domain, marker, or Fusion proteins further comprising a transgene that is heterologous to the target polynucleotide sequence or the cell from which said target polynucleotide sequence is obtained or derived.
[0680] In some embodiments, multiple Cas-alpha orthologs may be desired. In some embodiments, the plurality of Cas-alpha orthologs may include Cas-alpha orthologs from different biological sources or from different genetic loci within the same organism. In some embodiments, the plurality of Cas-alpha orthologs may comprise Cas-alpha orthologs that have different binding specificities for target polynucleotides. In some embodiments, the plurality of Cas-alpha orthologs may comprise Cas-alpha orthologs with different cleavage efficiencies. In some embodiments, the plurality of Cas-alpha orthologs may comprise Cas-alpha orthologs with different PAM properties. In some embodiments, the plurality of Cas-alpha orthologs may comprise orthologs of different molecular composition, ie, polynucleotide Cas-alpha orthologs and polypeptide Cas-alpha orthologs.
[0681] A guide polynucleotide can be provided as a single guide RNA (sgRNA), a chimeric molecule comprising tracrRNA, a chimeric molecule comprising crRNA, a chimeric RNA-DNA molecule, a DNA molecule, or a polynucleotide comprising one or more chemically modified nucleotides.
[0682] Storage conditions for Cas-alpha orthologs and / or guide polynucleotides include parameters relating to temperature, state of matter, and time. In some embodiments, Cas-alpha orthologs and / or guide polynucleotides are stored at about -80°C, about -20°C, about 4°C, about 20-25°C, or about 37°C. In some embodiments, Cas-alpha orthologs and / or guide polynucleotides are stored as liquids, frozen liquids, or lyophilized powders. In some embodiments, the Cas-alpha ortholog and / or guide polynucleotide is stable for at least 1 day, at least 1 week, at least 1 month, at least 1 year, or more than 1 year.
[0683] Any or all of the reactive polynucleotide components (e.g., guide polynucleotides, donor polynucleotides, optionally Cas-alpha polynucleotides) may be part of a vector, construct, linearized or circularized plasmid , or as part of a chimeric molecule. Each component can be fed to the reaction mixture separately or together. In some embodiments, one or more polynucleotide components are operably linked to heterologous non-coding regulatory elements that regulate their expression.
[0684] A method of modifying a target polynucleotide involves combining minimal elements to form a Cas-alpha orthologue (or variant, fragment, or other related molecule as described above), substantially complementary to the target polynucleotide sequence of the target polynucleotide. or a reaction mixture containing a guide polynucleotide containing a sequence that selectively hybridizes, and a target polynucleotide for modification. In some aspects, Cas-alpha orthologs are provided as polypeptides. In some aspects, Cas-alpha orthologs are provided as Cas-alpha polynucleotides. In some embodiments, guide polynucleotides are provided as RNA molecules, DNA molecules, RNA:DNA hybrids, or polynucleotide molecules comprising chemically modified nucleotides.
[0685] Storage buffers for any one of the components, or reaction mixtures, can be optimized for stability, efficacy, or other parameters. Further components of storage buffers or reaction mixtures are the buffer composition, Tris, EDTA, dithiothreitol (DTT), phosphate buffered saline (PBS), sodium chloride, magnesium chloride, HEPES, glycerol, BSA, salts, Emulsifiers, detergents, chelating agents, redox agents, antibodies, nuclease-free water, proteinases, and / or viscosity agents may be included. In some embodiments, the storage buffer or reaction mixture further comprises a buffer comprising at least one of the following components: HEPES, MgCl2, NaCl, EDTA, proteinase, proteinase K, glycerol, nuclease-free water.
[0686] Incubation conditions vary according to the desired results. The temperature is preferably at least 10°C, 10-15°C, at least 15°C, 15-17°C, at least 17°C, 17-20°C, at least 20°C, 20-22°C, at least 22°C, 22-25°C. , at least 25°C, 25-27°C, at least 27°C, 27-30°C, at least 30°C, 30-32°C, at least 32°C, 32-35°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, or greater than 40°C. 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 more than 10 minutes. .
[0687] The sequence of polynucleotides in a reaction mixture before, during, or after incubation can be determined by methods known in the art. In one aspect, modification of the target polynucleotide can be confirmed by comparing the sequence of the polynucleotide purified from the reaction mixture to the sequence of the target polynucleotide prior to combining with the Cas-alpha ortholog.
[0688] Any one or more of the compositions disclosed herein useful for polynucleotide detection, binding and / or modification in vitro or in vivo can be included in a kit. A kit comprises a Cas-alpha ortholog or a polynucleotide Cas-alpha ortholog encoding such, optionally a buffer component to allow for efficient storage, and said Cas-alpha ortholog or polynucleotide Cas-alpha ortholog. - further comprising one or more additional compositions that allow introduction of an alpha ortholog into a heterologous polynucleotide, wherein said Cas-alpha ortholog or polynucleotide Cas-alpha ortholog is Nucleotide modifications, additions, deletions, or substitutions can result. In a further aspect, the Cas-alpha orthologs disclosed herein can be used for enrichment of one or more polynucleotide target sequences from a mixed pool. In a further aspect, the Cas-alpha orthologs disclosed herein can be immobilized on a matrix for use in detecting, binding, and / or modifying target polynucleotides in vitro.
