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 claims the benefit of U.S. Provisional Patent Application Nos. 62 / 980750, filed February 24, 2020, and 63 / 030964, filed May 28, 2020, each of which is incorporated herein by reference in its entirety.

[0002] Reference to an electronically submitted sequence listing An official copy of the Sequence Listing has been submitted electronically via EFS-Web as an ASCII formatted Sequence Listing in a file named 8207-WO-PCT_SequenceListing_ST25.txt, created on February 4, 2021, and having a size of 158,138 bytes, and is being submitted contemporaneously herewith. The Sequence Listing contained in this ASCII format document is a part of the present specification and 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 a novel RNA-guided Cas endonuclease system and compositions and methods for editing or modifying the genome of a cell. [Background technology]

[0004] Recombinant DNA technology has made it possible to insert DNA sequences into targeted genomic locations and / or modify specific endogenous chromosomal sequences. Site-specific integration technology using site-specific recombination systems, as well as other types of recombinant technology, has been used to generate targeted insertions of genes of interest in various organisms. Genome editing technologies such as designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or homing meganucleases can be used to generate targeted genome perturbations, but these systems tend to use designer nucleases that have low specificity and need to be redesigned for each target site, which makes their production costly and time-consuming.

[0005] A newer technology has been identified that utilizes the archaeal or bacterial adaptive immune system, termed CRISPR (clustered regularly interspaced short palindromic repeats), which contains various domains of effector proteins encompassing diverse activities (recognition, binding and optionally cleavage of DNA).

[0006] Despite the identification and characterization of some of these systems, there remains a need to identify novel effectors and systems and demonstrate activity in eukaryotes, particularly animals and plants, for editing endogenous and previously introduced heterologous polynucleotides. Summary of the Invention [Problem to be solved by the invention]

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

[0008] Compositions and methods are provided 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.

[0009] In one aspect, a synthetic composition is provided comprising 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 the effector protein does not comprise an HNH domain; and a heterologous polynucleotide.

[0010] In one aspect, a synthetic composition is provided comprising a CRISPR-Cas effector protein comprising at least one domain upstream of a RuvC-I domain 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, wherein said effector protein does not comprise an HNH domain; and a heterologous polynucleotide.

[0011] In one aspect, a synthetic composition is provided comprising: a CRISPR-Cas effector protein comprising the set of domains 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-finger domain; and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide.

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

[0013] In one aspect, a CRISPR-Cas effector protein is provided that includes: (1) 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 the CRISPR-Cas effector protein does not include an HNH domain; (2) a CRISPR-Cas effector protein that includes at least one domain upstream of the RuvC-I domain 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, wherein the CRISPR-Cas effector protein does not include an HNH domain; and (3) a positively charged bridging helix-like domain, in this order. (3) a CRISPR-Cas effector protein comprising a set of domains including a bridge-helix-like domain, a DNA-binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridge-helix domain, a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide, the heterologous polynucleotide being derived from a different class of organism than that from which the effector protein is derived or is wholly or partially artificial.

[0014] In one aspect, (1) 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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain 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, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; (3) this Provided are synthetic compositions comprising, in order, a CRISPR-Cas effector protein comprising the following domains: 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 RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide, wherein the heterologous polynucleotide is an engineered guide RNA functional in a eukaryotic cell.

[0015] In one aspect, a CRISPR-Cas effector protein is provided, comprising: (1) 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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain 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, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a CRISPR-Cas effector protein comprising, in this order, (3) a CRISPR-Cas effector protein comprising a set of domains in the form of 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 RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide, wherein the CRISPR-Cas effector protein further comprises a nuclear localization signal.

[0016] In one aspect, a CRISPR-Cas effector protein is provided, which comprises: (1) at least two bridge-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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain selected from the group consisting of a positively charged bridge-helix-like domain and a DNA-binding domain optionally comprising a helix-turn-helix motif, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a CRISPR-Cas effector protein comprising at least two bridge-helix-like domains, a DNA-binding domain optionally comprising a helix-turn-helix motif, in this order. (3) a CRISPR-Cas effector protein comprising a set of domains: 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 RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide, wherein the CRISPR-Cas effector protein is Cas-beta or a functional fragment thereof.

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

[0018] In one aspect, a CRISPR-Cas effector protein is provided, comprising: (1) at least two bridge-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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain selected from the group consisting of a positively charged bridge-helix-like domain and a DNA-binding domain optionally comprising a helix-turn-helix motif, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a CRISPR-Cas effector protein comprising, in this order, a putative positively charged bridge-helix-like domain. (1) a CRISPR-Cas effector protein comprising a set of domains in (i) 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, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (2) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide, wherein the CRISPR-Cas effector protein is a fusion protein comprising a functional fragment of Cas-beta, and the fusion protein further comprises an additional nuclease domain.

[0019] In one aspect, a synthetic composition is provided comprising a fusion protein of Cas-beta or a functional fragment or functional 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.

[0020] In one aspect, a synthetic composition is provided comprising a fusion protein of Cas-beta or a functional fragment or functional 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; and further comprising a heterologous polynucleotide or polypeptide.

[0021] In one aspect, a synthetic composition is provided comprising: 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 the CRISPR-Cas effector protein does not comprise an HNH 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.

[0022] In one aspect, a synthetic composition is provided comprising, in that order: 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; and at least one protein that is a complex comprising Cas1, Cas2, and a Cas protein that is not Cas1 or Cas2.

[0023] In one aspect, any of the synthetic compositions described herein can have at least one component optimized for expression in a eukaryotic cell, hi some aspects, the cell is derived from an organism selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, and tomato.

[0024] In one aspect, a synthetic composition is provided 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 the effector protein does not comprise an HNH domain; and a heterologous polynucleotide.

[0025] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding a CRISPR-Cas effector protein comprising at least one domain upstream of a RuvC-I domain 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, including a CRISPR-Cas effector protein that does not include an HNH domain.

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

[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 aspect, a CRISPR-Cas effector protein comprising: (1) at least two bridge-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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain selected from the group consisting of a positively charged bridge-helix-like domain and a DNA-binding domain optionally comprising a helix-turn-helix motif, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a CRISPR-Cas effector protein comprising at least two bridge-helix-like domains, a DNA-binding domain optionally comprising a helix-turn-helix motif, in this order. and (3) a CRISPR-Cas effector protein comprising a set of domains in the following order: a RuvC-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 RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide encoding one of the following heterologous polynucleotides, the heterologous polynucleotide being derived from a different class of organism than that from which the effector protein is derived or is wholly or partially artificial.

[0029] In one aspect, a CRISPR-Cas effector protein is provided, comprising: (1) 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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain 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, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) in this order: Provided are synthetic compositions comprising: (1) a CRISPR-Cas effector protein comprising a set of domains: a positively charged putative bridge-helix-like domain, a DNA-binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridge-helix domain, a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (2) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide, wherein the heterologous polynucleotide encodes one of the heterologous polynucleotides that is an engineered guide RNA functional in a eukaryotic cell.

[0030] In one aspect, a CRISPR-Cas effector protein is provided, comprising: (1) at least two bridge-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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain selected from the group consisting of a positively charged bridge-helix-like domain and a DNA-binding domain optionally comprising a helix-turn-helix motif, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a CRISPR-Cas effector protein comprising at least two bridge-helix-like domains, a DNA-binding domain optionally comprising a helix-turn-helix motif, in this order. and (3) a CRISPR-Cas effector protein comprising a set of domains in the following order: a putative bridge-helix-like domain derived from a RuvC-I domain, a bridge-helix domain, a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, optionally including a helix-turn-helix motif; a putative bridge-helix-like domain derived from a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not include an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide encoding one of the following: a putative bridge-helix-like domain derived from a RuvC-II domain, a DNA-binding domain optionally including a helix-turn-helix motif, a RuvC-I domain, a bridge-helix domain, a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not include an HNH domain; and a heterologous polynucleotide, wherein the CRISPR-Cas effector protein further comprises a nuclear localization signal.

[0031] In one aspect, a CRISPR-Cas effector protein is provided, comprising: (1) 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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain 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, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a positively charged putative bridging helix-like domain, in this order. and (3) a CRISPR-Cas effector protein comprising a set of domains in the above sequence: a bridge-helix-like domain, a DNA-binding domain optionally comprising a helix-turn-helix motif, a RuvC-I domain, a bridge-helix domain, a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not contain an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a polynucleotide encoding one of the following heterologous polynucleotides, wherein the CRISPR-Cas effector protein is Cas-beta or a functional fragment thereof.

