A novel CRISPR-CAS system for genome editing

Abstract

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

Description

Technical field

[0001] Cross-reference to related applications This application is the subject of U.S. Provisional Patent Application No. 62 / 980750, filed February 24, 2020, and U.S. Provisional Patent Application No. 62 / 980,750, filed May 28, 2020, each of which is incorporated herein by reference in its entirety. Claims the benefit of 63 / 030964.

[0002] Reference to Sequence Listing Submitted Electronically An official copy of the Sequence Listing will be made available electronically via EFS-Web as a Sequence Listing in ASCII format in a file named 8207-WO-PCT_SequenceListing_ST25.txt, dated February 4, 2021, and having a size of 158,138 bytes. filed jointly and concurrently with this specification. The Sequence Listing contained in this ASCII format document is incorporated herein by reference in its entirety.

[0003] 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, as well as other types of 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 have low specificity. , tend to use engineered nucleases that need to be redesigned for each target site and are therefore expensive and time consuming to produce.

[0005] Archaeal or bacterial adaptive immunity 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 have been identified that utilize the system.

[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, particularly animals, remains a challenge. and to demonstrate activity in plants. [Outline of the Invention] [Problems to be solved by the invention]

[0007] Small and robust CRISPR-Cas nucleases are highly desirable for genome editing and related applications. Described herein are novel Cas endonucleases, "Cas-beta," exemplary proteins, and methods and compositions for their use. [Means for solving the problem]

[0008] Compositions for novel Cas polynucleotides and cas polypeptides and other elements derived from novel CRISPR-Cas loci including, but not limited to, additional proteins, CRISPR repeat sequences and CRISPR arrays containing CRISPR repeat sequences. An article and method are provided.

[0009] In one aspect, it comprises a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition is provided, wherein the effector protein is free of HNH domains; and heterologous polynucleotides.

[0010] In one aspect, a CRISPR-1 comprising at least one domain upstream of the RuvC-I domain is selected from the group consisting of a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif. A synthetic composition comprising a Cas effector protein is provided, said effector protein being free of HNH domains; and heterologous polynucleotides.

[0011] In one aspect, in this order, a positively charged putative bridging helix-like domain, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc - a CRISPR-Cas effector protein comprising a set of domains in the finger domain and the RuvC-III domain, wherein the CRISPR-Cas effector protein does not contain an HNH domain; and a synthetic composition comprising a heterologous polynucleotide.

[0012] In one aspect, a synthetic composition comprising a CRISPR-Cas effector protein from an organism of the phylum Armatimonadetes and a heterologous polynucleotide is provided.

[0013] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a heterologous polynucleotide from which the effector protein is derived. Synthetic compositions are provided that contain one of the heterologous polynucleotides that are derived from a different class of organism than the heterologous polynucleotide, or that are wholly or partially artificial.

[0014] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that is a protein and does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a heterologous polynucleotide that is functional in eukaryotic cells. A synthetic composition is provided that includes one heterologous polynucleotide that is an engineered guide RNA.

[0015] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain a CRISPR-Cas effector protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and one of the heterologous polynucleotides. Accordingly, a synthetic composition is provided wherein said CRISPR-Cas effector protein further comprises a nuclear localization signal.

[0016] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain a CRISPR-Cas effector protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and one of the heterologous polynucleotides. Accordingly, a synthetic composition is provided wherein said CRISPR-Cas effector protein is Cas-beta or a functional fragment thereof.

[0017] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain a CRISPR-Cas effector protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and one of the heterologous polynucleotides. Accordingly, a synthetic composition is provided, wherein said CRISPR-Cas effector protein is a fusion protein comprising a functional fragment of Cas-beta.

[0018] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain a CRISPR-Cas effector protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and one of the heterologous polynucleotides. Accordingly, a synthetic composition is provided wherein said CRISPR-Cas effector protein is a fusion protein comprising a functional fragment of Cas-beta, said fusion protein further comprising an additional nuclease domain.

[0019] In one aspect, a synthetic composition comprising a fusion protein of Cas-beta or a functional fragment or variant thereof and at least one additional protein selected from the group consisting of Cas1, Cas2 and a Cas protein that is not Cas1 or Cas2 provided.

[0020] In one aspect, a fusion protein of Cas-beta or a functional fragment or variant thereof and at least one additional protein selected from the group consisting of Cas1, Cas2 and a Cas protein that is not Cas1 or Cas2; Synthetic compositions are provided that further comprise nucleotides or polypeptides.

[0021] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. and a heterologous polynucleotide; and at least one protein selected from the group consisting of Cas1, Cas2 and a Cas protein that is not Cas1 or Cas2. A synthetic composition is provided further comprising:

[0022] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide; Further provided are synthetic compositions comprising the sequence.

[0023] In one aspect, any of the synthetic compositions described herein can have at least one component optimized for expression in eukaryotic cells. In some embodiments, the cells are corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanuts, potatoes. , tobacco, Arabidopsis, safflower and tomato.

[0024] In one embodiment, at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein comprises HNH domains); as well as synthetic compositions comprising heterologous polynucleotides are provided.

[0025] In one aspect, a CRISPR-1 comprising at least one domain upstream of the RuvC-I domain is selected from the group consisting of a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif. A synthetic composition is provided comprising a polynucleotide encoding a Cas effector protein, including a CRISPR-Cas effector protein that does not contain an HNH domain.

[0026] In one embodiment, a positively charged putative bridging helix-like domain, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and A synthetic composition comprising a polynucleotide encoding a CRISPR-Cas effector protein comprising a RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; as well as a heterologous polynucleotide is provided.

[0027] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding a CRISPR-Cas effector protein from Armatimonadetes and a heterologous polynucleotide.

[0028] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a heterologous polynucleotide from which the effector protein is derived. Synthetic compositions are provided that contain a polynucleotide that encodes one of the heterologous polynucleotides that are derived from a different class of organism than the heterologous polynucleotide, or that are wholly or partially artificial.

[0029] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that is a protein and does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a heterologous polynucleotide that is functional in eukaryotic cells. A synthetic composition is provided that includes a polynucleotide encoding one of the heterologous polynucleotides that is the engineered guide RNA.

[0030] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that is a protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a polynucleotide encoding one of the heterologous polynucleotides. A synthetic composition is provided, wherein said CRISPR-Cas effector protein further comprises a nuclear localization signal.

[0031] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that is a protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a polynucleotide encoding one of the heterologous polynucleotides. A synthetic composition is provided, wherein said CRISPR-Cas effector protein is Cas-beta or a functional fragment thereof.

[0032] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that is a protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a polynucleotide encoding one of the heterologous polynucleotides. A synthetic composition is provided, wherein said CRISPR-Cas effector protein is a fusion protein comprising a functional fragment of Cas-beta.

[0033] In one embodiment, (1) a CRISPR-Cas effector comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a protein selected from the group consisting of a CRISPR-Cas effector protein that does not contain an HNH domain; (2) a positively charged bridging helix-like domain and a DNA binding domain optionally comprising a helix-turn-helix motif; a CRISPR-Cas effector protein containing at least one domain upstream of the RuvC-I domain and not containing an HNH domain, (3) a positively charged putative bridging helix-like in this order A CRISPR-Cas effector comprising the set of domains in domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridging helix domain, a RuvC-II domain, a zinc-finger domain and a RuvC-III domain A CRISPR-Cas effector protein that is a protein that does not contain an HNH domain, and (4) a CRISPR-Cas effector protein from Armatimonadetes; and a polynucleotide encoding one of the heterologous polynucleotides. A synthetic composition is provided, wherein said CRISPR-Cas effector protein is a fusion protein comprising a functional fragment of Cas-beta, said fusion protein further comprising an additional nuclease domain. .

[0034] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. Accordingly, synthetic compositions are provided comprising a polynucleotide encoding a CRISPR-Cas effector protein that does not contain an HNH domain as well as a heterologous polynucleotide, further comprising at least one protein.

[0035] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. and a heterologous polynucleotide, further comprising a polynucleotide encoding at least one protein, wherein said polynucleotides are cas1, cas2 and A synthetic composition is provided which is selected from the group consisting of one additional cas gene.

[0036] In one aspect, it encodes a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. wherein said effector protein comprises a polynucleotide that does not contain an HNH domain, said polynucleotide encoding at least one protein, comprising a sequence of genes cas1, cas2 and one additional cas gene Synthetic compositions are provided that further comprise certain polynucleotides in that order.

[0037] In one aspect, any of the synthetic compositions described herein can have at least one component optimized for expression in eukaryotic cells. In some embodiments, the cells are corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanuts, potatoes. , tobacco, Arabidopsis, safflower and tomato.

[0038] In one aspect, it encodes a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. Synthetic compositions are provided comprising polynucleotides that do not contain an HNH domain, as well as heterologous polynucleotides.

[0039] In one aspect, at least 250, 250-500, at least 500, 500-600, at least 600, 600-700, at least 700, 700-750, at least 750, 750-800, at least 800, 800-825 or more than 825 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%~ A synthetic composition is provided comprising a CRISPR-Cas effector protein sharing 100% or 100% sequence identity; and a heterologous polynucleotide or polypeptide.

[0040] In one aspect, at least 250, 250-500, at least 500, 500-600, at least 600, 600-700, at least 700, 700-750, at least 750, 750-800, at least 800 of SEQ ID NO: 10, 11 or 12, 800-850, at least 850, 850-900, at least 900, 900-950, at least 950, 950-1000, at least 1000, 1000-1250, at least 1250, 1250-1500, at least 1500, 1500-1750, at least 1750, 1750 ~2000, at least 2000 or more than 2000 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%~ Synthetic compositions comprising polynucleotides encoding CRISPR-Cas effector proteins sharing 100% or 100% sequence identity are provided.

[0041] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. Also provided are polynucleotide sequences encoding CRISPR-Cas effector proteins that do not contain HNH domains; as well as engineered non-naturally occurring CRISPR-Cas systems that contain heterologous polynucleotides.

[0042] In one aspect, an engineered non-naturally occurring CRISPR-Cas system is provided that includes an effector protein system from Armatimonadetes and a heterologous polynucleotide.

[0043] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. and a heterologous polynucleotide, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is SEQ ID NO: 10, 11 or 12 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 450, 450~500, at least 500, 500~550, at least 550, 550~600, 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, 950-100 or more than 1000 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% , share at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100% or 100% sequence identity.

[0044] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. and a heterologous polynucleotide, wherein the CRISPR-Cas effector protein is SEQ ID NO: 13, 14, 15 or 57. 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 450, 450-500, at least 500, 500-525, at least 525, 525 amino acids to 540 amino acids or at least 540 contiguous amino acids and at least 50%, 50% to 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 100% sequence identity.

[0045] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. Also provided is a synthetic composition comprising a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide; and further comprising at least one gene encoding the protein.

[0046] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. and comprising a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and further comprising a plurality of genes encoding at least two proteins.

[0047] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a polynucleotide sequence encoding a CRISPR-Cas effector protein without the HNH domain; and a heterologous polynucleotide; and at least one gene encoding the protein, wherein said gene encoding the protein is selected from the group consisting of cas1, cas2 and cas genes that are not cas1 or cas2, and optionally cas4.

[0048] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide; and further comprising a plurality of genes encoding at least two different proteins; is selected from the group consisting of, in some order, cas1 flanking cas2, cas1 and cas1 genes not flanking cas genes that are not cas2, and cas2 flanking both genes encoding another Cas protein that is not Cas1 or Cas2. be.

[0049] In one aspect, a polynucleotide sequence encoding a CRISPR-Cas effector protein comprising at least two zinc finger-like domains, at least two bridging helix-like domains, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain; A synthetic composition is provided comprising a polynucleotide; further comprising a plurality of genes encoding at least two different proteins, wherein the plurality of genes are cas1, cas2 and genes that are not cas1 or cas2 in the following order.

[0050] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide; and at least one gene encoding the protein. Said polynucleotide sequence encoding is present upstream of at least one encoding protein.

[0051] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide; further comprising at least two genes encoding different proteins; is downstream of at least two genes encoding two different proteins selected from the group consisting of cas1, cas2 and genes that are not cas1 or cas2.

[0052] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide; further comprising at least three genes encoding different proteins; is downstream of at least three genes encoding three different proteins selected from the group consisting of cas1, cas2 and genes that are not cas1 or cas2.

[0053] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; and a heterologous polynucleotide comprising a regulatory element capable of effecting transcription of said polynucleotide sequence encoding the CRISPR-Cas effector protein. Synthetic compositions comprising heterologous polynucleotides are provided.

[0054] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. provided is a synthetic composition comprising a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; Sequence optimized for improved expression.

[0055] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. provided is a synthetic composition comprising a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; Sequence optimized for improved expression, said eukaryotic cells are plant cells, animal cells, fungal cells or protists.

[0056] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. provided is a synthetic composition comprising a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; Sequence optimized for improved expression, said eukaryotic cell further comprises a pre-existing heterologous polynucleotide.

[0057] In one aspect, a CRISPR-Cas effector protein comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. provided is a synthetic composition comprising a polynucleotide sequence encoding a CRISPR-Cas effector protein that does not contain an HNH domain; sequence optimized for improved expression, said eukaryotic cell being a plant cell, said plant cell being maize, soybean, cotton, wheat, canola, rape, sorghum, rice, rye, barley, millet, oat , sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower and tomato.

[0058] In one aspect, any of the synthetic compositions provided herein can be contained within a recombinant vector.

[0059] In one aspect, any of the synthetic compositions provided herein can be attached to a solid matrix. Said mechanism of attachment may be covalent or non-covalent.

[0060] In one aspect, any of the synthetic compositions provided herein can be further combined with microparticles, and in some aspects, said microparticles are from the group consisting of gold particles, tungsten particles and silicon carbide whisker particles. selected.

[0061] In one embodiment, a eukaryotic cell; a CRISPR-Cas comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain a CRISPR-Cas effector protein that does not contain an HNH domain; and a guide polynucleotide comprising a variable targeting domain that is substantially complementary to a target sequence in the genome of said cell; The guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence.

[0062] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition is provided comprising a Cas effector protein, wherein the CRISPR-Cas effector protein does not contain an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. , said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said heterologous CRISPR-Cas Effector proteins contain at least 500 amino acids.

[0063] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition is provided comprising a Cas effector protein, wherein the CRISPR-Cas effector protein does not contain an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. , said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said heterologous CRISPR-Cas Effector proteins contain up to 1000 amino acids.

[0064] In one embodiment, a eukaryotic cell; a heterologous CRISPR-Crispr- A synthetic composition is provided comprising a Cas effector protein, wherein the CRISPR-Cas effector protein does not contain an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. , said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said heterologous CRISPR-Cas Effector proteins contain less than 875 amino acids.

[0065] In one embodiment, a eukaryotic cell; a heterologous CRISPR-Crispr- A synthetic composition is provided comprising a Cas effector protein, wherein the CRISPR-Cas effector protein does not contain an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. , said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said heterologous CRISPR-Cas Effector proteins are at least 800 amino acids, but contain less than 900 amino acids.