[0689] The Cas-alpha endonuclease can be attached, associated, or immobilized to a solid matrix for storage, purification, and / or characterization purposes. Examples of solid matrices include, but are not limited to, filters, chromatography resins, assay plates, test tubes, cryogenic vials, and the like. The Cas-alpha endonuclease can be substantially purified and stored in a suitable buffered solution or lyophilized.
[0690] Detection method Methods for detecting Cas-alpha:guide polynucleotide complexes bound to target polynucleotides include any method known in the art, including, but not limited to, microscopy, chromatographic separation, electrophoresis, Immunoprecipitation, filtration, nanopore separations, microarrays, and those described below.
[0691] DNA electrophoretic mobility shift assays (EMSA) examine proteins binding to known DNA oligonucleotide probes to assess the specificity of the interaction. This 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. This assay is also called a gel retardation assay because the migration rate of DNA is slowed when bound to proteins. Addition of a protein-specific antibody to the binding component produces a larger complex (antibody-protein-DNA) that migrates more slowly during electrophoresis, known as supershift, and is used to determine protein identity. can be used to check the
[0692] DNA pull-down assays use a DNA probe labeled with a high affinity tag such as biotin to retrieve or immobilize the probe. DNA probes can be complexed with proteins from cell lysates in reactions similar to those used in EMSA, and then used to purify the complexes using agarose or magnetic beads. Proteins are then eluted from the DNA and detected by Western blot or identified by mass spectrometry. Alternatively, proteins can be labeled with affinity tags, or DNA-protein complexes can be isolated using antibodies against the protein of interest (similar to supershift assays). In this case, the unknown DNA sequence bound to the protein is detected by Southern blotting or PCR analysis.
[0693] Reporter assays provide an in vivo real-time readout of the translational activity of a promoter of interest. A reporter gene is a fusion of a target promoter DNA sequence and a reporter gene DNA sequence customized by the researcher, the DNA sequence identifying proteins with detectable properties such as firefly / renilla luciferase or alkaline phosphatase. code. These genes produce enzymes only when the promoter of interest is activated. The enzyme then catalyzes the substrate to produce a light or color change that can be detected by spectroscopy. Signals from reporter genes are used as indirect determinants of translation of endogenous proteins driven from the same promoter.
[0694] Microplate capture and detection assays use immobilized DNA probes to capture specific protein-DNA interactions and confirm protein identity and relative abundance with target-specific antibodies. Typically, DNA probes are immobilized on the surface of streptavidin-coated 96-well or 384-well microplates. A cell extract is prepared and added to bind the binding protein to the oligonucleotide. The extract is then removed and each well washed several times to remove non-specifically bound proteins. Finally, the protein is detected using a specific antibody labeled for detection. This method is extremely sensitive and can detect less than 0.2 pg of target protein per well. This method can also be applied to oligonucleotides labeled with other tags, such as primary amines, which can be immobilized on microplates coated with amino group-reactive surface chemistries.
[0695] DNA footprinting is one of the most widely used methods to obtain detailed information about individual nucleotides in protein-DNA complexes, even in living cells. In such methods, chemicals or enzymes are used to modify or digest DNA molecules. When sequence-specific proteins bind to DNA, they can protect the binding site from modification or digestion. This can then be visualized by denaturing gel electrophoresis, where unprotected DNA is roughly randomly cleaved. Thus, it appears as a 'ladder' of bands, and protein-protected sites appear like footprints in the pattern of bands without corresponding bands. Their footprints thus identify specific nucleosides in protein-DNA binding sites.
[0696] Microscopy techniques include optical microscopy, fluorescence microscopy, electron microscopy, and atomic force microscopy (AFM).
[0697] Chromatin Immunoprecipitation Assay (ChIP) covalently binds proteins to their DNA targets, then unbinds them and characterizes them separately.
[0698] Systematic evolution of ligands by selection in vitro (SELEX) exposes target proteins to random libraries of oligonucleotides. Those genes that bind are isolated and amplified by PCR.
[0699] The methods and compositions provided herein include, but are not limited to the following aspects.
[0700] Embodiment 1: (a) a guide polynucleotide; (b) a C-terminal three-split RuvC domain further comprising a bridge helix and at least one zinc finger domain, an alpha-helical bundle, and a Cas comprising multiple beta-sheets forming a wedge-shaped domain an endonuclease, wherein the Cas endonuclease is less than 650 amino acids in length; and (c) a target sequence comprising a nucleotide sequence that shares complementarity with the guide polynucleotide, wherein the guide polynucleotide and the Cas endonuclease are A synthetic composition that forms a complex that cleaves a double-stranded DNA polynucleotide containing a target sequence.