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

[0033] In one aspect, a CRISPR-Cas effector protein is provided, comprising: (1) at least two bridge-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 the CRISPR-Cas effector protein does not comprise an HNH domain; (2) a CRISPR-Cas effector protein comprising at least one domain upstream of the RuvC-I domain selected from the group consisting of a positively charged bridge-helix-like domain and a DNA-binding domain optionally comprising a helix-turn-helix motif, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (3) a CRISPR-Cas effector protein comprising, in this order, a putative positively charged bridge-helix-like domain, a helix-turn-helix motif, and a RuvC-II domain. (3) a CRISPR-Cas effector protein comprising a set of domains including a DNA-binding domain optionally comprising a turn-helix motif, a RuvC-I domain, a bridge helix domain, a RuvC-II domain, a zinc-finger domain, and a RuvC-III domain, wherein the CRISPR-Cas effector protein does not comprise an HNH domain; and (4) a CRISPR-Cas effector protein derived from Armatimonadetes; and a heterologous polynucleotide; wherein the CRISPR-Cas effector protein is a fusion protein comprising a functional fragment of Cas-beta, and the fusion protein further comprises an additional nuclease domain.

[0034] In one aspect, a synthetic composition is provided that comprises a polynucleotide encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain, and a heterologous polynucleotide, and further comprising at least one protein.

[0035] In one aspect, a synthetic composition is provided that comprises a polynucleotide of 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 the CRISPR-Cas effector protein does not comprise an HNH domain, and a heterologous polynucleotide, further comprising a polynucleotide encoding at least one protein, wherein the polynucleotide is selected from the group consisting of cas1, cas2, and one additional cas gene.

[0036] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding 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 does not comprise an HNH domain; and further comprising a polynucleotide encoding at least one protein comprising the set of genes cas1, cas2, and one additional cas gene, in that order.

[0037] In one aspect, any of the synthetic compositions described herein can have at least one component optimized for expression in a eukaryotic cell, hi some aspects, the cell is derived from an organism selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, and tomato.

[0038] In one aspect, a synthetic composition is provided comprising a polynucleotide encoding 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 the effector protein does not comprise an HNH domain, and a heterologous polynucleotide.

[0039] In one aspect, at least 250, 250 to 500, at least 500, 500 to 600, at least 600, 600 to 700, at least 700, 700 to 750, at least 750, 750 to 800, at least 800, 800 to 825, or more than 825 consecutive amino acids of SEQ ID NO: 13, 14, 15, or 57 and at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least Provided are synthetic compositions comprising a CRISPR-Cas effector protein that shares at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity; and a heterologous polynucleotide or a heterologous polypeptide.

[0040] In one aspect, at least 250, 250 to 500, at least 500, 500 to 600, at least 600, 600 to 700, at least 700, 700 to 750, at least 750, 750 to 800, at least 800, 800 to 850, at least 850, 850 to 900, at least 900, 900 to 950, at least 950, 950 to 1000, at least 1000, 1000 to 1250, at least 1250, 1250 to 1500, at least 1500, 1500 to 1750, at least 1750, 1750 to 2000, at least 2000 or more than 2000 consecutive nucleotides of SEQ ID NO: 10, 11 or 12 and at least 50%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 125%, 150%, 150%, 175 ... Provided are synthetic compositions comprising polynucleotides encoding CRISPR-Cas effector proteins that share between 0% and 55%, at least 55%, 55% and 60%, at least 60%, 60% and 65%, at least 65%, 65% and 70%, at least 70%, 70% and 75%, at least 75%, 75% and 80%, at least 80%, 80% and 85%, at least 85%, 85% and 90%, at least 90%, 90% and 95%, at least 95%, 95% and 96%, at least 96%, 96% and 97%, at least 97%, 97% and 98%, at least 98%, 98% and 99%, at least 99%, 99% and 100%, or 100% sequence identity.

[0041] In one aspect, there is provided an engineered non-naturally occurring CRISPR-Cas system comprising: a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide.

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

[0043] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is 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, 50 0 to 550, at least 550, 550 to 600, at least 600, 600 to 650, at least 650, 650 to 700, at least 700, 700 to 750, at least 750, 750 to 800, at least 800, 800 to 850, at least 850, 850 to 900, at least 900, 900 to 950, 950 to 100 or more than 1000 consecutive nucleotides and at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, sharing 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100% or 100% sequence identity.

[0044] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide, wherein the CRISPR-Cas effector protein comprises 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, ... 50 to 400, at least 400, 400 to 450, at least 450, 450 to 500, at least 500, 500 to 525, at least 525, 525 to 540 amino acids or at least 540 consecutive amino acids and at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70% , 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100% or 100% sequence identity.

[0045] In one aspect, a synthetic composition is provided comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; further comprising at least one gene encoding the protein.

[0046] In one aspect, a synthetic composition is provided comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and further comprising a plurality of genes encoding the at least two proteins.

[0047] In one aspect, a synthetic composition is provided comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; further comprising at least one gene encoding a protein, wherein the gene encoding the protein is selected from the group consisting of cas1, cas2, and a cas gene that is not cas1 or cas2, and optionally is cas4.

[0048] In one aspect, a synthetic composition is provided that comprises a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; further comprising a plurality of genes encoding at least two different proteins, the plurality of genes being selected from the group consisting of, in an order, cas1 adjacent to cas2, cas1 not adjacent to a cas gene that is not cas2, and cas2 adjacent to both a cas1 gene and a gene encoding another Cas protein that is not Cas1 or Cas2.

[0049] In one aspect, a synthetic composition is provided that comprises 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; and a heterologous polynucleotide; and further comprises a plurality of genes encoding at least two different proteins, wherein the plurality of genes are, in the following order: cas1, cas2, and a gene that is not cas1 or cas2.

[0050] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; further comprising at least one gene encoding a protein, wherein said polynucleotide sequence encoding the CRISPR-Cas effector protein is upstream of at least one of the encoding proteins.

[0051] In one aspect, a synthetic composition is provided comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; further comprising at least two genes encoding different proteins, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is downstream of at least two genes encoding two different proteins selected from the group consisting of cas1, cas2, and a gene that is not cas1 or cas2.

[0052] In one aspect, a synthetic composition is provided comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide; further comprising at least three genes encoding different proteins, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is downstream of at least three genes encoding three different proteins selected from the group consisting of cas1, cas2, and a gene that is not cas1 or cas2.

[0053] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide comprising regulatory elements capable of effecting transcription of said polynucleotide sequence encoding the CRISPR-Cas effector protein.

[0054] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein has been sequence-optimized for improved expression in eukaryotic cells.

[0055] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is sequence-optimized for improved expression in a eukaryotic cell, wherein the eukaryotic cell is a plant cell, an animal cell, a fungal cell, or a protist.

[0056] In one aspect, there is provided a synthetic composition comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is sequence-optimized for improved expression in a eukaryotic cell, and the eukaryotic cell further comprises an existing heterologous polynucleotide.

[0057] In one aspect, a synthetic composition is provided comprising a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a heterologous polynucleotide, wherein the polynucleotide sequence encoding the CRISPR-Cas effector protein is sequence-optimized for improved expression in a eukaryotic cell, wherein the eukaryotic cell is a plant cell, and the plant cell is derived from a plant 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, 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. The mechanism of attachment can be covalent or non-covalent.

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

[0061] In one aspect, a synthetic composition is provided comprising: a eukaryotic cell; 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, wherein the guide polynucleotide and 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 aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence, and wherein the heterologous CRISPR-Cas effector protein comprises at least 500 amino acids.

[0063] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence, and wherein the heterologous CRISPR-Cas effector protein comprises 1000 amino acids or less.

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

[0065] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave, the target sequence, and wherein the heterologous CRISPR-Cas effector protein comprises at least 800 amino acids but fewer than 900 amino acids.

[0066] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise 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 the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence, and wherein the heterologous CRISPR-Cas effector protein comprises at least 800 amino acids but fewer than 875 amino acids.

[0067] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; at least one protein; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in a genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence, and wherein the heterologous CRISPR-Cas effector protein comprises fewer than 875 amino acids.

[0068] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise 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 the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence, and wherein the heterologous CRISPR-Cas effector protein comprises at least 800 amino acids but fewer than 900 amino acids.

[0069] In one aspect, a synthetic composition is provided comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; at least one protein; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in a genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence, wherein the heterologous CRISPR-Cas effector protein comprises at least 800 amino acids, and the protein is selected from the group consisting of Cas1, Cas2, and another protein that is not Cas1 or Cas2.

[0070] In one aspect, a synthetic composition is provided comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; at least one protein; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in a genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence, wherein the heterologous CRISPR-Cas effector protein comprises fewer than 875 amino acids, and the protein is selected from the group consisting of Cas1, Cas2, and another protein that is not Cas1 or Cas2.