[0066] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition comprising a CRISPR-Cas effector protein, wherein the Cas effector protein does not contain an HNH domain; at least one protein and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. is provided, wherein said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said Heterologous CRISPR-Cas effector proteins are at least 800 amino acids but contain less than 875 amino acids.

[0067] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition comprising a CRISPR-Cas effector protein, wherein the Cas effector protein does not contain an HNH domain; at least one protein and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. is provided, wherein said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said Heterologous CRISPR-Cas effector proteins contain less than 875 amino acids.

[0068] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition comprising a CRISPR-Cas effector protein, wherein the Cas effector protein does not contain an HNH domain; at least one protein and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. is provided, wherein said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said Heterologous CRISPR-Cas effector proteins are at least 800 amino acids but contain less than 900 amino acids.

[0069] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition comprising a CRISPR-Cas effector protein, wherein the Cas effector protein does not contain an HNH domain; at least one protein and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. is provided, wherein said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said The heterologous CRISPR-Cas effector protein comprises at least 800 amino acids, said protein being selected from the group consisting of Cas1, Cas2 and another protein which is not Cas1 or Cas2.

[0070] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition comprising a CRISPR-Cas effector protein, wherein the Cas effector protein does not contain an HNH domain; at least one protein and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. is provided, wherein said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said The heterologous CRISPR-Cas effector protein comprises less than 875 amino acids, said protein being selected from the group consisting of Cas1, Cas2 and another protein which is not Cas1 or Cas2.

[0071] In one embodiment, a eukaryotic cell; a heterologous CRISPR-containing cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain. A synthetic composition comprising a CRISPR-Cas effector protein, wherein the Cas effector protein does not contain an HNH domain; at least one protein and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. is provided, wherein said guide polynucleotide and heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, said The heterologous CRISPR-Cas effector protein comprises at least 800 amino acids but less than 860 amino acids, said protein being selected from the group consisting of Casl, Cas2 and another protein which is not Casl or Cas2.

[0072] In one aspect, a eukaryotic cell; a cas-beta gene and a Cas-beta effector protein or functional variant or function thereof capable of expressing the Cas-beta effector protein or functional variant or functional fragment thereof in said eukaryotic cell. a heterologous molecule selected from the group consisting of target fragments; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell, wherein said guide polynucleotide and A Cas-beta effector protein can recognize the target sequence, bind to it, and form a complex that can optionally be nicked or cleaved.

[0073] In one aspect, a eukaryotic cell; a cas-beta gene and a Cas-beta effector protein or functional variant or function thereof capable of expressing the Cas-beta effector protein or functional variant or functional fragment thereof in said eukaryotic cell. a heterologous molecule selected from the group consisting of target fragments; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell, wherein said guide polynucleotide and The Cas-beta effector protein is capable of recognizing the target sequence, binding to it, and forming a complex capable of optionally nicking or cleaving; 10, 11 or 12 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 450, 450-500, at least 500, 500-550, at least 550, 550-600, 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, 950-100 or more than 1000 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%~ share 97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100% or 100% sequence identity.

[0074] In one aspect, a eukaryotic cell; a cas-beta gene and a Cas-beta effector protein or functional variant or function thereof capable of expressing the Cas-beta effector protein or functional variant or functional fragment thereof in said eukaryotic cell. a heterologous molecule selected from the group consisting of target fragments; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell, wherein said guide polynucleotide and The Cas-beta effector protein is capable of recognizing, binding to, and optionally forming a complex capable of nicking or cleaving said target sequence; Number 13, 14, 15 or 57 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 450, 450-500, at least 500, 500-550, at least 550, 550 amino acids to 600 amino acids or at least 600 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% , share 96%-97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100% or 100% sequence identity.

[0075] In one aspect, a eukaryotic cell; a cas1 gene capable of expressing a Cas1 protein in said eukaryotic cell, a cas2 gene capable of expressing a Cas2 protein in said eukaryotic cell, not Cas1 or Cas2 in said eukaryotic cell. a cas gene capable of expressing a Cas protein, a cas-beta gene capable of expressing a Cas-beta effector protein in said eukaryotic cell, a Cas1 protein, a Cas2 protein, a Cas protein that is not Cas1 or Cas2 and a Cas-beta effector a protein; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell; at least two different heterologous molecules selected from the group are provided; The nucleotide and Cas-beta effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence.

[0076] In one aspect, a eukaryotic cell; a cas1 gene capable of expressing a Cas1 protein in said eukaryotic cell, a cas2 gene capable of expressing a Cas2 protein in said eukaryotic cell, expressing a Cas protein in said eukaryotic cell. a cas-beta gene capable of expressing a Cas-beta effector protein in said eukaryotic cell; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. a Cas-beta effector protein encoded by the guide polynucleotide and the cas-beta gene recognizes and binds to the target sequence and optionally can form a complex that can be nicked or cleaved, wherein the plurality of heterologous molecules are cas-beta flanked by cas1, cas1 flanked by cas2, cas1 and cas1 or is selected from the group consisting of cas2 flanking both of another gene that is not cas2, cas-beta flanking cas1 flanking cas2, cas-beta flanking cas1 flanking cas2 flanking another cas gene Contained within a contiguous polynucleotide sequence comprising the following genes in order:

[0077] In one aspect, a eukaryotic cell; a Cas1 protein, a Cas2 protein, a Cas protein that is not Cas1 or Cas2 and a Cas-beta effector protein; and a guide poly comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. A synthetic composition is provided comprising a plurality of different heterologous molecules selected from the group consisting of nucleotides; said guide polynucleotides and Cas-beta effector proteins recognize and bind said target sequence and optionally cleave wherein the plurality of heterologous molecules are Cas-beta adjacent to Cas1, Cas1 adjacent to Cas2, Cas1 and another gene that is not Cas1 or Cas2. in an order selected from the group consisting of Cas2 flanking both, Cas-beta flanking Cas1 flanking Cas2, Cas-beta flanking Cas1 flanking Cas2 flanking another cas protein that is not Cas1 or Cas2 Contained within a contiguous polypeptide sequence comprising the following proteins:

[0078] In one aspect, the synthetic composition comprises eukaryotic cells and SEQ ID NO: 19, 20 or 21 and 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% , a heterologous CRISPR-Cas array comprising at least two repeats that are at least 98%, 98%-99%, at least 99%, 99%-100% or 100% identical. In some embodiments, the repeat is a spacer poly of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 nucleotides. separated by nucleotides.

[0079] In one aspect, the synthetic composition comprises eukaryotic cells and SEQ ID NO: 19, 20 or 21 and 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% , a heterologous CRISPR-Cas array comprising at least three repeats that are at least 98%, 98%-99%, at least 99%, 99%-100% or 100% identical. In some embodiments, the repeat is a spacer poly of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 nucleotides. separated by nucleotides.

[0080] In one aspect, eukaryotic cells and SEQ ID NO: 19, 20 or 21 and 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%, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 that are 98%-99%, at least 99%, 99%-100% or 100% identical, Heterologous CRISPR-Cas arrays comprising 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 repeats A synthetic composition is provided comprising: In some embodiments, the repeat is a spacer poly of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more than 35 nucleotides. separated by nucleotides.

[0081] In one aspect, in any of the disclosed compositions or methods, a eukaryotic cell is provided that further comprises a pre-existing heterologous polynucleotide.

[0082] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC -I domain, RuvC-II domain and RuvC-III domain (wherein said effector protein does not comprise a HNH domain); DNA binding comprising at least two bridging helix-like domains, optionally a helix-turn-helix motif a polynucleotide sequence encoding a domain, RuvC-I domain, RuvC-II domain and RuvC-III domain, wherein said effector protein does not include an HNH domain; cas-beta polynucleotide; Cas-beta a polypeptide or a functional variant or fragment thereof; comprising SEQ ID NO: 10, 11 or 12; and SEQ ID NO: 13, 14, 15 or 57; a variable targeting domain substantially complementary to a target sequence in the genome of said cell and incubating the cells at a temperature above 10° C. for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas Methods are provided wherein the effector protein is capable of recognizing, binding to, and optionally nicking or cleaving said target sequence to form a complex.

[0083] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain Polynucleotide sequences; cas-beta polynucleotides; Cas-beta polypeptides or functional variants or functional fragments thereof; polynucleotides sharing at least 70%-100% identity with SEQ ID NO: 10, 11 or 12; introducing a composition selected from the group consisting of polypeptides sharing at least 70% to 100% identity with numbers 13, 14, 15 or 57, wherein the guide polynucleotide and the CRISPR-Cas effector protein are , is capable of recognizing said target sequence, binding to it and forming a complex that can optionally be nicked or cleaved.

[0084] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain a composition selected from the group consisting of: a polynucleotide sequence; a cas-beta polynucleotide; a Cas-beta polypeptide or functional fragment or functional variant thereof; SEQ ID NO:3; and SEQ ID NO:4; Cas1 polypeptides, cas2 polynucleotides, Cas2 polypeptides, cas polynucleotides that are not cas1 or cas2, the polynucleotide sequences of the sequence listing and polypeptides of the sequence listing, Cas polypeptides that are not Cas1 or Cas2, heterologous polynucleotides and the cells introducing a composition further comprising at least one composition selected from the group consisting of a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome and a temperature above 10°C for at least about 4 hours. wherein the guide polynucleotide and CRISPR-Cas effector protein form a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. A method is provided that can be formed. In some embodiments, one or more of the compositions may be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments or variants of the composition may be substituted.

[0085] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain a polynucleotide sequence; a cas-beta polynucleotide; a Cas-beta polypeptide; a composition selected from the group consisting of the polynucleotide sequences of the sequence listing and the polypeptides of the sequence listing; A polynucleotide sharing at least 70%-100% identity with any of a polynucleotide, a Cas2 polypeptide, a cas4 polynucleotide, a Cas4 polypeptide, a heterologous polynucleotide, a polypeptide of the sequence listing; and a target sequence in the genome of said cell. and introducing a composition further comprising at least one composition selected from the group consisting of a guide polynucleotide comprising a variable targeting domain substantially complementary to incubating, wherein the guide polynucleotide and CRISPR-Cas effector protein form a complex capable of recognizing, binding, and optionally nicking or cleaving the target sequence. A method is provided. In some embodiments, one or more of the compositions may be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments of the composition may be replaced with respect to the entire molecule.

[0086] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain a composition selected from the group consisting of: a polynucleotide sequence; a cas-beta polynucleotide; a Cas-beta polypeptide or functional fragment or functional variant thereof; SEQ ID NO:3; and SEQ ID NO:4; Cas1 polypeptides, cas2 polynucleotides, Cas2 polypeptides, cas polynucleotides that are not cas1 or cas2, Cas polypeptides that are not Cas1 or Cas2, heterologous polynucleotides, polynucleotide sequences of the sequence listing and polypeptides of the sequence listing; and said cells and introducing a composition further comprising at least two compositions selected from the group consisting of guide polynucleotides comprising a variable targeting domain substantially complementary to a target sequence in the genome of and above 10°C for at least about 4 hours incubating said cell at a temperature, wherein said guide polynucleotide and CRISPR-Cas effector protein are complexes capable of recognizing, binding and optionally nicking or cleaving said target sequence. A method is provided, which can form a In some embodiments, one or more of the compositions may be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments or variants of the composition may be substituted.

[0087] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain a polynucleotide sequence; a cas-beta polynucleotide; a Cas-beta polypeptide; a polynucleotide sharing at least 70%-100% identity with SEQ ID NO: 10, 11 or 12; A composition selected from the group consisting of polypeptides sharing at least 70%-100% identity; cas1 polynucleotide, Cas1 polypeptide, cas2 polynucleotide, Cas2 polypeptide, cas4 polynucleotide, Cas1 or Cas2 a heterologous polynucleotide, a polynucleotide sequence of the sequence listing and a polypeptide of the sequence listing; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. introducing a composition further comprising at least two selected compositions and incubating said cells at a temperature above 10° C. for at least about 4 hours, wherein said guide polynucleotide and CRISPR-Cas effector protein are combined with said Methods are provided that are capable of recognizing a target sequence, binding to it, and forming a complex that can optionally be nicked or cleaved. In some embodiments, one or more of the compositions may be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments of the composition may be replaced with respect to the entire molecule.

[0088] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain a composition selected from the group consisting of: a polynucleotide sequence; a cas-beta polynucleotide; a Cas-beta polypeptide or functional fragment or functional variant thereof; SEQ ID NO:3; and SEQ ID NO:4; Cas1 polypeptides, cas2 polynucleotides, Cas2 polypeptides, cas polynucleotides that are not cas1 or cas2, Cas polypeptides that are not Cas1 or Cas2, heterologous polynucleotides, polynucleotide sequences of the sequence listing and polypeptides of the sequence listing; and said cells and introducing a composition further comprising at least three compositions selected from the group consisting of a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of and above 10°C for at least about 4 hours incubating said cell at a temperature, wherein said guide polynucleotide and CRISPR-Cas effector protein are complexes capable of recognizing, binding and optionally nicking or cleaving said target sequence. A method is provided, which can form a In some embodiments, one or more of the compositions may be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments or variants of the composition may be substituted.

[0089] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell, said cell comprising at least two bridging helix-like domains, a DNA binding domain optionally comprising a helix-turn-helix motif, RuvC - a CRISPR-Cas effector protein comprising the I domain, the RuvC-II domain and the RuvC-III domain, but without the HNH domain; at least two bridging helix-like domains, a helix-turn-helix motif; A polynucleotide sequence encoding a CRISPR-Cas effector protein comprising optionally a DNA binding domain, a RuvC-I domain, a RuvC-II domain and a RuvC-III domain, wherein said effector protein does not contain a HNH domain a polynucleotide sequence; a cas-beta polynucleotide; a Cas-beta polypeptide; a polynucleotide sharing at least 70%-100% identity with SEQ ID NO: 10, 11 or 12; A composition selected from the group consisting of polypeptides sharing at least 70%-100% identity; cas1 polynucleotide, Cas1 polypeptide, cas2 polynucleotide, Cas2 polypeptide, cas4 polynucleotide, Cas1 or Cas2 a heterologous polynucleotide, a polynucleotide sequence of the sequence listing and a polypeptide of the sequence listing; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell. introducing a composition further comprising at least three selected compositions and incubating said cells at a temperature above 10° C. for at least about 4 hours, wherein said guide polynucleotide and CRISPR-Cas effector protein are combined with said Methods are provided that are capable of recognizing a target sequence, binding to it, and forming a complex that can optionally be nicked or cleaved. In some embodiments, one or more of the compositions may be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments of the composition may be replaced with respect to the entire molecule.

[0090] Further provided in any of the methods or compositions provided herein are methods or compositions that further comprise a eukaryotic cell. In some embodiments, eukaryotic cells are animal cells. In some embodiments, eukaryotic cells are plant cells. In some embodiments, the plant cell is corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, It is of plant origin selected from the group consisting of potato, tobacco, Arabidopsis, safflower and tomato. In some embodiments, the eukaryotic cell may further comprise a pre-existing heterologous polynucleotide. In some embodiments, the animal cell can further comprise a pre-existing heterologous polynucleotide. In some embodiments, the plant cell may further contain a pre-existing heterologous polynucleotide.