[0701] Embodiment 2: (a) guide polynucleotide; (b) Archaea, Micrarchaeota, Acidibacillus sulfuroxidans, Candidatus Micrarchaeota archaeon, Clostridium to a class of organisms selected from the group consisting of Clostridium novyi, Parageobacillus thermoglucosidasius, Ruminococcus species, and Syntrophomonas palmitatica a Cas endonuclease from which the Cas endonuclease forms a complex with the guide polynucleotide; and (c) a double-stranded DNA polynucleotide comprising a target sequence that binds to the guide polynucleotide, the guide polynucleotide and A synthetic composition, wherein the Cas endonuclease forms a complex that cleaves a double-stranded DNA polynucleotide containing a target sequence.
[0702] Aspect 3: The synthetic composition of Aspect 1 or Aspect 2, wherein the Cas endonuclease further comprises a zinc zinc finger domain near the N-terminus.
[0703] Aspect 4: The synthetic composition of aspect 1 or aspect 2, wherein the double-stranded DNA polynucleotide further comprises PAM.
[0704] Embodiment 5: The synthetic composition of embodiment 4, wherein the PAM comprises multiple thymine nucleotides.
[0705] Embodiment 6: The synthetic composition of embodiment 1 or embodiment 2, further comprising a heterologous polynucleotide.
[0706] Aspect 7: The synthetic composition of aspect 1 or aspect 2, wherein the guide polynucleotide comprises a 20 nucleotide region of complementarity to the target sequence.
[0707] Embodiment 8: The synthetic composition of embodiment 1 or embodiment 2, wherein the guide polynucleotide is a double-stranded molecule comprising tracrRNA and crRNA.
[0708] Embodiment 9: The synthetic composition of embodiment 1 or embodiment 2, wherein the guide polynucleotide is a single guide polynucleotide comprising a Cas endonuclease recognition domain and a variable targeting domain.
[0709] Embodiment 10: The synthetic composition of embodiment 6, wherein the heterologous polynucleotide is an expression element.
[0710] Embodiment 11: The synthetic composition of embodiment 6, wherein the heterologous polynucleotide is a transgene.
[0711] Embodiment 12: The synthetic composition of embodiment 6, wherein the heterologous polynucleotide is a donor DNA molecule.
[0712] Embodiment 13: The synthetic composition of embodiment 6, wherein the heterologous polynucleotide is a modified polynucleotide template.
[0713] Embodiment 14: The synthetic composition of embodiment 1 or embodiment 2, wherein the CRISPR-Cas endonuclease further comprises a nuclear localization signal.
[0714] Embodiment 15: The synthetic composition of embodiment 1 or embodiment 2, wherein the CRISPR-Cas endonuclease is Cas-alpha or a functional fragment thereof.
[0715] Embodiment 16: The synthetic composition of embodiment 1 or embodiment 2, wherein the CRISPR-Cas endonuclease is catalytically inactive Cas-alpha.
[0716] Embodiment 17: A synthetic composition according to embodiment 1 or embodiment 2, wherein the CRISPR-Cas endonuclease is a fusion protein comprising a functional fragment of Cas-alpha.
[0717] Embodiment 18: The synthetic composition of embodiment 17, wherein the fusion protein further comprises another nuclease domain.
[0718] Embodiment 19: The synthetic composition of embodiment 1 or embodiment 2, further comprising at least one additional polypeptide.
[0719] Aspect 20: The synthetic composition of aspect 19, wherein the additional polypeptide is selected from the group consisting of Casl, Cas2, and Cas4:.
[0720] Aspect 21: The synthetic composition of Aspect 1 or Aspect 2, further comprising a cell.
[0721] Embodiment 22: The synthetic composition of embodiment 21, wherein the cell is a eukaryotic cell.
[0722] Embodiment 23: The synthetic composition of Embodiment 21, wherein the cells are plant cells.
[0723] Embodiment 24: A synthetic composition according to embodiment 23, wherein the plant cell is a monocotyledonous plant cell or a dicotyledonous plant cell.
[0724] Embodiment 25: The plant cell is corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis ( 24. The synthetic composition according to aspect 23, which is derived from an organism selected from the group consisting of: Arabidopsis), safflower, and tomato.
[0725] Embodiment 26: The synthetic composition of embodiment 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.
[0726] Embodiment 27: A polynucleotide encoding the synthetic composition of Embodiment 1 or Embodiment 2.
[0727] Embodiment 28: The polynucleotide of embodiment 27, further comprising at least one additional polynucleotide.
[0728] Embodiment 29: A polynucleotide according to embodiment 28, wherein the at least one further polynucleotide is an expression element.
[0729] Embodiment 30: A polynucleotide according to embodiment 28, wherein the at least one further polynucleotide is a gene.