[0071] In one aspect, a synthetic composition is provided comprising: a eukaryotic cell; a heterologous 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 the CRISPR-Cas effector protein does not comprise an HNH domain; at least one protein; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in a genome of the cell, wherein the guide polynucleotide and the heterologous effector protein are capable of forming a complex that recognizes, binds to, and optionally nicks or cleaves the target sequence, wherein the heterologous CRISPR-Cas effector protein comprises at least 800 amino acids but fewer than 860 amino acids, and wherein the protein is selected from the group consisting of Cas1, Cas2, and another protein that is not Cas1 or Cas2.

[0072] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous molecule selected from the group consisting of a cas-beta gene capable of expressing a Cas-beta effector protein or a functional variant or fragment thereof in said eukaryotic cell and a Cas-beta effector protein or a functional variant or fragment thereof; 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 Cas-beta effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence.

[0073] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a cas-beta gene capable of expressing a Cas-beta effector protein or a functional variant or functional fragment thereof in said eukaryotic cell; and a heterologous molecule selected from the group consisting of a Cas-beta effector protein or a functional variant or functional fragment thereof; 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 Cas-beta effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence; wherein said cas-beta gene is 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 up to 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 consecutive nucleotides and at least 50%, 50%-55%, at least 55%, sharing 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100% or 100% sequence identity.

[0074] In one aspect, there is provided a synthetic composition comprising: a eukaryotic cell; a heterologous molecule selected from the group consisting of a cas-beta gene and a Cas-beta effector protein or a functional variant or functional fragment thereof capable of expressing a Cas-beta effector protein or a functional variant or functional fragment thereof 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, wherein said guide polynucleotide and Cas-beta effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence; and wherein said Cas-beta effector protein is at least 50, 50-100, at least 100, 100-150, at least 150, 150-200, at least 200, 200-250 of SEQ ID NO: 13, 14, 15 or 57. 0, 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 consecutive amino acids and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, or at least or 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity.

[0075] In one aspect, there is provided a synthetic composition comprising at least two different heterologous molecules selected from the group consisting of 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, a cas gene capable of expressing a Cas protein that is not Cas1 or Cas2 in said eukaryotic cell, 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 protein; 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 Cas-beta effector protein are capable of forming a complex that is capable of recognizing, binding to, and optionally nicking or cleaving said target sequence.

[0076] In one aspect, a synthetic composition is provided comprising a plurality of different heterologous molecules selected from the group consisting of 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, a cas gene capable of 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; wherein said guide polynucleotide and the Cas-beta effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, and wherein said plurality of heterologous molecules are comprised within a contiguous polynucleotide sequence comprising the following genes in an order selected from the group consisting of cas-beta adjacent to cas1, cas1 adjacent to cas2, cas2 adjacent to both cas1 and another gene that is not cas1 or cas2, cas-beta adjacent to cas1 adjacent to cas2, and cas-beta adjacent to cas1 adjacent to cas2 that is adjacent to another cas gene.

[0077] In one aspect, a synthetic composition is provided comprising a plurality of different heterologous molecules selected from the group consisting of 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 polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of said cell; said guide polynucleotide and Cas-beta effector protein are capable of forming a complex that is capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, wherein said plurality of heterologous molecules are comprised within a contiguous polypeptide sequence comprising the following proteins in an order selected from the group consisting of Cas-beta adjacent to Cas1, Cas1 adjacent to Cas2, Cas2 adjacent to both Cas1 and another gene that is not Cas1 or Cas2, Cas-beta adjacent to Cas1 adjacent to Cas2, Cas-beta adjacent to Cas1 adjacent to Cas2, Cas-beta adjacent to Cas1 adjacent to Cas2 that is adjacent to another cas protein that is not Cas1 or Cas2.

[0078] In one aspect, a synthetic composition is provided comprising a eukaryotic cell and a heterologous CRISPR-Cas array comprising at least two repeats that are at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100% or 100% identical to SEQ ID NO: 19, 20 or 21. In some embodiments, the repeats are separated by spacer polynucleotides of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more than 35 nucleotides.

[0079] In one aspect, a synthetic composition is provided comprising a eukaryotic cell and a heterologous CRISPR-Cas array comprising at least three repeats that are at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100% or 100% identical to SEQ ID NO: 19, 20 or 21. In some embodiments, the repeats are separated by spacer polynucleotides of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more than 35 nucleotides.

[0080] In one embodiment, a eukaryotic cell and a nucleic acid sequence encoding SEQ ID NO: 19, 20 or 21 are linked to at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% In some embodiments, synthetic compositions are provided comprising heterologous CRISPR-Cas arrays comprising at least 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, or 37 repeats that are 99% to 99%, at least 99%, 99% to 100%, or 100% identical. In some embodiments, the repeats are separated by spacer polynucleotides of at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or more than 35 nucleotides.

[0081] In one aspect, any of the disclosed compositions or methods provides a eukaryotic cell further comprising a pre-existing heterologous polynucleotide.

[0082] In one aspect, there is provided a method for modifying a target sequence in the genome of a eukaryotic cell, comprising administering to said cell a polynucleotide sequence encoding at least two bridge-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 does not comprise an HNH domain; at least two bridge-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 does not comprise an HNH domain. a Cas-beta polynucleotide; a Cas-beta polypeptide or a functional variant or functional fragment thereof; SEQ ID NO: 10, 11, or 12; and SEQ ID NO: 13, 14, 15, or 57; further comprising a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell; and incubating the cell at a temperature above 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex that can recognize, bind to, and optionally nick or cleave the target sequence.

[0083] In one aspect, there is provided a method for modifying a target sequence in the genome of a eukaryotic cell, comprising administering to the cell a polypeptide encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; 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 polypeptide sharing at least 70% to 100% identity with SEQ ID NO: 13, 14, 15, or 57, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence.

[0084] In one aspect, there is provided a method for modifying a target sequence in the genome of a eukaryotic cell, comprising administering to the cell: 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 the CRISPR-Cas effector protein does not comprise an HNH domain; a polynucleotide sequence encoding 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 the effector protein does not comprise an HNH domain; a cas-beta polynucleotide; a Cas-beta polypeptide or functional fragment thereof or and a functional variant thereof; SEQ ID NO:3; and SEQ ID NO:4; further comprising at least one composition selected from the group consisting of a Cas1 polynucleotide, a Cas1 polypeptide, a Cas2 polynucleotide, a Cas2 polypeptide, a Cas polynucleotide that is not Cas1 or Cas2, a polynucleotide sequence in the Sequence Listing and a polypeptide in the Sequence Listing, a Cas polypeptide that is not Cas1 or Cas2, a heterologous polynucleotide, and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell; and incubating the cell at a temperature greater than 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. 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 functional variants of the compositions may be substituted.

[0085] In one aspect, there is provided a method for modifying a target sequence in the genome of a eukaryotic cell, comprising administering to the cell a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; a polynucleotide sequence encoding 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 the effector protein does not comprise an HNH domain; a cas-beta polynucleotide; a Cas-beta polypeptide; Methods are provided that include introducing a composition selected from the group consisting of a polynucleotide sequence and a polypeptide of a Sequence Listing; a polynucleotide that shares at least 70% to 100% identity with a cas1 polynucleotide, a cas1 polypeptide, a cas2 polynucleotide, a cas2 polypeptide, a cas4 polynucleotide, a cas4 polypeptide, a heterologous polynucleotide, or a polypeptide of a Sequence Listing; and 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 of the cell, and incubating the cell at a temperature above 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. In some embodiments, one or more of the compositions can be optimized for expression in a particular organism or cell type. In some embodiments, one or more functional fragments of the compositions can be substituted for the entire molecule.

[0086] In one aspect, there is provided a method for modifying a target sequence in the genome of a eukaryotic cell, comprising administering to the cell: 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 the CRISPR-Cas effector protein does not comprise an HNH domain; a polynucleotide sequence encoding 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 the effector protein does not comprise an HNH domain; a cas-beta polynucleotide; a Cas-beta polypeptide or a functional fragment thereof or and a composition selected from the group consisting of a functional variant; SEQ ID NO:3; and SEQ ID NO:4; further comprising at least two compositions selected from the group consisting of a Cas1 polynucleotide, a Cas1 polypeptide, a Cas2 polynucleotide, a Cas2 polypeptide, a Cas polynucleotide that is not Cas1 or Cas2, a Cas polypeptide that is not Cas1 or Cas2, a heterologous polynucleotide, a polynucleotide sequence in the Sequence Listing, and a polypeptide in the Sequence Listing; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell; and incubating the cell at a temperature above 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. 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 functional variants of the compositions may be substituted.