[0091] In one aspect, a polynucleotide modified template is provided comprising at least one region complementary to a PAM sequence that flanks a DNA target sequence recognized by a Cas-beta complex, wherein the at least one region corresponding to the PAM sequence comprises Contains at least one nucleotide mismatch.

[0092] In one aspect, a method of generating plants with modified genomes, wherein at least one of said plants comprises a Cas-beta effector protein or a cas-beta polynucleotide encoding a Cas-beta effector protein; a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome, said guide polynucleotide and Cas-beta endonuclease recognizing and binding to said target sequence, and optionally At least one region complementary to the PAM sequence flanking the guide polynucleotide and the DNA target sequence recognized by the Cas-beta complex, capable of forming a complex that can be nicked or cleaved. introducing a heterologous composition comprising a polynucleotide modified template comprising at least one region containing at least one nucleotide mismatch; incubating said cells at a temperature above 10°C for at least about 4 hours; and verifying at least one nucleotide alteration in the genome of at least one cell of said plant compared to a target sequence in the genome of said cell prior to introduction of said heterologous composition. provided.

[0093] In one aspect, a progeny of a plant having a modified genome, wherein in at least one of said plants a Cas-beta effector protein or a cas-beta polynucleotide encoding a Cas-beta effector protein, in the genome of said cell A guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence, said guide polynucleotide and Cas-beta endonuclease recognizing and binding to said target sequence and optionally forming a nick. at least one region complementary to the PAM sequence flanking the DNA target sequence recognized by the guide polynucleotide and the Cas-beta complex capable of forming a complex that can be inserted or cleaved, introducing a heterologous composition comprising a polynucleotide modified template comprising at least one region containing at least one nucleotide mismatch; incubating said cells at a temperature above 10° C. for at least about 4 hours; removing whole plants from said cells. and verifying at least one nucleotide alteration in the genome of at least one cell of said plant compared to a target sequence in the genome of said cell prior to introduction of said heterologous composition. offspring are provided.

[0094] In some embodiments, a progeny of a plant produced by any of the methods or produced with any of the compositions disclosed herein, wherein the plant element or plant derived from said plant or progeny Reproductive elements and a plurality of plants, progeny, plant elements and plant reproductive elements are provided.

[0095] In some aspects, the plants provided herein include corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turfgrass, switchgrass, alfalfa, Sunflowers, tobacco, peanuts, potatoes, tobacco, Arabidopsis, safflower and tomatoes.

[0096] In any embodiment, the Cas-beta protein may be identical to SEQ ID NO: 13, 14, 15 or 57, or may be a functional fragment or variant thereof, or 70-100% identical to said sequence. can share the identity of In either aspect, the cas-beta gene may encode a Cas-beta protein or functional fragment or variant thereof.

[0097] 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; any at least one nucleotide modification compared to the polynucleotide sequence in the cell; guide RNA; guide DNA; guide RNA-DNA hybrid; endonuclease; nuclear localization signal;

[0098] In one aspect, methods are provided for using any of the compositions disclosed herein. In some embodiments, methods are provided for binding a Cas-beta endonuclease to a polynucleotide target sequence, eg, in the genome of a cell or in vitro. In some embodiments, the Cas-beta endonuclease forms a complex with a guide polynucleotide, eg, guide RNA. In some embodiments, the complex recognizes, binds, and optionally generates a nick (single-stranded) or break (double-stranded) in the polynucleotide at or near the target sequence. . In some embodiments, the nick or break is repaired by non-homologous end joining (NHEJ). In some embodiments, nicks or breaks are repaired by homologous recombination repair (HDR) or homologous recombination (HR) using a polynucleotide modified template or donor DNA molecule.

[0099] The novel Cas endonucleases described herein are capable of producing double-strand breaks at or adjacent to a target polynucleotide that are guided by a guide polynucleotide in any prokaryotic or eukaryotic cell, containing a suitable PAM. 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.

[0100] The present disclosure can be understood in more detail from the following detailed description, which forms part of this application, and the accompanying drawings and sequence listing. [Brief description of the drawing]

[0101]

Figure 1

Figure 2

[0102] The sequence descriptions and sequence listing attached hereto comply with the rules governing 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.

[0103] SEQ ID NO: 1 is the Armatimonadetes bacterial PRT sequence for Cas1 encoded by the Cas-beta1 locus.

[0104] SEQ ID NO:2 is the Armatimonadetes bacterial PRT sequence for Cas1 encoded by the Cas-beta2 locus.

[0105] SEQ ID NO:3 is the Armatimonadetes bacterial PRT sequence for Cas1 encoded by the Cas-beta3 locus.

[0106] SEQ ID NO: 4 is the Armatimonadetes bacterial PRT sequence for Cas2 encoded in the Cas-beta1 locus.

[0107] SEQ ID NO:5 is the Armatimonadetes bacterial PRT sequence for Cas2 encoded at the Cas-beta2 locus.

[0108] SEQ ID NO: 6 is the Armatimonadetes bacterial PRT sequence for Cas2 encoded at the Cas-beta3 locus.

[0109] SEQ ID NO:7 is the Armatimonadetes bacterial PRT sequence for Cas4 encoded in the Cas-beta1 locus.

[0110] SEQ ID NO:8 is the Armatimonadetes bacterial PRT sequence for Cas4 encoded at the Cas-beta2 locus.

[0111] SEQ ID NO:9 is the Armatimonadetes bacterial PRT sequence for Cas4 encoded at the Cas-beta3 locus.

[0112] SEQ ID NO: 10 is the Armatimonadetes bacterial DNA sequence for the Cas-beta1 endonuclease gene.

[0113] SEQ ID NO: 11 is the Armatimonadetes bacterial DNA sequence for the Cas-beta2 endonuclease gene.

[0114] SEQ ID NO: 12 is the Armatimonadetes bacterial DNA sequence for the Cas-beta3 endonuclease gene.

[0115] SEQ ID NO: 13 is the Armatimonadetes bacterial PRT sequence for the Cas-beta1 endonuclease gene.

[0116] SEQ ID NO: 14 is the Armatimonadetes bacterial PRT sequence for the Cas-beta2 endonuclease gene.

[0117] SEQ ID NO: 15 is the Armatimonadetes bacterial PRT sequence for the Cas-beta3 endonuclease gene.

[0118] SEQ ID NO: 16 is the Armatimonadetes bacterial DNA sequence for the Cas-beta1 locus.

[0119] SEQ ID NO: 17 is the Armatimonadetes bacterial DNA sequence for the Cas-beta2 locus.

[0120] SEQ ID NO: 18 is the Armatimonadetes bacterial DNA sequence for the Cas-beta3 locus.

[0121] SEQ ID NO: 19 is the Armatimonadetes bacterial DNA sequence for the Cas-beta1 CRISPR repeat.

[0122] SEQ ID NO:20 is the Armatimonadetes bacterial DNA sequence for the Cas-Beta2 CRISPR repeat.

[0123] SEQ ID NO:21 is the Armatimonadetes bacterial DNA sequence for the Cas-beta3 CRISPR repeat.

[0124] SEQ ID NO:22 is the Armatimonadetes bacterial RNA sequence for Cas-beta1 CRISPR RNA.

[0125] SEQ ID NO:23 is the Armatimonadetes bacterial RNA sequence for Cas-beta2 CRISPR RNA.

[0126] SEQ ID NO:24 is the Armatimonadetes bacterial RNA sequence for Cas-Beta3 CRISPR RNA.

[0127] SEQ ID NO:25 is the Armatimonadetes bacterial RNA sequence for Cas-beta1 tracrRNA with a terminator-like hairpin.

[0128] SEQ ID NO:26 is the Armatimonadetes bacterial RNA sequence for Cas-beta1 tracrRNA without a terminator-like hairpin.

[0129] SEQ ID NO:27 is the Armatimonadetes bacterial RNA sequence for Cas-beta2 tracrRNA with a terminator-like hairpin.

[0130] SEQ ID NO:28 is the Armatimonadetes bacterial RNA sequence for Cas-beta2 tracrRNA without a terminator-like hairpin.

[0131] SEQ ID NO:29 is the Armatimonadetes bacterial RNA sequence for Cas-beta3 tracrRNA with a terminator-like hairpin.

[0132] SEQ ID NO:30 is the Armatimonadetes bacterial RNA sequence for Cas-beta3 tracrRNA without a terminator-like hairpin.

[0133] SEQ ID NO:31 is the artificial RNA sequence for Cas-beta1 sgRNA with a terminator-like hairpin.

[0134] SEQ ID NO:32 is the artificial RNA sequence for Cas-beta1 sgRNA without a terminator-like hairpin.

[0135] SEQ ID NO:33 is the artificial RNA sequence for Cas-beta2 sgRNA with a terminator-like hairpin.

[0136] SEQ ID NO:34 is the artificial RNA sequence for Cas-beta2 sgRNA without a terminator-like hairpin.

[0137] SEQ ID NO:35 is the artificial RNA sequence for Cas-beta3 sgRNA with a terminator-like hairpin.

[0138] SEQ ID NO:36 is the artificial RNA sequence for Cas-beta3 sgRNA without a terminator-like hairpin.

[0139] SEQ ID NO:37 is an artificial DNA sequence for the T2 PAM library target sequence.

[0140] SEQ ID NO:38 is the artificial PRT sequence for the 6X histidine tag.

[0141] SEQ ID NO:39 is the artificial PRT sequence for the 10X histidine tag.

[0142] SEQ ID NO:40 is an artificial PRT sequence for the maltose binding protein tag.

[0143] SEQ ID NO:41 is the tobacco etch virus PRT sequence for the tobacco etch virus cleavage site.

[0144] SEQ ID NO:42 is an artificial DNA sequence for the A1 oligonucleotide.

[0145] SEQ ID NO:43 is an artificial DNA sequence for the A2 oligonucleotide.

[0146] SEQ ID NO:44 is an artificial DNA sequence for the R0 oligonucleotide.

[0147] SEQ ID NO:45 is an artificial DNA sequence for the C0 oligonucleotide.

[0148] SEQ ID NO:46 is an artificial DNA sequence for the F1 oligonucleotide.

[0149] SEQ ID NO:47 is an artificial DNA sequence for the R1 oligonucleotide.

[0150] SEQ ID NO:48 is an artificial DNA sequence for the bridge amplification portion of the F1 oligonucleotide.

[0151] SEQ ID NO:49 is an artificial DNA sequence for the bridge amplification portion of the R1 oligonucleotide.

[0152] SEQ ID NO:50 is an artificial DNA sequence for the F2 oligonucleotide.

[0153] SEQ ID NO:51 is an artificial DNA sequence for the R2 oligonucleotide.

[0154] SEQ ID NO:52 is an artificial DNA sequence for the C1 oligonucleotide.

[0155] SEQ ID NO:53 is an artificial DNA sequence for the sequence resulting from cleavage at position 23 of the target and adapter ligation.

[0156] SEQ ID NO:54 is an artificial DNA sequence for the adapter portion of SEQ ID NO:53.

[0157] SEQ ID NO:55 is an artificial DNA sequence for the target portion of SEQ ID NO:53.

[0158] SEQ ID NO:56 is an artificial DNA sequence for the 5' sequence of PAM.

[0159] SEQ ID NO:57 is the protein sequence for Cas-beta4 from archaea.

[0160] SEQ ID NO:58 is the protein sequence for the 14x histidine tag.

[0161] SEQ ID NO:59 is the RNA sequence for the 24nt spacer sequence (targeting T2).

[0162] SEQ ID NO:60 is the RNA sequence for Cas-Beta2 sgRNA with a terminator-like hairpin that targets the T2 sequence.

[0163] SEQ ID NO:61 is the RNA sequence for a Cas-Beta2 sgRNA without a terminator-like hairpin targeting the T2 sequence.

[0164] SEQ ID NO:62 is the RNA sequence for Cas-beta3 sgRNA with a terminator-like hairpin that targets the T2 sequence.

[0165] SEQ ID NO:63 is the RNA sequence for a Cas-beta3 sgRNA without a terminator-like hairpin that targets the T2 sequence.

[0166] SEQ ID NO:64 is the DNA sequence for the T1 target sequence.

[0167] SEQ ID NO:65 is the RNA sequence for a Cas-beta2 sgRNA without a terminator-like hairpin that targets T1 sequences.

[0168] SEQ ID NO:66 is the RNA sequence for a Cas-beta2 sgRNA without a terminator-like hairpin targeting a 20 nt T2 sequence.

[0169] SEQ ID NO:67 is the DNA sequence for the oligonucleotide activator.

[0170] SEQ ID NO:68 is the DNA sequence for the oligonucleotide complementary to the activator.

[0171] SEQ ID NO:69 is the DNA sequence for Oligonucleotide 1 used to make the dsDNA non-specific activator.

[0172] SEQ ID NO:70 is the DNA sequence for Oligonucleotide 2 used to make the dsDNA non-specific activator.

[0173] SEQ ID NO:71 is the simian virus 40 PRT sequence for SV40 NLS.

[0174] SEQ ID NO: 72 is the Zea mays DNA sequence for the ubiquitin promoter.

[0175] SEQ ID NO: 73 is the Zea mays DNA sequence for the ubiquitin 5'UTR.

[0176] SEQ ID NO: 74 is the Zea mays DNA sequence for ubiquitin intron 1.

[0177] SEQ ID NO:75 is an artificial DNA sequence for the sequence encoding SV40 NLS.

[0178] SEQ ID NO:76 is an artificial DNA sequence for exon 1 of the maize-optimized Cas-beta1 nuclease.

[0179] SEQ ID NO:77 is an artificial DNA sequence for exon 2 of the maize-optimized Cas-beta1 nuclease.

[0180] SEQ ID NO:78 is the potato (Solanum tuberosum) DNA sequence for ST-LS1 intron 2.

[0181] SEQ ID NO: 79 is the potato (Solanum tuberosum) DNA sequence for the potato proteinase inhibitor II terminator.

[0182] SEQ ID NO:80 is the Zea mays DNA sequence for the U6 promoter with 3'G bp.

[0183] SEQ ID NO:81 is an artificial DNA sequence for the sequence encoding the Cas-beta affinity domain of sgRNA.

[0184] SEQ ID NO: 82 is the Zea mays DNA sequence for the sequence encoding the variable targeting domain of the sgRNA targeting the Zea mays male sterility 26 gene.

[0185] SEQ ID NO:83 is the hepatitis delta virus DNA sequence for the HDV ribozyme sequence.

[0186] SEQ ID NO:84 is the Zea mays DNA sequence for the U6 terminator.

[0187] SEQ ID NO:85 is the Armatimonadetes bacterial PRT sequence for Cas2 encoded at the Cas-beta4 locus.