[0730] Embodiment 31: The synthetic composition of embodiment 30, wherein the gene is selected from the group consisting of Casl, Cas2, and Cas4.
[0731] Embodiment 32: The polynucleotide of embodiment 28, wherein the at least one polynucleotide is contained within a recombinant construct.
[0732] Embodiment 33: The synthetic composition of embodiment 1 or embodiment 2, wherein at least one component is attached to a solid matrix.
[0733] Embodiment 34: A target double-stranded DNA polynucleotide, a guide polynucleotide that is complementary to a sequence in the double-stranded DNA polynucleotide, and SEQ ID NOs: 17, 18, 19, 20, 32, 33, 34, 35, 36, A synthetic composition comprising a Cas endonuclease that is at least 80% identical to a sequence selected from the group consisting of 37, and 38, or a functional fragment or variant thereof.
[0734] Embodiment 35: A target double-stranded DNA polynucleotide, a polynucleotide encoding a guide polynucleotide that is complementary to a sequence in the double-stranded DNA polynucleotide, and SEQ ID NOs: 13, 14, 15, 16, 25, 26, 27 A synthetic composition comprising a cas endonuclease gene, or a functional fragment or variant thereof, that is at least 80% identical to a sequence selected from the group consisting of: , 28, 29, 30, and 31.
[0735] Embodiment 36: A method for introducing a site-specific modification into a target sequence in the genome of a cell, comprising introducing into the cell the synthetic composition of any of embodiments 1-35.
[0736] Embodiment 37: A method of producing an organism with a modified genome, comprising: (a) i. a Cas-alpha endonuclease or a cas-alpha polynucleotide encoding a Cas-alpha endonuclease; A guide polynucleotide comprising a variable targeting domain substantially complementary to the target sequence, wherein the guide polynucleotide and the Cas-alpha endonuclease are capable of recognizing, binding, and optionally nicking or cleaving the target sequence. and iii. a polynucleotide modification template comprising at least one region complementary to the PAM sequence flanking the DNA target sequence recognized by the Cas-alpha complex (wherein (b) incubating the cells; generating a whole organism from the cells; and (d) prior to introduction of the heterologous composition of (a), at least one nucleotide in the genome of at least one cell of the organism as compared to a target sequence in the genome of the cell. A method that includes verifying that there is a qualification.
[0737] Aspect 38: A method according to aspect 36 or 37, wherein the cell is a eukaryotic cell.
[0738] Aspect 39: A method according to aspect 38, wherein the eukaryotic cell is derived from or obtained from an animal or plant.
[0739] Aspect 40: A method according to aspect 39, wherein the plant is a monocotyledonous plant or a dicotyledonous plant.
[0740] Embodiment 41: The plant is corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis ), safflower, and tomato.
[0741] Aspect 42: A method according to aspects 36 or 37, further comprising introducing a heterologous polynucleotide.
[0742] Embodiment 43: The method of embodiment 42, wherein the heterologous polynucleotide is a donor DNA molecule.
[0743] Embodiment 44: The method of embodiment 42, wherein the heterologous polynucleotide is a polynucleotide modified template comprising a sequence that is at least 50% identical to that of the cell.
[0744] Embodiment 45: A progeny of an organism obtained by the method of embodiment 37, the progeny retaining at least one nucleotide modification in at least one cell.
[0745] Embodiment 46: A method of modifying the genomic sequence of a target cell, wherein the genomic sequence is at least 95%-100% identical to one of SEQ ID NOS: 17, 18, 19, 20, 32, 33, 34, 35, 36, 37 and 38. providing a Cas endonuclease comprising an amino acid sequence and a guide polynucleotide that targets the genomic sequence of the target cell; a method comprising modifying the
[0746] Although the invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the relevant art will recognize that modifications and variations can be made without departing from the spirit and scope of the invention. It will be appreciated that various changes in detail may be made therein. For example, although the specific examples below may illustrate the methods and embodiments described herein using particular target sites or target organisms, the principles in these examples apply to any target site or It can be applied to target organisms. It will therefore be understood that the scope of the invention is encompassed by the embodiments of the invention enumerated herein rather than by the specific examples illustrated below. All cited patents, applications, and publications referenced in this application are hereby incorporated by reference in their entirety for all purposes to the same extent that each was individually and specifically incorporated by reference. incorporated into the specification.
Example
[0747] Below are examples of specific embodiments of some aspects of the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but, of course, some experimental errors and deviations should be allowed for.
[0748] Example 1: Identification and characterization of a novel class of Cas-alpha CRISPR-Cas system This example describes a method to identify novel Class 2 CRISPR (Clustered Regularly Arranged Short Palindromic Repeats)-Cas (CRISPR-associated) loci using operon-like gene structure identification and protein structure analysis. .