[0087] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell comprises administering to the cell a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; a polynucleotide sequence encoding 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 the effector protein does not comprise an HNH domain; a cas-beta polynucleotide; a Cas-beta polypeptide; or a polynucleotide sequence sharing at least 70% to 100% identity with SEQ ID NO: 10, 11, or 12. and a polypeptide sharing at least 70% to 100% identity with SEQ ID NO: 13, 14, 15, or 57; wherein the composition further comprises at least two compositions selected from the group consisting of a cas1 polynucleotide, a cas1 polypeptide, a cas2 polynucleotide, a cas2 polypeptide, a cas4 polynucleotide, a Cas polypeptide that is not Cas1 or Cas2, a heterologous polynucleotide, a polynucleotide sequence in the Sequence Listing, and a polypeptide in the Sequence Listing; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, and incubating the cell at a temperature greater than 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. 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 compositions may be substituted for the entire molecule.

[0088] In one aspect, there is provided a method for modifying a target sequence in the genome of a eukaryotic cell, comprising administering to the cell: 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 the CRISPR-Cas effector protein does not comprise an HNH domain; a polynucleotide sequence encoding 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 the effector protein does not comprise an HNH domain; a cas-beta polynucleotide; a Cas-beta polypeptide or a functional fragment thereof or and a composition selected from the group consisting of a Cas1 polynucleotide, a Cas1 polypeptide, a Cas2 polynucleotide, a Cas2 polypeptide, a Cas polynucleotide that is not Cas1 or Cas2, a Cas polypeptide that is not Cas1 or Cas2, a heterologous polynucleotide, a polynucleotide sequence in the Sequence Listing, and a polypeptide in the Sequence Listing; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell; and incubating the cell at a temperature above 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. 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 functional variants of the compositions may be substituted.

[0089] In one aspect, a method for modifying a target sequence in the genome of a eukaryotic cell comprises administering to the cell a polynucleotide sequence encoding 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 the CRISPR-Cas effector protein does not comprise an HNH domain; a polynucleotide sequence encoding 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 the effector protein does not comprise an HNH domain; a cas-beta polynucleotide; a Cas-beta polypeptide; or a polynucleotide sequence sharing at least 70% to 100% identity with SEQ ID NO: 10, 11, or 12. and a polypeptide sharing at least 70% to 100% identity with SEQ ID NO: 13, 14, 15, or 57; wherein the composition further comprises at least three compositions selected from the group consisting of a cas1 polynucleotide, a cas1 polypeptide, a cas2 polynucleotide, a cas2 polypeptide, a cas4 polynucleotide, a Cas polypeptide that is not Cas1 or Cas2, a heterologous polynucleotide, a polynucleotide sequence in the Sequence Listing, and a polypeptide in the Sequence Listing; and a guide polynucleotide comprising a variable targeting domain substantially complementary to a target sequence in the genome of the cell, and incubating the cell at a temperature greater than 10°C for at least about 4 hours, wherein the guide polynucleotide and CRISPR-Cas effector protein are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving the target sequence. 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 compositions may be substituted for the entire molecule.

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

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

[0092] In one aspect, a method of generating a plant having a modified genome is provided, comprising: introducing into at least one of said plants a heterologous composition comprising 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 of said cells, wherein said guide polynucleotide and Cas-beta endonuclease are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, and a polynucleotide modification template comprising at least one region complementary to a PAM sequence adjacent to a DNA target sequence recognized by the guide polynucleotide and Cas-beta complex, wherein the PAM sequence comprises at least one region comprising at least one nucleotide mismatch; incubating said cells for at least about 4 hours at a temperature greater than 10°C; producing whole plants from said cells; and verifying the at least one nucleotide modification in the genome of at least one cell of said plant compared to the target sequence in the genome of said cells prior to introduction of said heterologous composition.

[0093] In one aspect, provided are progeny of plants having a modified genome, the progeny being produced by a method comprising: introducing into at least one of said plants a heterologous composition comprising 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 of said cells, wherein said guide polynucleotide and Cas beta endonuclease are capable of forming a complex capable of recognizing, binding to, and optionally nicking or cleaving said target sequence, a polynucleotide modification template comprising at least one region complementary to a PAM sequence adjacent to a DNA target sequence recognized by the guide polynucleotide and Cas beta complex, wherein the PAM sequence comprises at least one region comprising at least one nucleotide mismatch; incubating said cells at a temperature greater than 10°C for at least about 4 hours; producing whole plants from said cells; and verifying the at least one nucleotide modification in the genome of at least one cell of said plant compared to the target sequence in the genome of said cells prior to introduction of said heterologous composition.

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

[0095] In some aspects, plants provided herein include corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, and tomato.

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

[0097] Any of the methods or compositions herein may further comprise a heterologous polynucleotide. The heterologous polynucleotide may be selected from the group consisting of: a non-coding expression regulatory element such as a promoter, intron, enhancer, or terminator; a donor polynucleotide; a polynucleotide-modified template optionally containing at least one nucleotide modification compared to the polynucleotide sequence in the cell; a transgene; a guide RNA; a guide DNA; a guide RNA-DNA hybrid; an endonuclease; a nuclear localization signal; and a cellular transit peptide.

[0098] In one aspect, methods for using any of the compositions disclosed herein are provided. In some embodiments, methods are provided for binding a Cas-beta endonuclease to a target sequence of a polynucleotide, for example, in the genome of a cell or in vitro. In some embodiments, the Cas-beta endonuclease forms a complex with a guide polynucleotide, e.g., a guide RNA. In some embodiments, the complex recognizes, binds to, and optionally creates 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, the nick or break is repaired by homology-directed repair (HDR) or homologous recombination (HR) using a polynucleotide-modified template or donor DNA molecule.

[0099] The novel Cas endonucleases described herein can generate double-stranded breaks in or adjacent to a target polynucleotide that contains an appropriate PAM and is guided by a guide polynucleotide in any prokaryotic or eukaryotic cell. In some examples, the cell is a plant cell, an animal cell, or a fungal cell. In some examples, the plant cell is selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, and tomato.

[0100] The present disclosure can be more fully understood from the following detailed description and the figures and sequence listing accompanying this specification, which form a part of this application. Furthermore, the present application relates to the invention described in the claims, but may also include the following as other aspects. (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 the source of the Cas endonuclease; and (c) a guide polynucleotide comprising a variable targeting domain that includes 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) The synthetic composition according to (1), wherein the Cas endonuclease is provided as a polynucleotide encoding the Cas endonuclease. (3) The synthetic composition according to (1), wherein the Cas endonuclease cleaves the double-stranded DNA polynucleotide. (4) The synthetic composition according to (1) above, further comprising a heterologous polynucleotide. (5) The synthetic composition according to (4) above, wherein the heterologous polynucleotide is an expression element. (6) The synthetic composition according to (4) above, wherein the heterologous polynucleotide is a transgene. (7) The synthetic composition according to (4) above, wherein the heterologous polynucleotide is a donor DNA molecule. (8) The synthetic composition according to (4) above, wherein the heterologous polynucleotide is a polynucleotide-modified template. (9) 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 includes 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. (10) The synthetic composition according to (1) above, further comprising a deaminase. (11) The synthetic composition according to (1), wherein the Cas endonuclease is part of a fusion protein. (12) The synthetic composition according to (11), wherein the fusion protein further comprises a heterologous nuclease domain. (13) The synthetic composition according to (1) above, further comprising a eukaryotic cell. (14) The synthetic composition according to (13) above, wherein the eukaryotic cell is a plant cell, an animal cell or a fungal cell. (15) The synthetic composition according to (14) above, wherein the plant cells are monocotyledonous or dicotyledonous plant cells. (16) The synthetic composition according to (14), wherein the plant cell is derived from an organism selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato. (17) 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 according to (1), wherein at least one component is attached to a solid matrix. (19) A method for introducing targeted editing 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 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: (20) The method of claim 22, 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 before introduction of the heterologous composition, and further comprising 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 the target sequence in the genome of the cell before introduction of the heterologous composition. (21) An offspring of an organism obtained by the method according to (23) above, the offspring comprising the Cas endonuclease or a functional fragment thereof. (22) The method according to (22) above, wherein the cell is a eukaryotic cell. (23) The method according to (25) above, wherein the eukaryotic cells are derived from or obtained from an animal, a fungus, or a plant. (24) The method according to (26), wherein the plant is a monocotyledonous plant or a dicotyledonous plant. (25) The method according to (26), wherein the plant is selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato. (26) The method according to (22) above, further comprising introducing a heterologous polynucleotide. (27) The method according to (29) above, 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. [Brief explanation of the drawings]