[0188] SEQ ID NO:86 is the Armatimonadetes bacterial PRT sequence for Cas4 encoded in the Cas-Beta4 locus.

[0189] SEQ ID NO:87 is the Armatimonadetes bacterial DNA sequence for the Cas-beta4 endonuclease gene.

[0190] SEQ ID NO:88 is the Armatimonadetes bacterial DNA sequence for the Cas-beta4 locus.

[0191] SEQ ID NO:89 is the Armatimonadetes bacterial RNA sequence for Cas-beta4 tracrRNA with a terminator-like hairpin.

[0192] SEQ ID NO:90 is the Armatimonadetes bacterial RNA sequence for Cas-beta4 tracrRNA without a terminator-like hairpin.

[0193] SEQ ID NO:91 is the artificial RNA sequence for Cas-beta4 sgRNA with a terminator-like hairpin.

[0194] SEQ ID NO:92 is the artificial RNA sequence for Cas-beta4 sgRNA without a terminator-like hairpin.

[0195] SEQ ID NO:93 is the Homo sapiens DNA sequence for WTAP site 1.

[0196] SEQ ID NO:94 is the Homo sapiens DNA sequence for WTAP site 2.

[0197] SEQ ID NO:95 is the Homo sapiens DNA sequence for RUNX1 site 1.

[0198] SEQ ID NO:96 is the Homo sapiens DNA sequence for RUNX1 site 2.

[0199] SEQ ID NO:97 is the artificial RNA sequence for Cas-beta2 sgRNA targeting WTAP site 1.

[0200] SEQ ID NO:98 is the artificial RNA sequence for Cas-beta2 sgRNA targeting WTAP site 2.

[0201] SEQ ID NO:99 is the artificial RNA sequence for Cas-beta2 sgRNA targeting RUNX1 site 1.

[0202] SEQ ID NO:100 is the artificial RNA sequence for Cas-beta2 sgRNA targeting RUNX1 site 2.

[0203] SEQ ID NO:101 is the artificial RNA sequence for Cas-beta3 sgRNA targeting WTAP site 1.

[0204] SEQ ID NO:102 is the artificial RNA sequence for Cas-beta3 sgRNA targeting WTAP site 2.

[0205] SEQ ID NO:103 is the artificial RNA sequence for Cas-beta3 sgRNA targeting RUNX1 site 1.

[0206] SEQ ID NO:104 is the artificial RNA sequence for Cas-beta3 sgRNA targeting RUNX1 site 2.

[0207] SEQ ID NO: 105 is the artificial DNA sequence for the 5'FAM labeled RUNX1 site 1 non-target strand.

[0208] SEQ ID NO: 106 is the artificial DNA sequence for the 5' ROX labeled RUNX1 site 1 target strand.

[0209] SEQ ID NO: 107 is the artificial DNA sequence for the 5'FAM labeled WTAP site 1 non-target strand.

[0210] SEQ ID NO: 108 is the artificial DNA sequence for the 5'ROX-labeled WTAP site 1 target strand.

[0211] SEQ ID NO: 109 is the Homo sapiens DNA sequence for WTAP site 3.

[0212] SEQ ID NO:110 is the Homo sapiens DNA sequence for WTAP site 4.

[0213] SEQ ID NO: 111 is the Homo sapiens DNA sequence for WTAP site 5.

[0214] SEQ ID NO:112 is the Homo sapiens DNA sequence for WTAP site 6.

[0215] SEQ ID NO: 113 is the Homo sapiens DNA sequence for WTAP site 7.

[0216] SEQ ID NO: 114 is the Homo sapiens DNA sequence for RUNX1 site 3.

[0217] SEQ ID NO:115 is the Homo sapiens DNA sequence for RUNX1 site 4.

[0218] SEQ ID NO:116 is the Homo sapiens DNA sequence for RUNX1 site 5.

[0219] SEQ ID NO:117 is the Homo sapiens DNA sequence for RUNX1 site 6.

[0220] SEQ ID NO:118 is the Homo sapiens DNA sequence for RUNX1 site 7.

[0221] SEQ ID NO: 119 is an artificial DNA sequence for the non-target strand (NTS).

[0222] SEQ ID NO: 120 is an artificial DNA sequence for the Sanger sequence from the reverse orientation (Cas-beta2).

[0223] SEQ ID NO: 121 is an artificial DNA sequence for the target strand (TS).

[0224] SEQ ID NO: 122 is an artificial DNA sequence for the Sanger sequence from the forward direction (Cas-beta2).

[0225] SEQ ID NO: 123 is an artificial DNA sequence for the Sanger sequence from the reverse orientation (Cas-beta3).

[0226] SEQ ID NO: 124 is an artificial DNA sequence for the Sanger sequence from the forward direction (Cas-beta3).

[0227] SEQ ID NO: 125 is the Homo sapiens DNA sequence for RUNX1 site 1 reference.

[0228] SEQ ID NO: 126 is the human (Homo sapiens) DNA sequence for Cas-beta2 RUNX1 site 1 mutation 1.

[0229] SEQ ID NO: 127 is the human (Homo sapiens) DNA sequence for Cas-beta2 RUNX1 site 1 mutation 2.

[0230] SEQ ID NO: 128 is the human (Homo sapiens) DNA sequence for Cas-beta2 RUNX1 site 1 mutation 3.

[0231] SEQ ID NO: 129 is the human (Homo sapiens) DNA sequence for Cas-beta2 RUNX1 site 1 mutation 4.

[0232] SEQ ID NO: 130 is the human (Homo sapiens) DNA sequence for Cas-beta2 RUNX1 site 1 mutation 5.

[0233] SEQ ID NO: 131 is the human (Homo sapiens) DNA sequence for Cas-beta2 RUNX1 site 1 mutation 6.

[0234] SEQ ID NO: 132 is the Homo sapiens DNA sequence for WTAP site 6 reference.

[0235] SEQ ID NO: 133 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 6 mutation 1.

[0236] SEQ ID NO: 134 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 6 mutation 2.

[0237] SEQ ID NO: 135 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 6 mutation 3.

[0238] SEQ ID NO: 136 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 6 mutation 4.

[0239] SEQ ID NO: 137 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 6 mutation 5.

[0240] SEQ ID NO: 138 is the Homo sapiens DNA sequence for WTAP site 7 reference.

[0241] SEQ ID NO: 139 is the human (Homo sapiens) DNA sequence for Cas-beta2 or 3 WTAP site 7 mutation 1.

[0242] SEQ ID NO: 140 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 7 mutation 2.

[0243] SEQ ID NO: 141 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 7 mutation 3.

[0244] SEQ ID NO: 142 is the human (Homo sapiens) DNA sequence for Cas-beta2 WTAP site 7 mutation 4.

[0245] SEQ ID NO: 143 is the artificial RNA sequence for RUNX1 site 1 (20 nt spacer) targeted to Cas-beta2 sgRNA.

[0246] SEQ ID NO: 144 is the artificial RNA sequence for WTAP site 6 (20 nt spacer) targeted to Cas-beta2 sgRNA.

[0247] SEQ ID NO: 145 is the artificial RNA sequence for WTAP site 7 (20 nt spacer) targeted to Cas-beta2 sgRNA.

[0248] SEQ ID NO: 146 is the artificial RNA sequence for WTAP site 7 (20 nt spacer) targeted to Cas-beta3 sgRNA.

[0249] SEQ ID NO: 147 is the Zea mays DNA sequence for reference MS26 site 1.

[0250] SEQ ID NO: 148 is the Zea mays DNA sequence for the MS26 site 1 mutation.

[0251] SEQ ID NO: 149 is the Zea mays DNA sequence for reference MS26 site 2.

[0252] SEQ ID NO: 150 is the Zea mays DNA sequence for the MS26 site 2 mutation.

[0253] SEQ ID NO: 151 is an artificial DNA sequence for the Cas-beta2 natural spacer 1 target.

[0254] SEQ ID NO: 152 is an artificial DNA sequence for the Cas-beta2 natural spacer2 target.

[0255] SEQ ID NO: 153 is an artificial DNA sequence for the Cas-beta2 natural spacer3 target.

[0256] SEQ ID NO: 154 is the artificial PRT sequence for D528A-cleaved inactive Cas-Beta2.

[0257] SEQ ID NO: 155 is the artificial PRT sequence for E634A-cleaved inactive Cas-Beta2.

[0258] Novel CRISPR effector systems and elements comprising such systems, including but not limited to novel guide polynucleotide / endonuclease complexes, guide polynucleotides, guide RNA elements, Cas proteins and endonucleases and endonuclease functions. Compositions and methods are provided for proteins containing 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 target sequence modification, gene editing, and insertion of polynucleotides of interest into the genome of cells.

[0259] The small novel Cas endonuclease described herein, "Cas-beta," is a unique gene containing, in this order: the cas-beta gene, the cas1 gene, the cas2 gene, a gene with low identity to cas4 and a CRISPR array. derived from the CRISPR locus with the structure of The cas-beta gene encodes a Cas-beta endonuclease that is less than 900 amino acids and has a unique protein structure.

[0260] 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" unless the context clearly dictates otherwise. It should be noted that it includes multiple referents.

[0261] 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. Accordingly, 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. Used interchangeably, optionally includes synthetic, non-natural or altered 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), cytosine or deoxy "C" for cytosine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine and "N" for any nucleotide.

[0262] The term "genome" when applied to prokaryotic and eukaryotic cells or biological cells includes not only chromosomal DNA found within the nucleus, but also organellar DNA found within the cellular components of the cell (e.g. mitochondria or plastids). do.

[0263] "Open reading frame" is abbreviated ORF.

[0264] The term "selectively hybridizes" includes specific nucleic acid target sequences relative to hybridization to non-target nucleic acid sequences and substantial exclusion of non-target nucleic acids under stringent hybridization conditions. Included is reference to a detectably large degree (eg, at least 2-fold over background) of hybridization to. Selectively hybridizing sequences typically have at least 80% sequence identity or 90% sequence identity and up to 100% sequence identity (ie, fully complementary) to each other.

[0265] The terms "stringent conditions" or "stringent hybridization conditions" include 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 some sequence mismatches such that lower similarities are detected (non-homologous probing). Generally, probes are less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length. 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, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C and 1-2 x SSC at 50-55°C. Washing in (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.

[0266] "Homology" means similar DNA sequences. For example, a "region of homology to a genomic region" found on donor DNA is a region of DNA that has sequence similarity to a given "genomic region" of a fungal cell genome. A homologous region can be of any length sufficient 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 and the sequence similarity of the polynucleotides. Sequence similarity includes percent sequence identity over the entire length of the sequence and / or percent 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 explained by

[0267] As used herein, a "genomic region" is a segment of a chromosome in the genome of a cell that is on either side of a target site, or alternatively a segment that also contains part of the target site. be. 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.

[0268] 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 the homologous region required for observation of homologous recombination also varies depending on the species. In many cases, homology of at least 5 kb has been exploited, but homologous recombination with homology as small as 25-50 bp has been observed. 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.

[0269] "Sequence identity" or "identity", in the context of nucleic acid or polypeptide sequences, refers to the nucleobases or Refers to amino acid residues.

[0270] The term "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein the portions of the polynucleotide or polypeptide sequences within the comparison window are those In order to optimally align two sequences, additions or deletions (ie, gaps) may be included as compared to a reference sequence (containing no additions or deletions). 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 multiply the result by 100 to obtain the percentage sequence identity. Useful examples of percent sequence identity include 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or any percentage from 50% to 100%. include, but are not limited to. These identities can be determined using any of the programs described herein.

[0271] 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. In the context of this application, when sequence analysis software is used for analysis, it will be understood that analysis results are based on the "default values" of the mentioned programs unless otherwise specified. As used herein, "default values" shall mean any set of values ​​or parameters that are initially loaded with the software when first initialized.

[0272] 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 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 use 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 use 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 used to find similar regions of biological sequences provided by the National Center for Biotechnology Information (NCBI). 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. This 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%. include, but are not limited to. Indeed any amino acid identity between 50% and 100%, e.g. 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% identity can be useful in describing the present disclosure.

[0273] 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". They refer to polypeptide or nucleic acid sequences 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. These 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 initial, unmodified nucleic acid. These modifications include deletions, substitutions and / or insertions of one or more nucleotides in the nucleic acid fragment. Substantially similar nucleic acid sequences encompass sequences exemplified herein or They can be defined by their ability to hybridize to any portion of the nucleotide sequence 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.

[0274] 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.

[0275] An "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide or protein, or biologically active portion thereof, refers to the polynucleotide or It is substantially or essentially free of components that co-occur with or interact with the protein. Thus, an isolated or purified polynucleotide or protein will be substantially free of other cellular material and culture medium when produced by recombinant techniques, or chemical precursors when chemically synthesized. Virtually free of body and 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, an isolated polynucleotide is about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb that naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide was derived. It may contain nucleotide sequences of less than 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.

[0276] The term "fragment" refers to a contiguous set of nucleotides or amino acids. In one embodiment, the fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 consecutive is a nucleotide with In one embodiment, the fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 consecutive It is an amino acid that Fragments may or may not exhibit the function of sequences sharing some percent identity over the entire length of the fragment.

[0277] 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, regardless of whether the fragment encodes an active protein. For example, fragments can be used to design genes that give rise to desired phenotypes in modified plants. A gene is designed to be used repressively by binding its nucleic acid fragment, whether or not it encodes an active enzyme, to a plant promoter sequence in a sense or antisense orientation. be able to.

[0278] A "gene" refers to a functional molecule such as, but not limited to, a specific protein, including, for example, regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. Included are expressed nucleic acid fragments. "Native gene" refers to a gene as found in its natural endogenous location with its own regulatory sequences.

[0279] 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 is not heterologous.

[0280] 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 are different, the plant is heterozygous at that locus.

[0281] A "coding sequence" refers to a polynucleotide sequence that encodes a specific amino acid sequence. A "regulatory sequence" refers to a nucleotide sequence located upstream (5'-noncoding sequence), within a coding sequence, or downstream (3'-noncoding sequence) of a coding sequence, which is responsible for transcription of the associated coding sequence. , affect RNA processing or 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. .

[0282] 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 changes resulting from the guide polynucleotide / Cas endonuclease system as disclosed herein. A mutant plant is a plant containing a mutant gene.

[0283] 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.

[0284] 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).

[0285] 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 suitable donor DNA polynucleotides). 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.

[0286] "Domain" means a contiguous stretch of nucleotides (which can be RNA, DNA and / or combined RNA-DNA sequences) or amino acids.

[0287] The term "conserved domain" or "motif" refers to a series of polynucleotides or amino acids that are conserved at specific positions along aligned sequences of 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. Identifiers or "signatures" that determine whether a protein with a newly determined sequence belongs to a previously identified protein family, as they are identified by the high degree of conservation of aligned sequences of families of protein homologues can be used as

[0288] 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.

[0289] An "optimized" polynucleotide is a sequence that has been optimized to improve expression in a particular heterologous host cell.

[0290] 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.

[0291] 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 a promoter-specific element or a heterologous element inserted to enhance the level or tissue specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, and / or 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 developmental stages, 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.