[0749] First, CRISPR arrays were analyzed using the software programs PILER-CR (Edgar, R. (2007) BMC Bioinformatics, 8:18) and MinCED (Bland, C. et al. (2007) BMC Bioinformatics, 8:209). was detected within the microbial sequence. Known CRISPR-Cas systems were then tested at site-specific genes encompassing all known Cas protein families, as described in Makarova, K. et al. (2015) Nature Reviews Microbiology, 13:722-736. Proteins encoded near the CRISPR array (20 kb 5' and 20 kb 3' (where possible)) for homology to known CRISPR-associated (Cas) proteins using a set of statistical scoring matrices (PSSM). was removed from the dataset by searching for Class 2 CRISPR-Cas endonucleases (e.g., Cas9, Cpf1 (Cas12a), C2c1 (Cas12b), C2c2 (Cas13), C2c3 (Cas12c)) to aid in complete removal of known class 2 CRISPR-Cas systems A multiple sequence alignment of protein sequences from collections of orthologs of each family of . was performed using MUSCLE (Edgar R. (2004) Nucleic Acids Res. 32:1792-1797). Alignments were examined, curated and profiled hidden Markov models (HMM) using HMMER (Eddy, S.R. (1998) Bioinformatics.14:755-763; Eddy, S.R. (2011) PLoS Comp.Biol.,7:e1002195)). used to build the The resulting HMM model was then utilized to further identify and remove known class 2 CRISPR-Cas systems from the dataset. Using PSSM-specific searches as described above, the remaining CRISPR loci were then assessed for the presence of genes encoding Cas1 and Cas2, proteins thought to be important for spacer insertion and adaptation (Makarova, K. et al. 2015, Nature Reviews Microbiology 13:722-736). A CRISPR locus containing the cas1 and cas2 genes was then selected and further investigated to determine the proximity, order and orientation of the cas1 and cas2 genes and the locus-encoded unidentified genes to the CRISPR array. Further analysis was performed by selecting only those CRISPR loci where large (≧1500 bp open reading frames) unidentified genes were adjacent to the cas1 and cas2 genes and formed an operon-like structure lying in the same transcriptional direction. Proteins encoded by unidentified genes were then analyzed for sequence and structural features indicative of a class 2 endonuclease capable of cleaving DNA. First, depending on the degree of similarity between a candidate sequence and a known protein, its conserved functional features can be identified using various bioinformatics tools, from pairwise comparisons to family profile searches, structural threading, and bioinformatics. A manual structural survey revealed this. Generally, homologous sequences of new candidate proteins are first subjected to PSI-BLAST (Altschul, SF. et al.) against the National Center for Biotechnology Information (NCBI) non-redundant (NR) protein collection. (1997) Nucleic Acids Res. 25:3389-3402) searches were collected with a cutoff e value of 0.01. After reducing redundancy at an identity level of about 90%, a multiple sequence alignment tool that aligns groups of homologous sequences with various member inclusion thresholds (e.g., greater than 60, 40, or 20% identity), MSAPRobs (Liu, Y. et al. (2010) Bioinformatics. 26:1958-1964) and Clustalw revealed conserved motifs. The most conserved homologous sequences are identified by HMMER (Eddy, S.R. (1998) Bioinformatics. 14:755-763) as Pfam, Superfamily and SCOP (Murzin, A.G. et al. (1995) J. Mol. Biol. 247:536). -540), as well as in-house structure-based profiles were subjected to sequence versus family profile searches. Alternatively, the candidate homologous sequence alignments obtained were also used to generate candidate protein profiles with the predicted secondary structures added. Candidate profiles were also used to perform a profile-profile search against the 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 are either threaded into a 3D structure template in MODELLER or manually into known structure references on DiscoveryStudio (BIOVIA) and Pymol (Schrodinger). mapped with Finally, to validate and confirm its potential biological relevance as a class 2 endonuclease, we manually examined the catalytic or most conserved residues and critical structural integrity of the protein. evaluated in light of biochemical function. After structural identification of key features indicative of class 2 endonucleases (e.g., the DNA cleavage domain), we then determined intralocus (5 kb 5' and 5 kb 3' from the end of the newly defined CRISPR-Cas system (possible case)) using InterProScan software (EMBL-EBI, UK) and by the BLAST program (Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410). Homology to known protein families was determined by comparison to the NCBI NR protein collection. Genes encoding proteins with similarity (at least 30% identity) to known proteins were annotated as such in the CRISPR-Cas locus.
[0750] Initially, four novel class 2 CRISPR-Cas systems were identified from unknown microorganisms (Table 1). As shown in Figures 1A and 1B, each locus encoded a complete CRISPR-Cas system containing all the components necessary for acquisition and interference. These include all proteins required for the acquisition and integration of spacers (Cas1, Cas2, and optionally Cas4), as well as a novel protein, Cas-alpha ( The gene co-encoding α) was included.