[0101] [Figure 1] Overview of the entire Cas-beta CRISPR locus, including, from 5' to 3': the cas-beta gene, the cas1 gene, the cas2 gene, the cas4 gene, and the CRISPR array. For Cas-beta 1, the locus contains 37 repeats of 37 nucleotides, three of which (numbered 5, 19, and 30 from the 5' end) share 97.3% identity with the array repeat sequence (SEQ ID NO: 19, 20, or 21), and the last repeat shares 78.4% identity with SEQ ID NO: 19, 20, or 21. For Cas-beta 3, only one repeat was found in the array. [Figure 2] Domain structure of the Cas-beta protein, showing the split RuvC domain, including an R / K / Q-rich bridging helix-like domain, a helix-turn-helix domain, and a bridging helix domain between RuvC-I and RuvC-II. [Figure 3]Maximum likelihood evolutionary analysis was used to demonstrate the phylogenetic relationships between Cas-beta proteins and other Cas endonucleases. The evolutionary history was inferred using a model based on maximum likelihood and the JTT matrix. The phylogenetic tree with the highest log-likelihood (-79975.80) is shown. The initial phylogenetic tree for the heuristic search was automatically obtained by applying neighbor-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, followed by selecting the topology with the most significant log-likelihood value. The phylogenetic tree was drawn to scale, with branch lengths measured as the number of substitutions per site. This analysis included 30 amino acid sequences. The final dataset contained a total of 1639 positions. Evolutionary analysis was performed in MEGA X. [Figure 4A] Figure 4: Showing conserved features in Cas-beta tracrRNA. [Figure 4B] (As mentioned above.) [Figure 5] 1 shows a method for Cas-beta expression and target strand recognition and cleavage detection. [Figure 6A] Figure 6: Cleavage of target polynucleotides by various Cas-beta compositions. Figure 6A shows no cleavage for the negative control. Figure 6B shows targeted double-stranded cleavage for the complete Cas-beta2 locus at 37°C. Figure 6C shows targeted double-stranded cleavage for the complete Cas-beta2 locus at 40°C. Figure 6D shows targeted double-stranded cleavage for the complete Cas-beta2 locus at 45°C. Figure 6E shows targeted double-stranded cleavage for the complete Cas-beta2 locus at 50°C. [Figure 6B] (As mentioned above.) [Figure 6C] (As mentioned above.) [Figure 6D] (As mentioned above.) [Figure 6E] (As mentioned above.) [Figure 7A]Figure 7: Figure 7A shows some sequence motifs and domains of Cas-beta as shown on the sequence of Cas-beta 1 (SEQ ID NO: 13). Figure 7B shows the domains and motifs of Cas-beta (as shown for Cas-beta 1). BH = positively charged K / R / H-rich bridging helix for DNA / RNA recognition; TSL = target strand load; Helical = helical bundle domain, DNA / RNA binding element; Wed = wedge-like DNA / RND binding domain at the separating strand fork point. [Figure 7B] (As mentioned above.) [Figure 8A] Figure 8: Figure 8A shows no cleavage for the negative control (Cas-beta proteins 1 and 3). Figure 8B shows targeted double-stranded cleavage for the complete Cas-beta 1 locus at 37°C. Figure 8C shows targeted double-stranded cleavage for the complete Cas-beta 3 locus at 37°C. [Figure 8B] (As mentioned above.) [Figure 8C] (As mentioned above.) [Figure 9A] Figure 9: Figure 9A shows in vitro reconstitution of double-stranded DNA target cleavage activity for sgRNAs targeting Cas-beta2 and T2 sites. Figure 9B shows in vitro reconstitution of double-stranded DNA target cleavage activity for sgRNAs targeting Cas-beta3 and T2 sites. Figure 9C shows in vitro reconstitution of double-stranded DNA target cleavage activity for sgRNAs targeting Cas-beta2 and T2 sites. Figure 9D shows that Cas-beta2 target cleavage (supercoiled plasmid DNA) is more efficient with sgRNAs composed of a 24-nt-long variable targeting domain compared to sgRNAs containing a 20-nt-long variable targeting domain. Figure 9E shows digestion of Cas-beta2 RNP confirms that both a C-rich PAM directly 5' of the protospacer target and an sgRNA with homology to the target are required for the observed site-specific cleavage. SEQ ID NOs: 65 and 61 correspond to the T1- and T2-targeting sgRNAs, respectively. [Figure 9B] (As mentioned above.) [Figure 9C] (As mentioned above.) [Figure 9D] (As mentioned above.) [Figure 9E] (As mentioned above.) [Figure 10] Figure 1 shows the characteristics of Cas-beta target site recognition. [Figure 11A] Figure 11: Figure 11A shows that Cas-beta2 is triggered to nonspecifically degrade single-stranded DNA after double- or single-stranded DNA target recognition. Figure 11B shows that Cas-beta3 is triggered to nonspecifically degrade single-stranded DNA after double- or single-stranded DNA target recognition. [Figure 11B] (As mentioned above.) [Figure 12] 1 shows an example of a plant-optimized Cas-beta expression construct. [Figure 13] Examples of plant-optimized gRNA expression constructs are shown. [Figure 14A] Figure 14: Figure 14A shows no cleavage for the negative control (Cas-beta4 protein). Figure 14B shows targeted double-strand cleavage using Cas-beta4 ribonucleoprotein (RNP) at 37°C. [Figure 14B] (As mentioned above.) [Figure 15] 1 shows robust cleavage by Cas-beta2 of a single target site in the RUNX1 gene when using linear double-stranded DNA as a substrate. [Figure 16] RUNX1 site 1 and WTAP site 1 substrates generated for analysis of Cas-beta cleavage preference by capillary electrophoresis are shown. Targets consisting of a PAM and a protospacer target are written in uppercase. The PAM is underlined. [Figure 17A] Figure 17: Figures 17A and 17B show that under 37°C conditions, both Cas-beta 2 and 3 cleaved RUNX1 site 1, while WTAP site 1 failed to produce appreciable levels of cleavage. [Figure 17B] (As mentioned above.) [Figure 18A]Figure 18: Figures 18A and 18B show that when incubated at 47°C, Cas-beta3 cleaved both substrates, while cleavage of WTAP site 1 by Cas-beta2 remained inefficient. [Figure 18B] (As mentioned above.) [Figure 19] We show that Cas-beta2 robustly cleaved both target sites when present in a supercoiled state and cleaved only RUNX1 site 1 in the linear form. [Figure 20A] Figure 20: Figure 20A shows that the Cas-beta2 RNP complex cleaved 5 of 10 targets (WTAP sites 6 and 7 and RUNX1 sites 3, 4, and 7). Figure 20B shows that at 37°C, only Cas-beta3 robustly cleaved WTAP site 7, while at 47°C, it cleaved 8 of 10 targets. [Figure 20B] (As mentioned above.) [Figure 21] We show that when using linear double-stranded DNA substrates, Cas-beta2 preferred sgRNAs with spacers 20 nt in length. [Figure 22] This indicates that Cas-beta3 was more tolerant of sgRNAs with longer spacer lengths. [Figure 23] We show that both Cas-beta2 and Cas-beta3 left 5' overhanging ends that varied in length from 6 to 8 nt upon cleavage of supercoiled targets. [Figure 24]We show that when nucleofected into HEK293 cells as an NLS-tagged RNP, Cas-beta2 resulted in targeted chromosomal DNA sequence modifications when paired with an sgRNA (SEQ ID NO: 143) targeting RUNX1 site 1. These included deletion mutations that coincided with the predicted cleavage site of Cas-beta2 and disrupted protospacer target recognition. The lack of similar mutations in control experiments confirmed that the changes were due to introduction of Cas-beta2 RNP, collectively providing the first evidence of double-stranded DNA target binding and cleavage activity in eukaryotic cells. Lowercase nucleotides represent additional bases inserted as part of double-strand break repair. Dashes represent deleted bases. [Figure 25] Figure 1 shows restoration of a deletion mutation spanning the predicted Cas-beta2 cleavage site when Cas-beta2 sgRNA (SEQ ID NOs: 144 and 145) can base-pair with the complementary strand of WTAP site 6. The deletion resulting from Cas-beta target recognition and cleavage repair was large when generated by T7 in vitro transcription, complexed with a Cas-beta2 NLS-tagged protein, and delivered to HEK293 cells using electroporation. To confirm alignment between the mutant read and the reference, some bases were removed from the reference. The position and number of removed bases are indicated between two " / "s. The sequence listing contains the complete reference sequence. Lowercase nucleotides represent additional bases inserted during double-strand break repair. Dashes represent deleted bases. [Figure 26]Figure 1 shows restoration of a deletion mutation spanning the predicted Cas-beta2 cleavage site when Cas-beta2 sgRNA (SEQ ID NOs: 144 and 145) can base-pair with the complementary strand of WTAP site 7. The deletion resulting from Cas-beta target recognition and cleavage repair was large when generated by T7 in vitro transcription, complexed with a Cas-beta2 NLS-tagged protein, and delivered to HEK293 cells using electroporation. To confirm alignment between the mutant read and the reference, some bases were removed from the reference. The position and number of removed bases are indicated between two " / "s. The sequence listing contains the complete reference sequence. Lowercase nucleotides represent additional bases inserted during double-strand break repair. Dashes represent deleted bases. [Figure 27] We demonstrate that Cas-beta 3 also functions in HEK293 cells to induce targeted mutations at WTAP site 7. The deletion resulting from Cas-beta target recognition and repair of the cleavage was large. To confirm alignment between the mutant read and the reference, some bases were removed from the reference. The position and number of removed bases are indicated between two " / ". The sequence listing contains the complete reference sequence. Dashes represent deleted bases. [Figure 28] We demonstrate that Cas-beta nuclease and sgRNA can function in other eukaryotic cells. Here, when an expression cassette encoding Cas-beta 2 and sgRNA were delivered to immature embryos (IEs) using a gene gun, targeted mutations were restored in both male sterility 26 (MS26) gene targets two days after transformation. Dashes represent deleted bases. [Figure 29] When the recipient strain, E. coli Arctic Express (DE3), harboring the Cas-beta 2 nuclease and guide RNA expression cassette was transformed with the target plasmid, no colonies were obtained, demonstrating that the Cas-beta nuclease and sgRNA were functional in heterologous prokaryotic cells. Dashes represent deleted bases. DETAILED DESCRIPTION OF THE INVENTION