[0292] 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. Refers to a promoter that selectively expresses a sequence or functional RNA. Inducible or regulated promoters include, for example, light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wound or ethanol, abscisic acid (ABA), jasmonic acid, salicylic acid or safeners. Promoters that are induced or regulated by chemical substances are included.

[0293] "Translation leader sequence" refers to the polynucleotide sequence 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 described (see, eg, Turner and Foster, (1995) Mol Biotechnol 3:225-236).

[0294] A "3' non-coding sequence," "transcription terminator," or "termination sequence" refers to a DNA sequence located downstream of a coding sequence, which includes polyadenylation recognition sequences and regulatory sequences capable of affecting mRNA processing or expression of a gene. Contains other sequences that encode signals. Polyadenylation signals are usually characterized by affecting the addition of a polyadenylate stretch 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.

[0295] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfectly complementary copy of a DNA sequence, it is called the primary transcript or pre-mRNA. 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 and can be translated into protein 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 blocks expression of a target gene (see, e.g., U.S. Patent No. 5,107,065). want to be). Complementarity of antisense RNA can be with any part 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.

[0296] 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; refers to chromosomes.

[0297] 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 (ie, the coding sequence is under the transcriptional control of the promoter) if the expression of the coding sequence can be controlled. 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 region is the 5' end of the target mRNA and its complement is the 3' end of the target mRNA.

[0298] 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.

[0299] The term "recombination" refers to the artificial combination of two otherwise separate sequence segments, eg, by manipulation of isolated segments of nucleic acids by chemical synthesis or genetic engineering techniques.

[0300] The terms "plasmid," "vector," and "cassette" refer to additional chromosomal elements, often linear or circular, that carry genes that are not part of the central metabolism of the cell, usually in the form of double-stranded DNA. Take. 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, some nucleotide sequences are linked or recombined into unique constructs that are capable of introducing 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.

[0301] 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 and coding sequences that are derived from different sources, or may be derived from the same source but are arranged in a different way that is found in nature. . Such constructs can be used by themselves or 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. Those skilled in the art are familiar with the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells. One skilled in the art will recognize that different independent transformation events result in 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), thus it will also be appreciated that usually multiple events are screened to obtain a strain exhibiting the desired expression level and pattern. 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 expression. Standard molecular biological, biochemical and other analytical methods, including typing, can be performed.

[0302] 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. Non-limiting examples include taxonomically derived differences (e.g., polynucleotide sequences from Zea mays may be different from the genome of the rice (Oryza sativa) plant or Zea mays). heterologous if inserted into a subspecies or cultivar; or derived from a polynucleotide obtained from a bacterium and introduced into a cell of a plant) or sequence divergence (e.g. from Zea mays, Polynucleotide sequences that have been isolated, modified, and reintroduced into maize plants). As used herein, "heterologous" in the context of a sequence is a sequence originating from a different species, subspecies, foreign species, or from the same species if the composition and / or genomic locus is intentional. It may refer to a sequence that has been substantially altered from its natural form by 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 presented 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.

[0303] 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).

[0304] 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).

[0305] 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.

[0306] "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). A CRISPR locus can consist of a CRISPR array containing short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.

[0307] 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. An 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.

[0308] 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-beta protein disclosed herein, 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 thereto. The Cas protein, when complexed with suitable polynucleotide components, can recognize, bind to, and optionally nick or cleave all or part of a specific polynucleotide target sequence. can be a "Cas endonuclease" or "Cas effector protein". Cas-beta 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 %, 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 100% sequence identity and retain at least a portion of the activity of the native sequence Further defined as protein.

[0309] "Functional fragment", "functionally equivalent fragment" and "functionally equivalent fragment" of Cas endonuclease are used interchangeably herein to recognize and bind to a target site, and optionally retained the ability to unwind, nick or cleave (introduce single- or double-stranded breaks within the target site) a portion or subsequence of a Cas endonuclease sequence of the present disclosure point to 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.

[0310] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" of the Cas endonuclease or Cas effector proteins, including Cas-beta, described herein refer to used interchangeably herein and disclosed herein that retain the ability to recognize, bind to, and optionally unwind, nick, or cleave all or part of a target sequence. Cas effector protein variants.

[0311] Cas endonucleases can also include multifunctional Cas endonucleases. 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). domain) and at least one other function, including 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 polypeptide. 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 any of these combination), comprising at least one additional protein domain.

[0312] 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 target nucleic acids complementary to the variable targeting domain of the guide polynucleotide.

[0313] 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 multi-subunit protein complexes that can assemble with polynucleotides to form polynucleotide-protein complexes (PNPs). Here, one of the cascade proteins is a Cas endonuclease capable of recognizing, binding to, and optionally unwinding, nicking or cleaving all or part of a target sequence.

[0314] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. A 7-methylguanylate residue is located on 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.

[0315] The terminology "5'-uncapped" is used herein to refer to RNAs that have, for example, a 5'-hydroxyl group instead of a 5'-cap. Such RNA can be referred to, for example, as "decapped RNA". Since 5'-capped RNA undergoes nuclear export, decapped RNA can more fully accumulate in the nucleus after transcription. One or more of the RNA components herein are decapped.

[0316] 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 can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence).

[0317] 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, respectively. or a portion or partial sequence of a guide RNA, crRNA or tracrRNA of the present disclosure that retains the ability to function as a tracrRNA, respectively.

[0318] The terms "functional variant", "functionally equivalent variant" and "functionally equivalent variant" of guide RNA, crRNA or tracrRNA (respectively) are used interchangeably herein, , crRNA or tracrRNA, respectively.

[0319] 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 and a tracrRNA or tracrRNA fragment of a type II CRISPR / Cas system capable of forming a complex with a type II Cas endonuclease, said guide RNA / Cas endonuclease complex comprising: A Cas endonuclease can be directed 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).

[0320] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and refer to 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.

[0321] 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 can 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 of these. It can be configured with a combination of

[0322] 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 Refers to 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 any Selection allows for nicking or cutting (introducing single- or double-stranded 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.

[0323] The terms "guide RNA / Cas endonuclease complex", "guide RNA / Cas endonuclease system", "guide RNA / Cas complex", "guide RNA / Cas system", "gRNA / Cas complex", "gRNA / Cas system", "RNA-guided endonuclease", "RGEN" are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease capable of forming a complex; wherein said guide RNA / Cas endonuclease complex is capable of guiding the Cas endonuclease to a DNA target site, wherein the Cas endonuclease recognizes, binds to and optionally nicks the DNA target site, or Allows cleavage (introduces single-strand or double-strand breaks).

[0324] The terms "target site", "target sequence", "target site sequence", "target DNA", "target locus", "genomic target site", "genomic target sequence", "genomic target locus" and "protospacer is used interchangeably herein and is recognized by, but not limited to, the guide polynucleotide / Cas endonuclease complex, and optionally nicks or cleaves A polynucleotide sequence, such as a nucleotide sequence on a chromosome, episome, locus, or any other DNA molecule in the genome of a cell (e.g., chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA) Point. The target site may be an endogenous site in the genome of the cell, or alternatively, the target site may be heterologous to the cell and thus naturally occurring in the genome of the cell, or A target site may be found in a heterologous genomic location relative to the location where it occurs in nature. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein and are endogenous or naturally occurring in the genome of the cell, It refers to a target sequence in its endogenous or natural location in the genome. "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that is 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.

[0325] 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 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.

[0326] "Altered target site", "altered target sequence", "altered target site", "altered target sequence" are used interchangeably herein and refer to at least one alteration as compared to the unaltered target sequence. Refers to a target sequence disclosed herein comprising a Such "changes" include, for example, (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical modification of at least one nucleotide. changes, or (v) any combination of (i) to (iv).

[0327] "Modified nucleotide" or "edited nucleotide" refers to a nucleotide sequence of interest that contains at least one change when compared to its unmodified nucleotide sequence. Such "changes" include, for example, (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) Chemical change or any combination of (v) (i) to (iv) can be mentioned.

[0328] Methods for "altering a target site" and "altering a target site" are used interchangeably herein and refer to a method for generating a modified target site.

[0329] As used herein, "donor DNA" is a DNA construct containing a polynucleotide of interest to be inserted into the Cas endonuclease target site.

[0330] The term "polynucleotide modification template" includes polynucleotides that contain at least one nucleotide modification compared to the nucleotide sequence to be edited. Nucleotide alterations can be substitutions, additions or deletions 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.

[0331] 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.

[0332] "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.

[0333] 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 a cell wall. The term "plant organ" refers to a plant tissue or a group of tissues that constitute both morphologically and functionally independent parts of a plant. As used herein, "plant element" is synonymous with "part" of a plant and refers to any part of a plant, which may include separate tissues and / or organs and may be referred to throughout by the term "tissue". may be used interchangeably. Similarly, a "plant reproductive element" generally refers to any part of a plant that is capable of initiating other plants by sexual or asexual reproduction of that plant, including but not limited to seeds, seedlings, It is intended to refer to roots, shoots, cuttings, scions, grafts, stolons, bulbs, tubers, corms, cakes or buds. Plant elements can be in plants or plant organs, tissue cultures or cell cultures.

[0334] "Progeny" includes any subsequent generation of a plant.

[0335] 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. It refers to plant cells such as ovules, seeds, leaves, flowers, branches, fruits, grains, panicles, cobs, pods, stalks, roots, root tips, anthers, and parts thereof themselves. Grain is intended to mean mature seed produced by a grower for purposes other than seed cultivation or propagation. Progeny, variants and mutants of regenerated plants are also included within the scope of the present invention, provided that some of them contain the introduced polynucleotide.

[0336] The terms "monocotyledonous" or "monocotyledonous" refer to the subclass of angiosperms, also known as the "monocotyledoneae," whose seeds generally have only one early leaf or cotyledon. including. The term includes references to whole plants, plant elements, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells and their progeny.

[0337] The term "dicotyledonous" or "dicotyledonous" refers to the subclass of angiosperms, also known as the "dicotyledoneae," whose seeds generally have only two early leaves or cotyledons. including. The term includes references to whole plants, plant elements, plant organs (eg, leaves, stems, roots, etc.), seeds, plant cells and their progeny.

[0338] 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.

[0339] 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).

[0340] In the context of the present disclosure, the term "crossed" or "hybrid" or "crossing" means the fusion of gametes by pollination to produce progeny (i.e. cells, seeds or plants). . The term includes sexual crossing (pollination of one plant by another plant) and selfing (self-pollination, i.e. pollen and ovules (endoplasmic reticulum and megaspores) of the same plant or genetically identical plants). of the body).

[0341] 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 parent plants, wherein at least one parent plant The body has 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.

[0342] 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 were grown into two different embryos, one undergoing a treatment (such as the introduction of a CRISPR-Cas effector endonuclease) and a control without such treatment. can be divided into groups. Thus, the phenotypic differences between the two groups may be due only to treatments and not to any peculiarities of the plant's endogenous genetic makeup.

[0343] "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.

[0344] A "polynucleotide of interest" includes a nucleotide sequence that encodes a protein or polypeptide that improves crop desirability, ie, traits of agronomic benefit. Polynucleotides of interest include agronomically important traits, herbicide resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, virus resistance, fertility or sterility. , crop traits, commercial products, phenotypic markers or other traits of agricultural or commercial importance. Polynucleotides of interest may further be utilized in the sense or antisense orientation. Additionally, two or more polynucleotides of interest may be utilized together or "stacked" to provide additional benefits.

[0345] A "complex trait locus" includes a genomic locus having multiple transgenes that are genetically linked together.

[0346] The compositions and methods herein can provide improved "agronomic traits" or "agronomically important traits" or "agronomically beneficial traits" to plants and such traits may include, but are not limited to: disease tolerance, drought tolerance, heat tolerance compared to isogenic lines of plants not modified by the methods or compositions described herein; cold tolerance, salinity tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water efficiency, improved nitrogen utilization, improved nitrogen fixation, pest tolerance, herbivore tolerance, pathogen tolerance, increased yield, enhanced health, Improving vitality, improving growth, improving photosynthesis, fortifying, modifying protein content, modifying oil content, increasing biomass, increasing root length, improving root structure, modulating metabolites, modulating proteome, seeds Weight gain, modified seed carbohydrate composition, modified seed oil composition, modified seed protein composition, modified seed nutritional composition.

[0347] 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.

[0348] As used herein, the terms "reduced", "less", "slower" and "increased", "faster", "enhanced", "greater" are modified Refers to a reduction or increase in the properties of a modified plant element or resulting plant as compared to a plant element or resulting plant that does not contain the modified plant element. 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 the 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 less higher than treated controls.

[0349] As used herein, the term “before” refers to the presence of one sequence upstream or 5′ to another sequence with respect to sequence position.

[0350] The meaning of the abbreviations is as follows: "sec" means seconds, "min" means minutes, "h" means hours, "d" means days, "μL" 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.

[0351] Classification of CRISPR-Cas systems CRISPR-Cas systems have been classified according to component sequence and structural analysis. Multiple, including 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) A CRISPR / Cas system has 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, Molecular Cell 60, 1-13; Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6):e60; and Koonin et al. 2017, Curr Opinion Microbiology 37:67-78).

[0352] 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 contains an array of identical repeats interspersed with DNA targeting spacers that encode crRNA components and an operon-like unit of the cas gene that encodes the 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).

[0353] RNA transcripts (pre-crRNA) of CRISPR loci are cleaved specifically at repeat sequences by CRISPR-associated (Cas) endoribonucleases in type I and type III systems or RNase III in type II systems. The number of CRISPR-associated genes at a given CRISPR locus can vary by species.

[0354] 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 may serve more than one purpose, for example Cas9, among others, contains a domain for endonuclease function and a domain for targeted cleavage.

[0355] The Cas endonuclease is induced by a single CRISPR RNA (crRNA) through direct RNA-DNA base pairing and recognizes DNA target sites adjacent to the protospacer adjacent motif (PAM) (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).

[0356] 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 include 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 many 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).

[0357] 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 , functions as a non-catalytic RNA-binding protein, whereas in the type I-C system, crRNA processing can be catalyzed by Cas5 (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). are divided into seven subtypes (Makarova et al. 2011, Nat Rev Microbiol. 2011 9(6):467-477; Koonin et al. 2017, Curr Opinion Microbiology 37:67-78), at least protein subunits. A modified type I CRISPR-associated complex (cascade) for adaptive antiviral defense comprising Cas7, Cas5 and Cas6, one of these subunits synthetically fused to the Cas3 endonuclease or the modified restriction endonuclease FokI. (International Publication No. 2013098244 pamphlet published on July 4, 2013).

[0358] 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.

[0359] 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.

[0360] 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.

[0361] Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to direct the Cas endonuclease to a DNA target. The crRNA base-pairs with a spacer region complementary to one strand of the 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. , including 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).

[0362] 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 responsible for targeted cleavage. It is an active RNA-guided endonuclease that does not necessarily require additional trans-activating CRISPR (tracr) RNA for activation.

[0363] 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.

[0364] 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-beta.

[0365] 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.