[0751]
table 1
[0752] We then compared the Cas-alpha endonuclease to the NCBI NR protein collection using BLAST, followed by analysis by MinCED, which found proteins in the vicinity (≤5 kb) of the CRISPR array, and seven additional CRISPR systems. was obtained (Table 2). The genetic structure of the loci revealed for these new proteins is shown in Figures 1C and 1D. The locus encoding Cas-alpha6 contains the complete cas2 and cas4 genes in addition to the partial cas1 gene (Fig. 1C), whereas Cas-alpha5, 7, 8, 9, 10, and 11 , contained only the endonuclease gene flanking the CRISPR array (Fig. 1D). The loci of Cas-alpha 18 and 19 are shown in Figure 21A and the mechanism of action is shown in Figure 21B.
[0753] [Table 2]
[0754] Structural examination of these proteins revealed that they differ from previously described class 2 CRISPR-Cas endonucleases capable of recognizing and cleaving double-stranded DNA targets. First, the endonuclease size (422-613 amino acids) was remarkably compact compared to other known class 2 CRISPR-Cas systems. Second, the first amino (N)-terminal half of the protein was highly variable in sequence composition as evidenced by the lack of conservation of even a single amino acid (except the initiating methionine). Despite this, secondary structure predictions (PSIPRED (Jones, J.T. (1999) J. Mol. Biol. 292:195-202)) show a mixture of beta strands and alpha helices, with all Cas-alpha It was suggested that a wedge-shaped (WED) or oligonucleotide binding domain (OBD) structure and helical bundles are present in the N-terminal region of the protein. In the carboxyl (C)-terminal half of the protein, the structure was conserved, including key catalytic residues and a tripartite RuvC domain (Fig. 2). In addition, all proteins contained a bridge helix domain and a zinc finger domain inserted between I-II and II-III of the RuvC subdomain, respectively (Fig. 2). Of note, additional zinc finger-like motifs were detected in Cas-alpha-1, 2, 3, 4, and 10 proteins. In Cas-alpha-1, 2, 3, and 4, a second zinc finger motif is located near the N-terminus (e.g., amino acid positions 70-96 and 63-111 of Cas-alpha-1 and 2, respectively) ( 8A-8D), while in Cas-alpha-10 two additional zinc finger motifs were identified in the C-terminal half of the protein (FIG. 8J). Here one of the additional zinc finger domains is located in tandem with the first between the RuvC II and RuvC III subdomains (Cas-alpha-10 amino acid positions 376-422) and the third was found after RuvC subdomain III (Cas-alpha-10 amino acid positions 466-482) (Fig. 8J). Examples of recovered Cas-alpha sequences and motifs are shown in Figures 8A-8K for Cas-alpha 1-11, respectively. Figure 9 shows how some of the Cas-alpha domains interact with the hybrid double-stranded target DNA / guide RNA, using the Cas12b (C2c1) protein backbone (PDB: 5wti) as a reference.
[0755] Sequence analysis of Cas-alpha 1-129, aligned with the MUSCLE multiple sequence alignment, revealed a unique motif characteristic of Cas-alpha endonucleases for amino acid position numbers in SEQ ID NO: 17 (Table: 337). glycine (G) at position 341, glycine (G) at position 430, glutamic acid (E) at position 430, leucine (L) at position 432, cysteine (C) at position 487, cysteine (C) at position 490 and / or 507 Cysteine (C) at position Cas-alpha endonuclease contains the following motifs: GxxxG, ExL, Cx n C, and Cx n (C,H)(where x n = 2-4 residues). Cas-alpha endonucleases contain one or more zinc finger domains. Table 2b contains some of the conserved motifs present in Cas-alpha endonucleases.
[0756] [Table 3]
[0757] [Table 4]
[0758] [Table 5]
[0759] [Table 6]
[0760] Example 2: Cas-alpha guide RNA solution In this example, a novel group of class 2 CRISPR (clustered regularly arranged short palindromic repeats)-Cas (CRISPR-associated) endonucleases, Cas-alpha, guide RNAs that support recognition and cleavage of double-stranded DNA targets. Describe how to determine
[0761] One method utilizes computational prediction to determine the sRNA required to form a functional complex with the Cas-alpha endonuclease. Briefly, CRISPR arrays are used to determine the possible transcriptional orientations of the CRISPR array, as well as various arrangements of repeats and spacers that may be favored by endonucleases (e.g., repeat:spacer, spacer:repeat or repeat:spacer:repeat). ) can be generated. Additionally, transcoding CRISPR-associated RNA (tracrRNA) can be identified at loci computationally as described in Karvelis, T. et al (2015) Genome Biology. 16:253). Briefly, alignments can be performed between CRISPR repeat consensus sequences and locus sequences using BLAST or manually. Then, by analyzing the possible transcriptional orientations of the tracrRNA putative for secondary structure and possible termination signals present in the RNA versions of the sense and antisense genomic DNA sequences surrounding the antirepeats (CRISPR regions of homology (distinct from the array) can be examined. This tracrRNA can then be made double stranded using various crRNA predictions or engineered to form chimeric non-natural single guide RNAs (sgRNAs). crRNA, tracrRNA and sgRNA can be synthesized (IDT equivalent) or T7 transcribed using the TranscriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific) or equivalent for further experiments.