[0102] The sequence descriptions and the sequence listing attached hereto comply with the rules governing the disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.825. The sequence descriptions include the three-letter codes 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 in the Cas-beta1 locus.

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

[0105] SEQ ID NO: 3 is the Armatimonadetes bacterial PRT sequence for Cas1 encoded in 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 in the Cas-beta2 locus.

[0108] SEQ ID NO: 6 is the Armatimonadetes bacterial PRT sequence for Cas2 encoded in 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 in the Cas-beta2 locus.

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

[0112] SEQ ID NO: 10 is the Armatimonadetes bacterial DNA sequence for the Cas-beta 1 endonuclease gene.

[0113] SEQ ID NO: 11 is the Armatimonadetes bacterial DNA sequence for the Cas-beta 2 endonuclease gene.

[0114] SEQ ID NO: 12 is the Armatimonadetes bacterial DNA sequence for the Cas-beta 3 endonuclease gene.

[0115] SEQ ID NO: 13 is the Armatimonadetes bacterial PRT sequence for the Cas-beta 1 endonuclease gene.

[0116] SEQ ID NO: 14 is the Armatimonadetes bacterial PRT sequence for the Cas-beta 2 endonuclease gene.

[0117] SEQ ID NO: 15 is the Armatimonadetes bacterial PRT sequence for the Cas-beta 3 endonuclease gene.

[0118] SEQ ID NO: 16 is the Armatimonadetes bacterial DNA sequence for the Cas-beta 1 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-beta 3 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-beta 3 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 the 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 the 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 the terminator-like hairpin.

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

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

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

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

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

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

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

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

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

[0142] SEQ ID NO: 40 is an artificial PRT sequence for a 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 the artificial DNA sequence for the A1 oligonucleotide.

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

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

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

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

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

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

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

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

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

[0154] SEQ ID NO: 52 is the 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 24 nt spacer sequence (targeting T2).

[0162] SEQ ID NO: 60 is the RNA sequence for a Cas-beta2 sgRNA with a terminator-like hairpin targeting 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 a Cas-beta3 sgRNA with a terminator-like hairpin targeting the T2 sequence.

[0165] SEQ ID NO: 63 is the RNA sequence for a Cas-beta3 sgRNA without a terminator-like hairpin targeting 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 targeting the T1 sequence.

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

[0169] SEQ ID NO: 67 is the DNA sequence for an 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 generate a dsDNA non-specific activator.

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

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

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

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

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

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

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

[0179] SEQ ID NO: 77 is an artificial DNA sequence for exon 2 of the maize-optimized Cas-beta 1 nuclease.

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

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

[0182] SEQ ID NO: 80 is the DNA sequence of Zea mays 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 the sgRNA.

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

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

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

[0187] SEQ ID NO: 85 is the Armatimonadetes bacterial PRT sequence for Cas2 encoded in 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-beta 4 endonuclease gene.

[0190] SEQ ID NO: 88 is the Armatimonadetes bacterial DNA sequence for the Cas-beta 4 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 the terminator-like hairpin.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0207] SEQ ID NO: 105 is an artificial DNA sequence for 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 an artificial DNA sequence for 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 human (Homo sapiens) DNA sequence for WTAP site 3.

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

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

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

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

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

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

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

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

[0220] SEQ ID NO: 118 is the human (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 the 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 human (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 human (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 human (Homo sapiens) DNA sequence for WTAP site 7 reference.

[0241] SEQ ID NO: 139 is the human (Homo sapiens) DNA sequence for Cas-beta 2 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 by Cas-beta2 sgRNA.

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

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

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

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

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

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

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

[0253] SEQ ID NO: 151 is the 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 spacer 2 target.

[0255] SEQ ID NO: 153 is an artificial DNA sequence for the Cas-beta2 natural spacer 3 target.

[0256] SEQ ID NO: 154 is an artificial PRT sequence for D528A cleavage-inactive Cas-beta2.

[0257] SEQ ID NO: 155 is an artificial PRT sequence for E634A cleavage-inactive Cas-beta2.

[0258]

[0003] Compositions and methods are provided for novel CRISPR effector systems and elements comprising such systems, including, but not limited to, novel guide polynucleotide / endonuclease complexes, guide polynucleotides, guide RNA elements, Cas proteins, and endonucleases, as well as proteins comprising endonuclease functionality (domains). Also provided are compositions and methods for the direct delivery of endonucleases, cleavage-ready complexes, guide RNAs, and guide RNA / Cas endonuclease complexes. The present disclosure further includes compositions and methods for modifying target sequences in the genome of a cell, gene editing, and inserting a polynucleotide of interest into the genome of a cell.

[0259] The small novel Cas endonuclease described herein, "Cas-beta," is derived from a CRISPR locus with a unique structure that includes, in this order: the cas-beta gene, the cas1 gene, the cas2 gene, a gene with low identity to cas4, and the CRISPR array. 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. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0261] As used herein, "nucleic acid" refers to a polynucleotide, including single- or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can also include fragments and modified nucleotides. Thus, the terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," and "nucleic acid fragment" are used interchangeably to refer to polymers of RNA and / or DNA and / or RNA-DNA that are single- or double-stranded, and optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by the single letter designation: "A" for adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" for cytosine or deoxycytosine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "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" as applied to prokaryotic and eukaryotic cells or organismal cells encompasses not only chromosomal DNA found in the nucleus, but also organelle DNA found within cellular components of the cell (e.g., mitochondria or plastids).

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

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

[0265] The term "stringent conditions" or "stringent hybridization conditions" includes conditions under which a probe selectively hybridizes to its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will vary in various circumstances. By controlling the stringency of hybridization and / or washing conditions, it is possible to identify a target sequence that is 100% complementary to a probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some sequence mismatch such that lower similarities are detected (heterologous probing). Generally, probes are less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length. Typically, stringent conditions are those in which the salt concentration is about 1.5 M Na ion concentration, typically about 0.01 to 1.0 M Na ion concentration, at a temperature of at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than 50 nucleotides), at a pH of 7.0 to 8.3. Stringent conditions can also be achieved by adding destabilizing substances such as formamide. Examples of low stringency conditions include hybridization at 37°C in a buffer consisting of 30 to 35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing in 1 to 2×SSC (20×SSC = 3.0 M NaCl / 0.3 M sodium citrate) at 50 to 55°C. Examples of moderate stringency conditions include hybridization in 40-45% formamide, 1 M NaCl, and 1% SDS at 37° C., followed by washing in 0.5-1×SSC at 55-60° C. Examples of high stringency conditions include hybridization in 50% formamide, 1 M NaCl, and 1% SDS at 37° C., followed by washing in 0.1×SSC at 60-65° C.

[0266] "Homology" refers to a similar DNA sequence. For example, a "region homologous to a genomic region" found on donor DNA refers to a region of DNA that has a similar sequence to a given "genomic region" of the fungal cell genome. The homologous region can be of any length sufficient to promote homologous recombination at the target site of cleavage. For example, the homologous region can be 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, or 5-800 nucleotides long, such that the homologous region has sufficient homology to undergo homologous recombination with the corresponding genomic region. , 5 to 800, 5 to 900, 5 to 1000, 5 to 1100, 5 to 1200, 5 to 1300, 5 to 1400, 5 to 1500, 5 to 1600, 5 to 1700, 5 to 1800, 5 to 1900, 5 to 2000, 5 to 2100, 5 to 2200, 5 to 2300, 5 to 2400, 5 to 2500, 5 to 2600, 5 to 2700, 5 to 2800, 5 to 2900, 5 to 3000, 5 to 3100 or more bases. "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 total length of each polynucleotide fragment and the sequence similarity of the polynucleotides. Sequence similarity can be described as percent sequence identity over the entire length of the sequence and / or percent sequence identity over conserved regions and portions of the length of the sequence, including localized similarities such as consecutive nucleotides with 100% sequence identity.