[0366] Described herein are newly identified CRISPR-Cas systems that include novel sequences of cas genes. Further novel cas genes and proteins are described.

[0367] One distinguishing feature of the novel Cas-beta system is the locus structure as described in FIG. The order of the polynucleotides on the complementary strand is as follows: an unclassified cas gene that shares low sequence identity with the cas-beta gene, cas1 gene, cas2 gene and cas4. The unclassified cas gene was followed by a CRISPR array containing 37 repeats of 37 nucleotide sequences, 33 of which were identical, 3 of which were 97.3% identical, and one at the end. exists with 78.4% identity. The spacer polynucleotide between each array repeat is 35 or 36 nucleotides long.

[0368] 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

[0369] 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 ).

[0370] 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).

[0371] 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.

[0372] The novel Cas-beta proteins disclosed herein include effector proteins (endonucleases) and adaptation proteins.

[0373] 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.

[0374] 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 complexed with the Cas effector protein (hereinafter Optionally, at least one DNA strand is cleaved when mediated by recognition of the target sequence by, but not limited to, crRNA or guide RNA. Such recognition and cleavage of a target sequence by a Cas endonuclease generally requires that the correct protospacer adjacent motif (PAM) is located at or at the 3' end of the DNA target sequence. Occurs when adjacent. 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 DNA target sequences. (See also 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).

[0375] Cas endonucleases can occur as individual effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).

[0376] 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.

[0377] 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 (by deletion, knockout of the Cas3-helicase domain, or by mutagenesis of key residues, or as previously described (Sinkunas, T, et al., 2013, EMBO J.32:385-394) by abolishing the cleavage readiness cascade involving the modified Cas3 endonuclease by assembling the reaction in the absence of ATP as can be converted to a nickase (because the HD domain is still functional) Disabling HD endonuclease activity includes, but is not limited to, mutagenesis of key residues in the HD domain, known in the art. and can convert a cleavage-ready cascade comprising a modified Cas3 endonuclease into a helicase.The abrogation of both Cas helicase and Cas3 HD endonuclease activity includes, but is not limited to: A cleavage-ready cascade comprising a modified Cas3 endonuclease, which can be achieved by methods known in the art, such as mutagenesis of key residues in both the helicase and HD domains, binds to the target sequence. It can be converted into protein.

[0378] Cas9 (previously called Cas5, Csn1 or Csx12) complexes with cr and tracr nucleotides or a single guide polynucleotide to specifically recognize and cleave all or part of a DNA target sequence. is a Cas endonuclease that 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 strand of DNA at the target sequence (cooperative action of both domains results in DNA double-strand breaks, while activity of one domain leads to nicking. bring). 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 flanked by HNH domains. (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 exist in a complex with a single guide RNA (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).

[0379] Cas12 (previously called Cpf1 and variants c2c1, c2c3, CasX and CasY) contained the RuvC nuclease domain and generated a staggered 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 mutational studies in other Cas12 proteins demonstrated that the Nuc domain contributes to induction and target binding along with the RuvC domain involved in cleavage (Swarts et al., Mol Cell 2017, 66:221-233 e224). .

[0380] 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. Cas endonucleases can also be engineered to function as RNA-guided recombinases and through 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).

[0381] Cas-beta endonuclease A Cas-beta endonuclease is defined as a functional RNA-guided PAM-dependent dsDNA cleavage protein of less than 900 amino acids and contains at least one of the motifs listed in Table 1B.

[0382] The RuvC domain has been demonstrated in the literature to contain endonuclease functionality. Cas-beta endonucleases can be isolated or identified from genetic loci comprising arrays containing cas-beta genes and multiple repeats that encode effector proteins. In some embodiments, the cas-beta locus may further comprise part or all of the cas1 gene, the cas2 gene and / or the cas4 gene.

[0383] 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).

[0384] Cas-beta endonucleases are RNA-guided endonucleases that can bind and cleave double-stranded DNA targets that contain (1) sequences that share homology with the nucleotide sequence of the guide RNA and (2) PAM sequences. is. In some aspects, the PAM is T-rich. In some aspects, the PAM is C-rich.

[0385] A Cas-beta endonuclease functions as a double-strand break inducer and can be a nickase or a single-strand break inducer. In some embodiments, a catalytically inactive Cas-beta endonuclease can be used to target or recruit to a target DNA sequence, but does not induce cleavage. In some embodiments, a catalytically inactive Cas-beta protein can be used with a functional endonuclease that cleaves the target sequence. In some embodiments, a catalytically inactive Cas-beta 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.

[0386] The Cas-beta endonuclease is at least 50, 50-100, at least 100, 100-150 of any of SEQ ID NO: 13, 14, 15 or 57, or a functional fragment or functional variant thereof that retains at least a portion of the activity , 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 continuously 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 It is further defined as a sex-sharing RNA-induced double-stranded DNA-breaking protein. A "functional fragment" of a Cas-beta endonuclease is capable of recognizing or binding or nicking a single strand of a double-stranded polynucleotide or cleaving both strands of a double-stranded polynucleotide. Possess abilities or any combination thereof.

[0387] Cas-beta endonucleases contain one or more motifs listed in Table 1B.

[0388] The Cas-beta endonuclease is any of the sequences in the sequence listing 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, 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 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 100% sequence identity. .

[0389] Cas endonucleases, effector proteins or functional fragments thereof for use in the disclosed methods may be from natural sources or genetically modified host cells which have been modified to express nucleic acid sequences encoding the proteins. It can be isolated from alternative sources. Alternatively, the Cas protein can be produced using cell-free protein expression systems or produced synthetically. Effector Cas nucleases can be isolated and introduced into heterologous cells, or can 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.

[0390] 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. No. 2016186953 and WO2016186946 published Nov. 24, 2016, but are not limited thereto.

[0391] 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. 20140068797, published Mar. 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.

[0392] The Cas endonuclease can 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: methylation Enzyme 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 are also fused to proteins that 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. can.

[0393] Catalytically active and / or inactive Cas endonucleases can be fused to heterologous sequences (US20140068797, published Mar. 6, 2014). Suitable fusion partners include, but are not limited to, activities that indirectly increase transcription by acting directly on target DNA or polypeptides associated with target DNA (e.g., histones or other DNA binding proteins). and polypeptides that provide 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-beta 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. al. Nature biotechnology, volume 32, number 6, June 2014).

[0394] Catalytically active or inactive Cas proteins, such as the Cas-beta proteins described herein, are molecules that direct the editing of single or multiple bases in a polynucleotide sequence, e.g. can also be fused with a site-specific deaminase that can be changed, for example, from C G to T A or from A T to G C (Gaudelli et al., Programmable base editing of A T to G C in genomic DNA without DNA cleavage.”Nature(2017);Nishida et al.“Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.”Science 353(6305)(2016);Komor et al.“Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.”Nature 533(7603)(2016):420-4. Base-editing fusion proteins, for example, are active (double-strand-cleaving), partially active (nickases) or inactivated (catalytically inactive) Cas-beta 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.

[0395] Cas endonucleases described herein can be obtained by methods known in the art, e.g. It can be expressed and purified.

[0396] To date, certain PAM sequences have been recognized (WO 2016186953 published November 24, 2016, WO 2016186946 published November 24, 2016 and Zetsche B et al. 2015. Cell 163, 1013), describes a number of Cas endonucleases capable of cleaving target DNA at specific positions. It will be appreciated that those skilled in the art will be able to use any inducible endonuclease system based on the methods and embodiments described herein using the novel inducible Cas system. It is understood that it is possible to adapt

[0397] 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 (uninode) or multiple (e.g., binodes) of basic, positively charged residues (e.g., lysine and / or arginine). ) and can be positioned anywhere in the Cas amino acid sequence so that it is exposed on the protein surface. The NLS can be operably linked, for example, 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 binode VirD2 nuclear localization signal of the Cas codon region (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442- 6) is operatively connected to; Non-limiting examples of suitable NLS sequences herein include those disclosed in US Pat. Nos. 6,660,830 and 7,309,576.

[0398] guide polynucleotide The guide polynucleotide allows target recognition, binding and optionally cleavage of the target by the Cas endonuclease, and can be single or double molecules. A guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide may comprise at least one nucleotide, phosphodiester linkages or linkage modifications such as 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 molecules, linkage to polyethylene glycol molecules, linkage to spacer 18 (hexaethylene glycol chain) molecules or cyclization The resulting 5' to 3' covalent bond includes, but is not limited to. Guide polynucleotides comprising only ribonucleic acid are also referred to as "guide RNA" or "gRNA" (U.S. Patent Application Publication Nos. 20150082478, published March 19, 2015 and published February 26, 2015). US Patent Application Publication No. 20150059010). Guide polynucleotides can be engineered or synthesized.

[0399] 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 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.

[0400] 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.

[0401] The cr nucleotides are part of 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. Contains a nucleotide sequence (also called a 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 contiguous stretch of DNA nucleotides) or "crRNA" (consisting of a contiguous stretch of RNA nucleotides). when composed), 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 , 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:22, 23 and 24.

[0402] In some embodiments, the tracr nucleotide sequence is "tracrRNA" (when composed of a contiguous stretch of RNA nucleotides), or "tracrDNA" (when composed of a contiguous stretch of DNA nucleotides), or " called 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, the Cas endonuclease recognizing the target site, binding to the target site, and optionally nicking or cleaving the target site (introducing single- or double-strand breaks). ). (U.S. Patent Application Publication No. 20150082478 published March 19, 2015 and U.S. Patent Application Publication No. 20150059010 published February 26, 2015).

[0403] In some embodiments, the tracrRNA molecule is selected from the group consisting of SEQ ID NOs:25-30.

[0404] 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 complementary to the nucleotide sequence of the target DNA. A sequence domain and a second nucleotide sequence domain that interacts with the Cas endonuclease polypeptide.

[0405] 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 from the group consisting of DNA sequences, RNA sequences and combinations thereof. A guide polynucleotide of choice.

[0406] 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. 20150082478, published February 26, 2015).

[0407] The guide RNA comprises a duplex molecule comprising a chimeric non-natural crRNA linked to at least one tracrRNA. Chimeric non-naturally occurring 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.

[0408] 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 within the target DNA and a Cas endonuclease recognition domain (CER domain) that interacts with the Cas endonuclease polypeptide. including. In some embodiments, the sgRNA molecule is selected from the group consisting of SEQ ID NOs:31-36.

[0409] 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 nucleotides and tracr nucleotides is called "single guide RNA" (if it is composed of a continuous stretch of RNA nucleotides) or "single guide DNA" (a continuous stretch 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. the 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 enable the (U.S. Patent Application Publication No. 20150082478 published March 19, 2015 and U.S. Patent Application Publication No. 20150059010 published February 26, 2015).

[0410] 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).

[0411] The nucleotide sequence linking the cr and tracr nucleotides of the single 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") linking 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, It can be 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides long. In another embodiment, the nucleotide sequence linking the cr and tracr nucleotides of the single guide polynucleotide can include, but is not limited to, a tetraloop sequence, such as a GAAA tetraloop sequence.

[0412] A guide polynucleotide can be obtained by chemically synthesizing a guide polynucleotide (including, but not limited to, Hendel et al. 2015, Nature Biotechnology 33, 985-989), in vitro generation of a guide polynucleotide and / or guide RNA. can be produced by any method known in the art, including but not limited to self-splicing of (Xie et al., 2015, PNAS 112:3570-3575).

[0413] 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.

[0414] "Randomized PAM" and "randomized protospacer-adjacent motif" are used interchangeably herein and refer to 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.

[0415] Guide polynucleotide / Cas endonuclease complex The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding to, and optionally nicking, unwinding or cleaving all or part of a target sequence.

[0416] 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 each variants thereof that retain the activity of part or all of the 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 may be used in both DNA target sequences. It is a good example of a Cas protein that can cut strands.

[0417] 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 typically 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. A non-limiting example of a Cas9 nickase suitable for use herein is disclosed in US Patent Application Publication No. 20140189896, 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 in close proximity for 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 then result in incomplete repair leading to non-homologous end-joining NHEJ (mutations). easy) 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 to 50, at least 50, 50 to 60, at least 60, 60 to 70, at least 70, 70 to 80, at least 80, 80 to 90, at least 90, 90 to 100, or 100 or more (or any integer from 5 to 100 ), 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 (ie, Cas9 HNH+ / RuvC−) that has a mutated RuvC domain but a functional HNH domain 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.

[0418] 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) (eg, a repressor or activator, such as any of those disclosed herein).

[0419] 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 functional fragment thereof is PGEN covalently or non-covalently linked to or assembled with.

[0420] 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, attached to, and optionally scored, unwound or cut.

[0421] The Cas effector protein can be the Cas-beta effector protein disclosed herein.

[0422] 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-beta effector protein, wherein said guide polynucleotide The nucleotide / Cas effector protein complex can recognize, bind to, and optionally cleave, unwind or cleave all or part of the target sequence.

[0423] 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 may 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.

[0424] The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein comprises at least three Cas proteins selected from the group consisting of Cas1, Cas2 and optionally one further Cas protein, including Cas4. It further comprises different protein subunits or functional fragments thereof.

[0425] In one aspect, the guide polynucleotide / Cas effector protein complex (PGEN) described herein is a PGEN, wherein said Cas effector protein is covalently bound to at least one protein subunit or functional fragment thereof or PGEN that is non-covalently linked. The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein polypeptide comprises a Cas1 protein subunit, a Cas2 protein subunit and optionally one further Cas protein subunit comprising 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. The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein comprises at least two Cas proteins selected from the group consisting of Casl, Cas2 and optionally one further Cas protein, including Cas4. Covalently or non-covalently linked or assembled into different protein subunits. The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein said Cas effector protein is selected from the group consisting of Casl, Cas2 and optionally one additional Cas protein comprising Cas4 and combinations thereof. covalently or non-covalently linked to at least three different protein subunits.

[0426] 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 isolated from heterologous cells or cells by any method known in the art. It can be introduced into organisms.

[0427] Recombinant constructs for cell transformation those further comprising a disclosed guide polynucleotide, a Cas endonuclease, a polynucleotide modified template, donor DNA, a guide polynucleotide / Cas endonuclease system disclosed herein and optionally one or more polynucleotides of interest 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 seeds produced by the methods described herein. is mentioned.

[0428] 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.

[0429] Vectors and constructs include circular plasmids and linear polynucleotides, which include the polynucleotide of interest and optionally other components, including linkers, adapters, regulatory elements or analytical elements. In some examples, recognition sites and / or target sites may be contained within introns, coding sequences, 5'UTRs, 3'UTRs and / or regulatory regions.

[0430] Components for the expression and utilization of novel CRISPR-Cas systems in prokaryotic and eukaryotic cells The present invention provides guide RNAs capable of recognizing, binding to, and optionally nicking, unwinding or cleaving all or part of a target sequence in prokaryotic or eukaryotic cells / organisms. Further provided are expression constructs for expressing the / Cas system.

[0431] In one embodiment, the expression construct of the present disclosure is 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.