[0762] Another method utilizes sequencing of small RNAs (sRNA-seq) produced from novel Class 2 CRISPR-Cas loci. This can be done similarly to the method described in Zetsche, B. et al. (2015) Cell.163:1-13. Briefly, the CRISPR-Cas locus was placed on E. coli plasmid DNA and subsequent cultures containing the CRISPR-Cas locus on the plasmid were harvested by centrifugation and isolated from TRIzol Max bacteria. Total RNA was extracted using the Isolation Kit (Thermo Fisher Scientific), small RNA was isolated using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific), and TruSeq Small RNA Library Prep Kit (Illumina) was used. prepare the library for sequencing. Locus expression can be enhanced using known E. coli promoters. After sequencing on a MiSeq instrument (Illumina) or equivalent, the resulting sequence data is mapped to loci (Bowtie 2 software (Langmead, B. et al. (2012) Nat. Methods. 9:357-359) or corresponding) to determine the transcription and maturation patterns of the sRNA encoded by the locus.
[0763] Another method utilizes sequencing (sRNA-seq) of small RNA co-purified with Cas-alpha protein from a novel class 2 CRISPR-Cas locus. Sinkunas, T. et al. (2013) EMBO J.32, except that Illumina deep sequencing is used to sequence the small RNAs required to induce recognition and cleavage of double-stranded DNA targets. :385-394. Briefly, the CRISPR-Cas locus is placed on E. coli plasmid DNA. The Cas-alpha gene in the locus can be modified to also encode protein purification tags. Examples include, but are not limited to, histidine (His), streptavidin (Strep), and / or maltose binding protein (MBP) tags. Alternatively, a "single" Cas-alpha expression cassette encoding a His-, Strep-, and / or MBP-tagged version of the Cas-apha protein can be co-transformed with the locus on the plasmid. The plasmid is then transformed into E. coli (eg, but not limited to Artic Express (DE3) (ThermoFisher Scientific)) and the culture is then harvested by centrifugation. Cells are then lysed and the tagged Cas-alpha protein purified by chromatography. Finally, the small RNA bound to the Cas-alpha protein is extracted using the TRIzol Max Bacterial Isolation Kit (Thermo Fisher Scientific) or other suitable method and processed as above.
[0764] crRNA, tracrRNA, and sgRNA solutions are listed in Table 3 for selection of the Cas-alpha system described herein.
[0765] [Table 7]
[0766] Example 3: Bacterial Cas-alpha expression plasmid In this example, plasmid DNA expression constructs are generated to study double-stranded DNA target recognition and cleavage by Cas-alpha in the heterologous host, E. coli.
[0767] First, the CRISPR array of the native Cas-alpha CRISPR-Cas locus (Figure 1) (SEQ ID NO: 21) encoding the first Cas-alpha endonuclease, Cas-alpha 1 (SEQ ID NO: 17), is examined here. Modified. This was achieved by reducing the number of CRISPR units (repeat (SEQ ID NO:46):spacer:repeat (SEQ ID NO:46)) to three. The inter-repeat spacer sequence was then base-paired with the antisense strand of the double-stranded target sequence T2 flanked by a 7 bp randomized region from the plasmid DNA PAM library described in Karvelis et al., 2015. (SEQ ID NO: 78). The resulting 'complete' CRISPR-Cas locus engineered to target T2 (SEQ ID NO...
Claims
1. (a) a Cas endonuclease that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 20, 35, 37, and 38, wherein the Cas endonuclease further comprises: (i) a C-terminal three-split RuvC domain; (ii) the following amino acid motifs: GxxxG, ExL, CxnC, and Cxn(C or H), where G=glycine, E=glutamic acid, C=cysteine, H=histidine, x=any amino acid, and n=an integer from 0 to 11; (iii) an alpha helix, and (iv) multiple beta sheets forming a wedge-shaped domain a Cas endonuclease comprising: (b) a target double-stranded DNA polynucleotide, wherein the target double-stranded DNA polynucleotide is heterologous to the Cas endonuclease source; and (c) a guide polynucleotide comprising a variable targeting domain that comprises a region complementary to the target double-stranded DNA polynucleotide. Includes: The Cas endonuclease recognizes a PAM sequence on the target double-stranded DNA polynucleotide, and the guide polynucleotide and the Cas endonuclease form a complex that binds to the target double-stranded DNA polynucleotide. Synthetic composition.
2. 2. The synthetic composition of claim 1, wherein the Cas endonuclease comprises less than 800 amino acids.
3. 2. The synthetic composition of claim 1, wherein the Cas endonuclease is provided as a polynucleotide encoding the Cas endonuclease.
4. 2. The synthetic composition of claim 1, wherein the Cas endonuclease cleaves the double-stranded DNA polynucleotide.