[0267] As used herein, a "genomic region" is a segment of a chromosome in the genome of a cell that is present on either side of a target site, or alternatively, a segment that also includes a portion of the target site. This genomic region may be at least 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 200, 5 to 300, 5 to 400, 5 to 500, 5 to 600, 5 to 700, 5 to 800, 5 to 900, 5 to 1000, 5 to 1100, 5 to 1200, 5 to 1300, 5 to 1400, 5 to 1500, 5 to 2000, 5 to 2500, 5 to 3000, 5 to 3500, 5 to 4000, 5 to 500, 5 to 600, 5 to 700, 5 to 800, 5 to 900, 5 to 1000, 5 to 1100, 5 to 1200, 5 to 1300, 5 to 2500, 5 to 2600, 5 to 3000, 5 to 3500, 5 to 4000, 5 to 4500, 5 to 5000, 5 to 5000, 5 to 14 The genomic region may comprise 1,400, 5 to 1,500, 5 to 1,600, 5 to 1,700, 5 to 1,800, 5 to 1,900, 5 to 2,000, 5 to 2,100, 5 to 2,200, 5 to 2,300, 5 to 2,400, 5 to 2,500, 5 to 2,600, 5 to 2,700, 5 to 2,800, 5 to 2,900, 5 to 3,000, 5 to 3,100 or more bases, such that the genomic region has sufficient homology to undergo homologous recombination with a 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 among different organisms. In general, the length of the homologous region affects the frequency of homologous recombination events; the longer the region of homology, the higher the frequency. The length of the homologous region required to observe homologous recombination also varies among species. While at least 5 kb of homology has often been utilized, homologous recombination has been observed using as little as 25-50 bp. For example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. Natl. Acad. Sci. USA 82:4768-72, Sugawara and Haber, (1992) Mol Cell Biol. 12:563-75; Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., 1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay et al., (1987) Genetics 115:161-7.

[0269] With respect to nucleic acid sequences or polypeptide sequences, "sequence identity" or "identity" refers to the nucleic acid bases or amino acid residues in two sequences that are identical when aligned for maximum correspondence over a specified comparison window.

[0270] The term "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) to optimally align the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Useful examples of percent sequence identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage between 50% and 100%. These identities can be determined using any of the programs described herein.

[0271] Sequence alignments and percent identity or similarity calculations may be determined using a variety of comparison methods designed to detect homologous sequences, including, but not limited to, the MegAlign™ program in the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). In the context of this application, when sequence analysis software is used for analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program, 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 designated Clustal V (as 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, WI). For multiple alignments, the default values ​​correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. The default parameters for pairwise alignment and percent identity calculation of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5, and DIAGONALS SAVED=5. For nucleic acids, these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4, and DIAGONALS SAVED=4. After alignment of sequences using the Clustal V program, a "percent identity" can be obtained by consulting the "sequence distance" table in the same program. The "Clustal W method of alignment" is designated 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, WI). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Sequences (%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB).After aligning sequences using the Clustal W program, "percent identity" can be obtained by consulting the "sequence distance" table in the program. Unless otherwise specified, the sequence identity / similarity values ​​presented herein refer to values ​​obtained using GAP version 10 (GCG, Accelrys, San Diego, CA) with the following parameters: nucleotide sequence identity and similarity percentages are obtained using a gap creation penalty weight of 50, a gap extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; amino acid sequence identity and similarity percentages are obtained using a gap creation penalty weight of 8, a gap extension penalty weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). GAP uses the algorithm of Needleman and Wunsch (1970) J Mol Biol 48:443-53 to find a global alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and uses gap creation and extension penalties in units of matched bases to create an alignment with the maximum number of matched bases and the fewest gaps. "BLAST" is a search algorithm provided by the National Center for Biotechnology Information (NCBI) used to find similar regions of biological sequences. This program compares nucleotide or protein sequences with sequence databases and calculates the statistical significance of matches to identify sequences sufficiently similar to a query sequence that the similarity is not expected to occur randomly. BLAST reports the identified sequences and their local alignment to the query sequence. It will be appreciated by those skilled in the art that 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.Useful examples of percent identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or any percentage between 50% and 100%. Indeed, any amino acid identity between 50% and 100%, e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity may be useful in describing the present disclosure.

[0273] Polynucleotide and polypeptide sequences, their variants, and the structural relationships of these sequences can be described by the terms "homology," "homologous," "substantially identical," "substantially similar," and "substantially corresponding," which are used interchangeably herein. These refer to polypeptide or nucleic acid sequences in which changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as its ability to mediate gene expression 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 compared to the initial, unmodified nucleic acid. These modifications include the deletion, substitution, and / or insertion of one or more nucleotides in the nucleic acid fragment. Substantially similar nucleic acid sequences can be defined by their ability to hybridize (under moderate stringency conditions, e.g., 0.5×SSC, 0.1% SDS, 60°C) to any portion of the sequences exemplified herein or the nucleotide sequences disclosed herein, and are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for fragments with moderate similarity, such as homologous sequences from distantly related organisms, against fragments with high similarity, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.

[0274] A "centimorgan" (cM) or "map unit" is the distance between two polynucleotide sequences, linked genes, markers, target sites, loci, or any pair thereof, where 1% of meiotic outcomes are recombinations. Thus, a centimorgan is equivalent to the distance that equals 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, is substantially or essentially free from components that normally co-occur with or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein, if produced by recombinant techniques, is substantially free of other cellular material and culture medium, or, if chemically synthesized, is substantially free of chemical precursors and other chemicals. Optimally, an "isolated" polynucleotide (optimally, a sequence encoding a protein) is free of sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various embodiments, an isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Isolated polynucleotides can be purified from cells in which they naturally occur. Conventional nucleic acid purification methods known to those of skill in the art can be used to obtain isolated polynucleotides, and the term also encompasses recombinant and chemically synthesized polynucleotides.

[0276] The term "fragment" refers to a contiguous set of nucleotides or amino acids. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 contiguous nucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 contiguous amino acids. A fragment may or may not exhibit function of a sequence that shares 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 a portion or subsequence of an isolated nucleic acid fragment or polypeptide that exhibits the same activity or function as the longer sequence from which it is derived. In one example, a 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 engineer genes that produce a desired phenotype in an altered plant. A gene can be engineered to be used in an inhibitory manner by linking the nucleic acid fragment, whether it encodes an active enzyme or not, to a plant promoter sequence in a sense or antisense orientation.

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

[0279] The term "endogenous" refers to a sequence or other molecule that is naturally present in a cell or organism. In one aspect, an endogenous polynucleotide is one that is normally found in the genome of the cell, i.e., 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 locus on a chromosome are the same, the plant is homozygous at that locus. If alleles present at a given 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 of a coding sequence (5'-non-coding sequences), within a coding sequence, or downstream of a coding sequence (3'-non-coding sequences), that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5'-untranslated sequences, 3'-untranslated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0282] A "mutated gene" is a gene that has been altered by human intervention. Such a "mutated gene" has a sequence that differs from that of the corresponding non-mutated gene by the addition, deletion, or substitution of at least one nucleotide. In certain embodiments of the present disclosure, the mutant gene comprises an alteration resulting from a guide polynucleotide / Cas endonuclease system as disclosed herein. A mutant plant is a plant that comprises a mutant gene.

[0283] As used herein, a "targeted mutation" is a mutation in a gene (referred to as a target gene), including a native gene, that is made by altering a target sequence within the target gene using any method known to those of skill in the art, including methods involving the inducible Cas endonuclease system disclosed herein.

[0284] The terms "knockout," "gene 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; for example, the DNA sequence prior to the knockout may have encoded an amino acid sequence or had a regulatory function (e.g., a 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 using a Cas protein (e.g., by homologous recombination (HR), also using a suitable donor DNA polynucleotide). Examples of knock-ins are the specific insertion of a heterologous amino acid coding sequence in the coding region of a gene or the specific insertion of a transcriptional regulatory element at a gene locus.

[0286] "Domain" means a contiguous stretch of nucleotides (which may 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 the aligned sequences of evolutionarily related proteins. While amino acids at other positions may vary between homologous proteins, highly conserved amino acids at specific positions represent amino acids that are essential for the structure, stability, or activity of the protein. Because they are identified by the high degree of conservation in the aligned sequences of a family of protein homologs, they can be used as identifiers or "signatures" to determine whether a protein having a newly determined sequence belongs to a previously identified protein family.