[0432] Nucleotide sequence modifications of the guide polynucleotide, the VT domain and / or the CER domain include 5′ caps, 3′ polyadenylation tails, riboswitch sequences, stable regulatory sequences, sequences that form dsRNA duplexes, and guide polynucleotides. Modifications or sequences that target internal positions, modifications or sequences that provide tracking, modifications or sequences that provide 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, linkage to cholesterol molecule, linkage to polyethylene glycol molecule, linkage to spacer 18 molecule, 5' to 3' or any combination thereof, including, but not limited to: These modifications may confer at least one additional advantageous characteristic, which may be modification or modulation of stability, intracellular targeting, tracking, fluorescent labeling, for proteins or protein complexes. is selected from the group of binding sites for , modified binding affinity for complementary target sequences, modified resistance to cellular degradation and increased cell permeability.

[0433] 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 (US20150082478, published March 19, 2015). A method for expressing RNA components without a 5' cap has been described (WO 2016 / 025131, published February 18, 2016).

[0434] A variety of methods and compositions can be used to obtain cells or organisms 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, the polynucleotide of interest is introduced into the biological cell by means of a donor DNA construct.

[0435] The donor DNA construct further comprises first and second regions of homology that flank the polynucleotide of interest. The first and second homologous regions of the donor DNA have homology to the first and second genomic regions, respectively, that are present in or adjacent to the target site of the genome of the cell or organism.

[0436] Donor DNA can be bound to a guide polynucleotide. Tethered donor DNA may allow co-localization of target and donor DNA useful for genome editing, gene insertion and regulation of the target genome, and the function of the endogenous HR machinery is greatly enhanced. It may be useful for targeting postmitotic cells that are thought to be depressed (Mali et al., 2013, Nature Methods Vol. 10:957-963).

[0437] 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 with unit integer values ​​within the range up to the full length of the target site and / or Includes all areas. 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 expressed 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 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) Laboratory Techniques in See Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0438] 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 genome is such that those sequences undergo homologous recombination, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0439] A homologous region 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 have homology to fragments of the target site as well as downstream genomic regions.

[0440] 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, the first fragment and the second fragment being different.

[0441] Polynucleotide of interest Polynucleotides of interest are described in detail herein and include polynucleotides that reflect commercial market interests related to commercial markets and crop development. Target crops and markets will change, and new crops and technologies will 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.

[0442] 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. Polynucleotides of more specific 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 or pesticides). genes encoding proteins that confer resistance to biological stresses (such as those that confer resistance to toxins such as pesticides and herbicides); including, but not limited to, genes that encode proteins that confer resistance to disease progression, such as cancer.

[0443] 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.

[0444] 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. Pest resistance genes can encode resistance to pests that inhibit high yields, such as liceworm, cutworm, European corn borer. Disease and insect resistance genes such as lysozyme or cecropins that protect antibacterial properties or proteins such as defensins, glucanases or chitinases that protect antifungal properties or Bacillus thuringiensis endotoxins, proteases that control nematodes or insects. 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). Insect resistance genes can encode resistance to pests that inhibit high yields, such as liceworm, cutworm, 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).

[0445] A protein obtained by expression of a "herbicide resistance protein" or a "nucleic acid molecule encoding herbicide resistance" has the ability to tolerate higher concentrations of herbicides than cells that do not express the protein, or the ability to tolerate higher concentrations of herbicides than cells that do not express the protein. Proteins that confer on cells the ability to tolerate specific concentrations of herbicides for a period of time are included. 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 Introduced into plants by encoding genes (e.g. EPSP synthase gene and GAT gene), genes encoding resistance to HPPD inhibitors (e.g. HPPD gene) or other such genes known in the art can do. For example, U.S. Pat. See US Patent 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.

[0446] 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. Modifications of the antisense sequence can be made so long as the sequence hybridizes to the corresponding mRNA and prevents expression of the corresponding mRNA. Antisense constructs with 70%, 80% or 85% sequence identity to the corresponding antisense sequence can be used in this method. 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 may be used.

[0447] Furthermore, the polynucleotide of interest can be used in a sense orientation to suppress expression of endogenous genes in the plant. Methods for suppressing gene expression in plants by polynucleotides in the sense orientation are known in the art. The method generally involves transforming a plant with a DNA construct comprising a promoter driving expression in the plant, operably linked to at least a portion of a nucleotide sequence corresponding to transcription of an endogenous gene. is included. Typically, such nucleotide sequences have 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. have sex. See US Pat. Nos. 5,283,184 and 5,034,323.

[0448] A polynucleotide of interest can 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 favorably or against favorably, a molecule or cell containing a molecule or cell under certain conditions. contains a DNA segment that allows 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.

[0449] 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 modifying enzymes. , the inclusion of DNA sequences that have been unaffected or subjected to effects such as chemicals, as well as the inclusion of DNA sequences that are required for specific modifications (eg, methylation) that allow their identification.

[0450] 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).

[0451] 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. 20130263324, published Oct. 3, 2013 and International Publication No. WO 2013 / 112686, published Aug. 1, 2013 can be stacked together into compound trait loci as shown.

[0452] Polypeptides of interest include any protein or polypeptide encoded by the polynucleotides of interest described herein.

[0453] 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. 20090133152, published May 21, 2009. The method also includes recovering plants from plant cells that contain the polynucleotide of interest integrated into the genome. Plants can 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.

[0454] Sequence optimization for expression in plants Methods for synthesizing plant-preferred 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 gene Removal of such other well-characterized sequences that may be detrimental to 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.

[0455] 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 can be "minimal," meaning a short sequence derived from a natural source that still functions as an expression regulator or modifier. Alternatively, an expression element can be "optimized," meaning that its polynucleotide sequence has been altered from its natural state in order to function with more desirable characteristics in a particular host cell. (eg, but not limited to, a bacterial promoter can be "maize optimized" to improve its expression in maize plants). Alternatively, the expression element can be "synthetic," meaning that the expression element is designed in silico and synthesized for use in the host cell. Synthetic expression elements can be wholly synthetic or partially synthetic (including fragments of naturally occurring polynucleotide sequences).

[0456] Certain promoters have been shown to be 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 at low levels in other tissues. It is often referred to as a "tissue-specific promoter" or "tissue-preferred promoter" when it induces RNA synthesis in .

[0457] 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. .

[0458] 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);

[0459] Tissue-preferred promoters can be used to target enhanced expression within specific plant tissues. Tissue-preferred promoters include, for example, International Publication No. 2013103367 pamphlet 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.

[0460] 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); 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 WO2000012733, published Mar. 9, 2000. This document discloses seed-preferred promoters from the END1 and END2 genes.

[0461] 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 can be a chemically inducible promoter, in which application of a chemical induces gene expression, or a chemically repressible promoter, in which 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 1993001294 published Jan. 21, 1993) and salicylic acid. The activated tobacco PR-1a promoter (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).

[0462] 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.

[0463] Stress-inducible promoters include the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91). Those skilled in the art are familiar with protocols for simulating stress conditions such as drought, osmotic stress, salt stress and temperature stress, as well as protocols for assessing stress tolerance of plants subjected to simulated or naturally occurring stress conditions. Familiar with.

[0464] Another example of an inducible promoter useful for plant cells is the ZmCAS1 promoter described in US Patent Application Publication No. 20130312137, published Nov. 21, 2013.

[0465] 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.

[0466] Genome modification with novel CRISPR-Cas system components As described herein, the derived Cas endonuclease 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).

[0467] 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).

[0468] 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 at nicks (single-strand breaks) may occur by a different mechanism than HDR for double-strand breaks (Davis and Maizels. PNAS (0027-8424), 111(10), p.E924-E932) .

[0469] 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, homology of at least 150-200 bp 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.).

[0470] gene targeting The guide polynucleotide / Cas system described herein can be used for gene targeting.

[0471] In general, DNA targeting can be carried out by cleaving one or both strands at a specific polynucleotide sequence in a cell in which the Cas protein associates with a suitable polynucleotide component. 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.

[0472] The length of the target site DNA sequence may vary, e.g. , 28, 29, 30 or more than 30 nucleotides. The target site may be palindromic, ie, it is further possible that a sequence on one strand reads 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, resulting in single-stranded overhangs (5 ' overhang or 3' overhang). Active mutants 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.

[0473] Assays for measuring target site single- or double-strand cleavage 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.

[0474] 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. is implemented by the targeting method of

[0475] gene editing The process of editing a genome sequence that combines a DSB and a modified template generally involves recognizing a target sequence in a chromosomal sequence and introducing a DSB inducer or a nucleic acid encoding a DSB inducer capable of introducing a DSB into a genomic sequence and a nucleic acid 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. A polynucleotide modification template can further comprise a nucleotide sequence that flanks the 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. 20150082478 published March 19, 2015; Publication No. 2015026886, WO2016007347 published January 14, 2016 and WO2016 / 025131 published February 18, 2016.

[0476] Several uses of guide RNA / Cas endonuclease systems have been described (e.g., US Patent Application Publication No. 20150082478A1, published March 19, 2015, published February 26, 2015). See International Publication No. WO2015026886 and U.S. Patent Application Publication No. 20150059010, published Feb. 26, 2015), which include alterations or substitutions of nucleotide sequences of interest (such as regulatory elements), Insertion of polynucleotide, gene knockout, gene knock-in, splicing site modification and / or introduction of alternative splicing site, modification of the nucleotide sequence encoding the target protein, amino acid and / or protein fusion and inverted repeats in the target gene including, but not limited to, gene silencing by expressing .

[0477] Proteins can be altered 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 evaluated 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.

[0478] 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. To facilitate optimal expression and nuclear localization (for eukaryotic cells), genes containing complexes are optimized as described in WO2016186953 published Nov. 24, 2016. , and subsequently delivered to cells as a DNA expression cassette by methods known in the art. The components necessary to contain the active complex are 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 (WO2017070032 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 an uncapped mRNA (Zhang et al.2016 Nat.Commun.7:12617) or as a Cas protein guide polynucleotide complex (WO2017070032 published April 27, 2017), or any of these can be delivered as a combination of To generate crRNA in vivo, a tRNA-derived element is used to direct the crRNA transcript to its DNA target site, e.g., as described in WO2017105991, published June 22, 2017. endogenous RNAse can be recruited to cleave to the mature form that can induce the complex to 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), The result is an RNA-guided helicase that can be used to enhance homologous recombination repair, induce transcriptional activation, or remodel DNA local structures. Furthermore, the Cas cleavage domain and helicase domain activities are both knocked out and other DNA cleavages, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA reassembly, DNA deamination, DNA unwinding, DNA It can be used in combination with recombination enhancement, DNA integration, DNA inversion and DNA repair agents.

[0479] 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 Nov. 24, 2016. It can be estimated as described in WO2016186946 and WO2016186953 published on November 24, 2016.

[0480] 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. ., 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 the respective complexes. can be examined.

[0481] 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 (ii) deletion of at least one nucleotide; (iii) insertion of at least one nucleotide; chemical alteration of at least one nucleotide; and (v) any combination of (i) to (iv) selected from the group consisting of

[0482] The edited nucleotide can be located within or outside the target site recognized and cleaved by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not at the target site recognized and cleaved by the Cas endonuclease. In another embodiment, between at least one nucleotide to be edited and the genomic target site, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides.

[0483] Knockouts can 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.

[0484] 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.

[0485] Methods for editing nucleotide sequences in the genome of cells may be methods that do not use exogenous selectable markers by restoring the function of non-functional gene products.

[0486] 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, said donor DNA comprising a polynucleotide of interest and optionally at or near said target site. identifying at least one cell that has integrated said polynucleotide of interest into.

[0487] In one aspect, the methods disclosed herein may employ homologous recombination (HR) to provide for integration of the polynucleotide of interest at the target site.

[0488] Various methods and compositions can be used to generate 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 means of 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 homologous regions of the donor DNA have homology to the first and second genomic regions, respectively, that are present in or adjacent to the target site of the genome of the cell or organism.

[0489] Donor DNA can be tethered to a guide polynucleotide. Tethered donor DNA may allow co-localization of target and donor DNA useful for genome editing, gene insertion and regulation of the target genome, and the function of the endogenous HR machinery is greatly enhanced. It may be useful for targeting postmitotic cells that are thought to be depressed (Mali et al., 2013, Nature Methods Vol. 10:957-963).

[0490] 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 with unit integer values ​​within the range up to the full length of the target site and / or Includes all areas. 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 as percent sequence identity over the fully aligned length of the two polynucleotides, including at least about 50%, 55%, 60%, 65%, 70% , 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 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) Laboratory Techniques in See Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0491] Episomal DNA molecules can also ligate within double-strand breaks, e.g., integrate T-DNA within 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 maturation of the double-strand break, the gene conversion pathway may be altered, for example, by nondividing somatic cells or sister chromatids after DNA replication. The original structure can be restored if homologous sequences, such as bodies, are available (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA modification templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0492] In one embodiment, the disclosure includes a method of editing a nucleotide sequence in the genome of a cell, comprising introducing into at least one PGEN and a polynucleotide modified template described herein (herein wherein said polynucleotide modified template comprises at least one nucleotide modification of said nucleotide sequence), optionally further comprising selecting at least one cell containing the edited nucleotide sequence.

[0493] 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. 20150082478, published March 19, 2015 and International Publication No. WO2015026886, published February 26, 2015).

[0494] The polynucleotide and / or trait of interest may be at a composite trait locus, as described in WO2012129373 published September 27, 2012 and WO2013112686 published August 1, 2013. Can be stacked together. 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.

[0495] The guide polynucleotide / Cas system described herein mediating gene targeting is described in Methods for Directing Heterologous Gene Insertion and / or for Generating Complex Trait Loci Containing Multiple Heterologous Genes, September 2012 It can be used in a manner similar to that disclosed in WO2012129373, published on 27th, in which the use of a double-strand break-inducing agent is used to introduce the gene of interest. Instead, 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 (e.g. , U.S. Patent Application Publication No. 20130263324 published Oct. 3, 2013 or WO 2012129373 published Mar. 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 bred with both transgenic traits as a single locus. By repeating this process, any number of desired traits can be accumulated.

[0496] Further uses of the guide RNA / Cas endonuclease system have been described (e.g., US Patent Application Publication No. 20150082478 published March 19, 2015; No. 2015026886, U.S. Patent Application Publication No. 20150059010 published February 26, 2015, International Publication No. 2016007347 published January 14, 2016 and February 18, 2016 PCT Application No. WO 2016025131), which includes alterations or substitutions of nucleotide sequences of interest (such as regulatory elements), insertions of polynucleotides of interest, gene knockouts, gene knockins, splicing site modifications. Gene silencing by introducing modifications and / or 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. is not limited to

[0497] Characteristics obtained 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 drawn to encompass the markers 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 obscures 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 are co-segregating with a desired phenotypic trait, it is often difficult to determine whether each of these markers specifies the same QTL or two different QTLs. , it is ambiguous. 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 specific window.