5. The synthetic composition of claim 1 further comprising a heterologous polynucleotide.
6. The synthetic composition of claim 5 , wherein the heterologous polynucleotide is an expression element.
7. The synthetic composition of claim 5 , wherein the heterologous polynucleotide is a transgene.
8. The synthetic composition of claim 5 , wherein the heterologous polynucleotide is a donor DNA molecule.
9. The synthetic composition of claim 5 , wherein the heterologous polynucleotide is a modified polynucleotide template.
10. The synthetic composition of claim 1, wherein the CRISPR-Cas endonuclease is catalytically inactive.
11. 2. The synthetic composition of claim 1, wherein the Cas endonuclease recognizes a PAM sequence that contains multiple T or C nucleotides.
12. 11. The synthetic composition of claim 10, wherein the PAM sequence is selected from the group consisting of TTAT, TTTR, N(T>V)TTR, N(W>S)TTTR, N(Y>R)N(Y>S>R)TTN(A>G>Y), N(W>S)N(Y>R)TTTR, CTT, N(T>W>C)TTC, and CCD.
13. 2. The synthetic composition of claim 1, wherein the Cas endonuclease is part of a fusion protein.
14. 12. The synthetic composition of claim 11, further comprising a deaminase.
15. The synthetic composition of claim 11 , wherein the fusion protein further comprises a heterologous nuclease domain.
16. The synthetic composition of claim 1, further comprising a non-human eukaryotic cell.
17. 17. The synthetic composition of claim 16, wherein the eukaryotic cell is a plant cell, an animal cell, or a fungal cell.
18. 18. The synthetic composition of claim 17, wherein the plant cell is a monocotyledonous or dicotyledonous plant cell.
19. 18. The synthetic composition of claim 17, wherein the plant cell is derived from an organism selected from the group consisting of corn, soybean, cotton, wheat, canola, oilseed rape, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.
20. The synthetic composition of claim 1 , wherein at least one component is attached to a solid matrix.
21. (a) a Cas endonuclease that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 20, 35, 37, and 38; (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 that comprises a region complementary to the target double-stranded DNA polynucleotide. Includes: The Cas endonuclease recognizes a PAM sequence on the target double-stranded DNA polynucleotide, and the guide polynucleotide and the Cas endonuclease form a complex that binds to the target double-stranded DNA polynucleotide. Synthetic composition.
22. 1. A method for introducing a targeted edit into a target polynucleotide, comprising: (a) a Cas endonuclease that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 20, 35, 37, and 38, wherein the Cas endonuclease further comprises: (i) a C-terminal three-split RuvC domain; (ii) the following amino acid motifs: GxxxG, ExL, CxnC, and Cxn(C or H), where G=glycine, E=glutamic acid, C=cysteine, H=histidine, x=any amino acid, and n=an integer from 0 to 11; (iii) an alpha helix, and (iv) multiple beta sheets forming a wedge-shaped domain Cas endonuclease, wherein the Cas endonuclease recognizes a PAM sequence on the target polynucleotide; (b) a guide polynucleotide comprising a variable targeting domain substantially complementary to a portion of the target polynucleotide, wherein the guide polynucleotide and the Cas-alpha endonuclease form a complex capable of recognizing and binding to the target polynucleotide. providing a heterogeneous composition comprising: The method, wherein the target polynucleotide is in at least one biological cell, the cell being a non-human cell.
23. The method of claim 22, further comprising: introducing at least one nucleotide modification into said target polynucleotide of said at least one biological cell compared to said target polynucleotide of said at least one biological cell prior to introduction of said heterologous composition; incubating said at least one cell and generating a whole organism from said at least one cell; 23. The method of claim 22, further comprising confirming the presence of said at least one nucleotide modification in said target polynucleotide of at least one cell of said organism.
24. 23. The method of claim 22, wherein the Cas endonuclease recognizes a PAM sequence containing multiple T or C nucleotides.
25. 23. The method of claim 22, wherein the PAM sequence is selected from the group consisting of TTAT, TTTR, N(T>V)TTR, N(W>S)TTTR, N(Y>R)N(Y>S>R)TTN(A>G>Y), N(W>S)N(Y>R)TTTR, CTT, N(T>W>C)TTC, and CCD.
26. 24. The method of claim 23, wherein the cell is a eukaryotic cell.
27. 27. The method of claim 26, wherein the eukaryotic cell is derived from or obtained from an animal, fungus, or plant.
28. 28. The method of claim 27, wherein the plant is a monocotyledonous or dicotyledonous plant.
29. 28. The method of claim 27, wherein the plant is selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.
30. 24. The method of claim 23, further comprising introducing a heterologous polynucleotide.
31. 31. The method of claim 30, wherein the heterologous polynucleotide is a donor DNA molecule.
32. 31. The method of claim 30, wherein the heterologous polynucleotide is a modified polynucleotide template comprising a sequence that is at least 50% identical to a sequence of the cell.