[0498] Introduction of CRISPR-Cas system components into cells The methods and compositions disclosed herein do not rely on the particular method of introducing the sequences into an organism or cell, simply entering the polynucleotide or polypeptide inside at least one cell of this organism. be. Introduction includes reference to integration of a nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid can be integrated into the genome of the cell, and of a nucleic acid, protein or polynucleotide-protein complex (PGEN, RGEN). Reference to transient (direct) delivery to cells is included.

[0499] 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) Including, but not limited to, mediated direct protein delivery, topical application, sexual mating, sexual breeding and any combination thereof.

[0500] 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 a guide RNA (or crRNA + tracrRNA) that is operably linked to a specific promoter within the cell that can transcribe the guide RNA (crRNA + tracrRNA molecule) within the cell. can also be introduced indirectly by introducing a recombinant DNA molecule containing a heterologous nucleic acid segment encoding ). 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.

[0501] Plant cells differ from animal cells (such as human cells), fungal cells (such as yeast cells) and protoplasts, eg plant cells contain a plant cell wall that can act as a barrier to the delivery of components.

[0502] 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.

[0503] A Cas endonuclease, such as the Cas endonucleases described herein, may be a Cas polypeptide itself (referred to as direct delivery of the Cas endonuclease), an mRNA encoding a Cas protein and / or a guide polynucleotide / Cas endonuclease complex itself. can be introduced into cells by direct introduction using any method known in the art. A Cas endonuclease can also be introduced into cells indirectly 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 is facilitated using cell penetrating peptides (CPPs) as described in WO2016073433 published May 12, 2016. be able to. 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

[0504] 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 methods, cell permeable peptides or mesoporous silica nanoparticles (MSN) mediated direct protein delivery can be successfully used for delivery of polynucleotide modified templates in eukaryotic cells such as plant cells. .

[0505] 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. The donor DNA may exist transiently within the cell or it may 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.

[0506] 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 Direct delivery (co-delivery) of other mRNAs ). For example, the guide polynucleotide / Cas endonuclease component (and / or the guide polynucleotide / Cas endonuclease complex itself) may be used as 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 mRNAs encoding transcriptional activators such as ), as described in WO2017070032, published April 27, 2017. The function of the sex gene product can be restored, allowing selection and enrichment of cells without the use of exogenous selectable markers.

[0507] 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 guide and protein and protein subunits for Cas endonuclease, or functional fragments thereof) or as recombinant constructs expressing 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 cells (described herein). can be assembled by any means known in the art, prior to being targeted for genomic modification as is done).

[0508] 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).

[0509] 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) (27 Apr 2017). See also WO2017070032, published on 2017). The delivery matrix may comprise any one of the components, eg Cas endonuclease, which is attached to a solid matrix (eg impacting particles).

[0510] 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.

[0511] In one aspect, the guide polynucleotide / Cas endonuclease complex is a guide RNA and a Cas endonuclease protein that form a guide RNA / Cas endonuclease complex and at least one protein subunit of the complex is a cellular is a complex that is introduced into

[0512] In one aspect, the guide polynucleotide / Cas endonuclease complex comprises guide RNA and a Cas endonuclease protein forming a guide RNA and Cas endonuclease complex (cleavage-ready complex) and at least one protein subunit of the complex. is a complex that is preassembled in vitro and introduced into cells as a ribonucleotide-protein complex.

[0513] 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; Sanford et al., (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al., (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen, (1991) In vitro Cell Dev Biol 27P:175-82 (soybean); et al., (1998) Theor Appl Genet 96:319-24 (soybean); Datta et al., (1990) Biotechnology 8:736-40 (rice); Klein et al., (1988) Proc. Natl. USA 85:4305-9 (corn); Klein et al., (1988) Biotechnology 6:559-63 (corn); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646. Specification; Klein et al., (1988) Plant Physiol 91:440-4 (maize); Fromm et al., (1990) Biotechnology 8:833-9 (maize); Hooykaas-Van Slogteren et al., (1984) ) Nature 311:763-4; U.S. Patent No. 5,736,369 (cereals); Bytebier et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al, (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al., (Longman, New York), pp.197-209 (pollen); Kaeppler et al., (1990) Plant Cell Rep. 9:415-8) and Kaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al., (1992) Plant Cell 4:1495-505 (electroporation ); Li et al., (1993) Plant Cell Rep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al., (1996) Nat Biotechnol 14:745- 50 (corn via Agrobacterium tumefaciens).

[0514] Alternatively, polynucleotides can be introduced into a plant or plant cell by contacting the cell or organism 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.

[0515] 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.

[0516] 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. 20110035836, published February 10, 2011 and European Patent Application Publication No. 2821486A1, published January 7, 2015.

[0517] Other methods of introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts can be used, such as plastid transformation methods and methods of introducing polynucleotides into tissues from seedlings or mature seeds.

[0518] 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.

[0519] 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.

[0520] 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 plants produced by the methods described herein. Includes body and seed. Any plant, including monocotyledonous and dicotyledonous plants and plant elements, can be used with the compositions and methods described herein.

[0521] Examples of monocotyledonous plants that can be used include, but are not limited to, maize (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.

[0522] 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) · Hippogaea (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum).

[0523] 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, ornamentals and conifers.

[0524] 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 cucumbers such as cucumber (C. sativus), cantaloupe (C. cantalupensis) and muskmelon (C. melo) (Cucumis) members. 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.

[0525] 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 Contorta (Pinus contorta) and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesii); American hemlock (Tsuga canadensis); redwoods (Sequoia sempervirens); Sakhalin firs, such as European fir (Abies amabilis) and balsam fir (Abies balsamea); cedars such as Thuja plicata) and Alaskan Yellow Cedar (Chamaecyparis nootkatensis).

[0526] 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.

[0527] The disclosure finds use in breeding plants containing one or more introduced traits or edited genomes.

[0528] 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.

[0529] 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 fish. organisms of the class containing 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 monoploid cells, diploid cells, germ cells, neurons, muscle cells, endocrine or exocrine cells, epithelial cells, muscle cells, tumor cells, germ cells, hematopoietic 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.

[0530] 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.

[0531] Genome modification with 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 phenotypic or physiologically important trait of interest 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 phenotypic or physiologically important trait of interest is selected from the following group: improved overall health, reversal of disease, amelioration of disease, stabilization of disease, prevention of disease, parasitism Treatment of worm 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 (e.g., 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, cystic fibrosis, Durham disease, Down syndrome, Duane's 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, palatal-cardiofacial syndrome, WAGR syndrome and Wilson's disease), treatment of congenital immune disorders (e.g., but not limited to immunoglobulin subclass deficiency), acquired immunity Treatment of disorders (such as, but not limited to, AIDS and other HIV-related disorders), treatment of cancer, and treatment of diseases, including rare or "orphan" conditions, for which there are no other effective treatment options. .

[0532] 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 as antiviral, antipathogenic or anticancer therapeutics, agricultural can be implanted into a subject for the production of genetically engineered organisms or for biological research.

[0533] 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.

[0534] 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-beta orthologue polypeptide. . Insertions may be performed by non-homologous end joining or homologous recombination.

[0535] 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-beta orthologue. In one aspect, the sequence of the target polynucleotide is unknown prior to modification, and treatment with the Cas-beta orthologue is used as part of the method to determine the sequence of said target polynucleotide.

[0536] Polynucleotide modification with Cas-beta orthologues 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-betatorsolog is obtained from or derived from an Armatimonadetes bacterium. In some embodiments, the Cas-beta orthologue is a polypeptide sharing at least 80% identity with SEQ ID NOs:13-15. In some embodiments, the Cas-beta orthologue is a functional variant of any of SEQ ID NOs: 13-15. In some embodiments, the Cas-beta orthologue is a functional fragment of any of SEQ ID NOS: 13-15. In some embodiments, the Cas-beta orthologue is a Cas-beta polypeptide encoded by a polynucleotide selected from the group consisting of SEQ ID NOs: 13-15. In some embodiments, the Cas-beta orthologue is a Cas-beta polypeptide that recognizes a PAM sequence listed in any of Tables 4-83. In some embodiments, the Cas-beta orthologue is a Cas-beta polypeptide identified from an organism listed in the sequence listing.

[0537] In some aspects, Cas-beta orthologues are provided as Cas-beta polynucleotides. In some embodiments, the Cas-beta polynucleotide is at least 80%, 85%, 90%, 95%, 97%, 99% or 100% any one of SEQ ID NOs: 1-85 or SEQ ID NOs: 10-12. It is selected from the group consisting of arrays that share %.

[0538] In some embodiments, the Cas-beta tolusolog is an unmodified wild-type Cas-beta tolutosolog, a functional Cas-beta tolutosolog variant, a functional Cas-beta tolutosolog fragment, a fusion protein comprising an active or inactivated Cas-beta tolusolog, Cas-beta-tolusologs, biotinylated Cas-betatolusologs, Cas-betatolusolognicases, Cas-, further comprising one or more nuclear localization sequences (NLS) at the C-terminus or N-terminus or both the N- and C-termini It may be selected from the group consisting of a beta tol solog endonuclease, a Cas-beta tol solog further comprising a histidine tag, and mixtures of any two or more thereof.

[0539] In some embodiments, the Cas-beta orthologue 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 target A fusion protein that further comprises a polynucleotide sequence or a transgene that is heterologous to the cell from which said target polynucleotide sequence is obtained or derived.

[0540] In some embodiments, multiple Cas-beta orthologues may be desired. In some embodiments, the plurality of Cas-beta torusologs may comprise Cas-beta torusologs from different biological sources or from different genetic loci within the same organism. In some embodiments, the plurality of Cas-beta torusologs may comprise Cas-beta torusologs that have different binding specificities to a target polynucleotide. In some embodiments, the plurality of Cas-beta tolusologs may comprise Cas-beta tolusologs with different cleavage efficiencies. In some embodiments, the plurality of Cas-beta tolusologs may comprise Cas-beta tolusologs with different PAM specificities. In some embodiments, the plurality of Cas-beta tolsologs may comprise orthologs of different molecular composition, ie, polynucleotide Cas-beta tolsologs and polypeptide Cas-beta tolsologs.

[0541] 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.

[0542] Cas-beta orthologue and / or guide polynucleotide storage conditions include parameters relating to temperature, state of matter and time. In some embodiments, the Cas-beta orthologues 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, the Cas-beta orthologues and / or guide polynucleotides are stored as liquids, frozen liquids or lyophilized powders. In some embodiments, the Cas-beta orthologue 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.

[0543] any or all of the reactive polynucleotide components (e.g., guide polynucleotides, donor polynucleotides, optionally Cas-beta polynucleotides) as part of a vector, construct, linearized or circularized plasmid, or It can be provided 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.

[0544] A method for modification of a target polynucleotide involves combining minimal elements to form a Cas-beta orthologue (or variant, fragment or other related molecule as described above), substantially complementary to the target polynucleotide sequence of the target polynucleotide. creating a reaction mixture containing guide polynucleotides containing sequences that hybridize selectively, and target polynucleotides for modification. In some aspects, Cas-beta orthologues are provided as polypeptides. In some aspects, Cas-beta orthologues are provided as Cas-beta polynucleotides. In some embodiments, guide polynucleotides are provided as RNA molecules, DNA molecules, RNA:DNA hybrids or polynucleotide molecules comprising chemically modified nucleotides.

[0545] The storage buffer or reaction mixture for any one of the components 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.

[0546] 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 above 40°C. The i...

Claims

1. 1. A synthetic composition comprising: (a) a Cas endonuclease comprising an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, and 57, or a functional fragment thereof; (b) a target double-stranded DNA polynucleotide, the target double-stranded DNA polynucleotide being heterologous to a 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. wherein the Cas endonuclease or said functional fragment thereof recognizes a PAM sequence on the target double stranded DNA polynucleotide, the guide polynucleotide and the Cas endonuclease or said functional fragment thereof form a complex that binds to the target double stranded DNA polynucleotide, and the Cas endonuclease or said functional fragment thereof comprises DNA binding activity.

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

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

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

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

6. The synthetic composition of claim 4 , wherein the heterologous polynucleotide is a transgene.

7. The synthetic composition of claim 4 , wherein the heterologous polynucleotide is a donor DNA molecule.

8. The synthetic composition of claim 4 , wherein the heterologous polynucleotide is a modified polynucleotide template.

9. 1. A synthetic composition comprising: (a) a Cas protein or a functional fragment thereof comprising an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, and 57; (b) a target double-stranded DNA polynucleotide, the target double-stranded DNA polynucleotide being heterologous to the source of the Cas protein; and (c) a guide polynucleotide comprising a variable targeting domain that comprises a region complementary to the target double-stranded DNA polynucleotide. wherein the Cas protein recognizes a PAM sequence on the target double-stranded DNA polynucleotide, the guide polynucleotide and the Cas protein form a complex that binds to the target double-stranded DNA polynucleotide, and the Cas protein comprises DNA binding activity and lacks double-stranded DNA cleavage activity; The functional fragment is a portion or subsequence of the Cas protein that retains the ability to recognize, bind to, and optionally unwind, nick or cleave a target site.

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

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

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

13. 10. The synthetic composition of claim 1, further comprising a eukaryotic cell, said cell being non-human.

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

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

16. 15. The synthetic composition of claim 14, 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.

17. 1. A construct comprising a polynucleotide encoding a Cas protein operably linked to a heterologous expression element, wherein the Cas protein shares at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, and 57, and wherein the Cas protein comprises DNA binding activity.

18. The synthetic composition of claim 1 , wherein at least one component is attached to a solid matrix.

19. 1. A method for introducing a targeted edit into a target polynucleotide, comprising: (a) a Cas endonuclease or a functional fragment thereof comprising a polypeptide at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, and 57, wherein the Cas endonuclease recognizes a PAM sequence on the target polynucleotide, and the Cas endonuclease comprises a DNA binding activity; and (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 endonuclease form a complex capable of recognizing and binding to the target polynucleotide. providing a heterogeneous composition comprising: the target polynucleotide is not present in a human cell; The method, wherein the functional fragment is a portion or subsequence of the Cas endonuclease sequence that retains the ability to recognize, bind to, and optionally unwind, nick or cleave a target site.

20. 20. The method of claim 19, further comprising introducing at least one nucleotide modification into the genome of at least one cell of an organism, compared to a target sequence in the genome of the cell prior to introduction of the heterologous composition, incubating the cells and generating a whole organism from the cells, and confirming the presence of the at least one nucleotide modification in the genome of at least one cell of the organism, compared to a target sequence in the genome of the cell prior to introduction of the heterologous composition.

21. 21. The method of claim 20, wherein the cell is a eukaryotic cell.

22. 22. The method of claim 21 , wherein the eukaryotic cell is derived or obtained from an animal, fungus or plant.

23. 23. The method of claim 22, wherein the plant is a monocotyledonous or dicotyledonous plant.

24. 23. The method of claim 22, wherein the plant is 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.

25. 21. The method of claim 20, further comprising introducing a heterologous polynucleotide.

26. 26. The method of claim 25, wherein the heterologous polynucleotide is either a donor DNA molecule or a polynucleotide modified template comprising a sequence that is at least 50% identical to a sequence of the cell.

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