[0288] A "codon-modified gene" or "codon-preferred gene" or "codon-optimized gene" is a gene whose codon usage is designed to mimic the preferred codon usage of a host cell.

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

[0290] A "plant-optimized nucleotide sequence" is a nucleotide sequence that has been optimized for improved expression in plants, particularly for increased expression in plants. Plant-optimized nucleotide sequences include codon-optimized genes. Plant-optimized nucleotide sequences can be synthesized by modifying a nucleotide sequence encoding a protein, such as a Cas endonuclease disclosed herein, using one or more plant-preferred codons for improved expression. See, for example, 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. A promoter sequence consists of proximal and more distal upstream elements, the latter 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 the promoter. A promoter may be derived entirely from a native gene or may be composed of different elements derived from different promoters found in nature and / or may contain synthetic DNA segments. It is understood by those skilled in the art that different promoters can induce the expression of a gene in different tissue or cell types, at different developmental stages, or in response to different environmental conditions. It is further recognized that because the exact boundaries of regulatory sequences in most cases have not been completely defined, DNA fragments of some variation may have identical promoter activity.

[0292] Promoters that cause the expression of most genes in most cell types are commonly referred to as "constitutive promoters." The term "inducible promoter" refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, for example, by a chemical compound (chemical inducer), or in response to environmental, hormonal, chemical, and / or developmental signals. Inducible or regulatable promoters include, for example, promoters that are induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonic acid, salicylic acid, or safeners.

[0293] "Translation leader sequence" refers to a polynucleotide sequence between the promoter sequence and coding sequence of a gene. Translation leader sequences are present upstream of the translation initiation sequence of an mRNA. Translation leader sequences can affect processing of the primary transcript into mRNA, mRNA stability, or translation efficiency. Examples of translation leader sequences have been described (see, e.g., Turner and Foster, (1995) Mol Biotechnol 3:225-236).

[0294] "3' non-coding sequence," "transcription terminator," or "termination sequence" refers to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of a 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 an RNA transcript is a perfectly complementary copy of a DNA sequence, it is called a primary transcript or pre-mRNA. When an RNA transcript is an RNA sequence resulting from post-transcriptional processing of a primary transcript pre-RNA, it is called a mature RNA or mRNA. "Messenger RNA" or "mRNA" refers to RNA that is free of introns and can be translated into protein by a cell. "cDNA" refers to DNA that is complementary to an mRNA template and synthesized from the mRNA template using reverse transcriptase. cDNA can 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 mRNA and can be translated into protein in cells 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. Pat. No. 5,107,065). The complementarity of an antisense RNA can be with any part of a specific gene transcript, i.e., with the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated but affects cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to an mRNA transcript and are intended to define an antisense RNA of the message.

[0296] The term "genome" refers to the entire body of genetic material (genes and non-coding sequences) present in each cell or virus or organelle of an organism; and / or the set of chromosomes inherited as a (haploid) unit from one parent.

[0297] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence if it is capable of controlling the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. In another example, complementary RNA regions can be operably linked, directly or indirectly, to or within the 5' end of a target mRNA or the 3' end of a target mRNA, or a first complementary region is at the 5' end of the target mRNA and its complement is at 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, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell), or a cell from a multicellular organism (e.g., a cell line), cultured as a unicellular entity, in vivo or in vitro, into which a heterologous polynucleotide or polypeptide has been introduced. In some embodiments, the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a unicellular eukaryotic organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algae cell, an animal cell, an invertebrate cell, a vertebrate cell, a fish cell, a frog cell, an avian cell, an insect cell, a mammalian cell, a porcine cell, a bovine cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell. In some examples, the cell is in vitro. In some examples, the cell is in vivo.

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

[0300] The terms "plasmid," "vector," and "cassette" refer to linear or circular supplemental chromosomal elements, usually in the form of double-stranded DNA, that often carry genes that are not part of the cell's central metabolism. Such elements can be autonomously replicating sequences, genome-integrating sequences, phages, or nucleotide sequences of any origin, of single- or double-stranded DNA or RNA, in linear or circular form, in which several nucleotide sequences are linked or recombined into a unique construct capable of introducing a polynucleotide of interest into a cell. A "transformation cassette" refers to a specific vector containing a gene and having elements in addition to the gene that facilitate transformation of a specific host cell. An "expression cassette" refers to a specific vector containing a gene and having elements in addition to the gene that cause 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 fragments, such as regulatory and coding sequences, that are not necessarily found together in nature. For example, a recombinant DNA construct may include regulatory and coding sequences from different sources, or may include regulatory and coding sequences from the same source but arranged differently than they are found in nature. Such a construct may be used by itself or in conjunction with a vector. When a vector is used, the choice of vector will depend on the method used to introduce the vector into a host cell, as is well known to those skilled in the art. For example, a plasmid vector may be used. Those skilled in the art are familiar with the genetic elements that must be present on a vector in order to successfully transform, select, and propagate a host cell. Those skilled in the art will also recognize that different independent transformation events can 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), and therefore multiple events are typically screened to obtain strains exhibiting the desired expression levels and patterns. Such screening can be performed by standard molecular biological, biochemical, and other analytical methods, including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblotting of protein expression, enzyme or activity assays, and / or phenotypic assays.

[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 differences in taxonomic origin (e.g., a polynucleotide sequence obtained from Zea mays is heterologous if inserted into the genome of a rice (Oryza sativa) plant or a different subspecies or variety of Zea mays; or a polynucleotide obtained from a bacterium is introduced into the cells of a plant) or sequence differences (e.g., a polynucleotide sequence obtained from Zea mays, isolated, modified, and reintroduced into a maize plant). As used herein, "heterologous" in reference to a sequence can refer to a sequence originating from a different species, subspecies, or alien species, or, if from the same species, a sequence whose composition and / or genomic locus has been substantially altered from its native form by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide may be from a species different from the species from which the polynucleotide was derived, or, if from the same / similar species, one or both may be substantially altered from their original form and / or genomic locus, or the promoter may not be the native promoter of the operably linked polynucleotide. Alternatively, one or more of the regulatory regions and / or polynucleotides set forth herein may be entirely synthetic. In another example, a target polynucleotide for cleavage by a Cas endonuclease may be from a different organism than the Cas endonuclease. In another example, the Cas endonuclease and guide RNA may be introduced into a target polynucleotide along with an additional polynucleotide that acts as a template or donor for insertion into the target polynucleotide, where 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 a functional end-product (e.g., mRNA, guide RNA, or protein) in precursor or mature form.

[0304] A "mature" protein refers to a post-translationally processed polypeptide (i.e., a polypeptide from which any pre- or propeptides present in the primary translation product have been removed).

[0305] "Precursor" protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). The pre- and propeptides may be, but are not limited to, subcellular localization signals.

[0306] "CRISPR" (clustered regularly interspaced short palindromic repeats) loci refer to specific loci that encode components of DNA cleavage systems used, for example, by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327:167-170; International Publication WO 2007 / 025097, published March 1, 2007). CRISPR loci can consist of CRISPR arrays containing short direct repeats (CRISPR repeats) separated by short, variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.

[0307] As used herein, an "effector" or "effector protein" is a protein whose activities include recognizing, binding to, and / or cleaving or nicking a polynucleotide target. An effector or effector protein can also be an endonuclease. The "effector complex" of a CRISPR system includes Cas proteins that are involved in recognizing and binding the crRNA to the target. Some of the component Cas proteins may further include domains involved in target polynucleotide cleavage.

[0308] The term "Cas protein" refers to proteins encoded by Cas (CRISPR-associated) genes. Cas proteins include proteins encoded by genes in the cas locus and include adaptation molecules and interference molecules. Interference molecules of bacterial adaptive immune complexes include endonucleases. Cas endonucleases described herein contain one or more nuclease domains. Cas endonucleases include, but are not limited to, the novel Cas-beta proteins disclosed herein, Cas9 proteins, Cpf1 (Cas12) proteins, C2c1 proteins, C2c2 proteins, C2c3 proteins, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or combinations or complexes thereof. Cas proteins can be "Cas endonucleases" or "Cas effector proteins" that, when complexed with a suitable polynucleotide component, can recognize, bind to, and optionally nick or cleave all or part of a specific polynucleotide target sequence. Cas-beta endonucleases of the present disclosure include those having one or more RuvC nuclease domains.The Cas protein may be a functional fragment or functional variant of a naturally occurring Cas protein, or a fragment of 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 consecutive amino acids of a naturally occurring Cas protein, and at least 50%, 50%-55%, at least 55%, 55%-60%, at least It is further defined as a protein having 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100% or 100% sequence identity and retaining at least some activity of the native sequence.

[0309] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a Cas endonuclease are used interchangeably herein to refer to a portion or subsequence of a Cas endonuclease sequence of the present disclosure that retains the ability to recognize, bind to, and optionally unwind, nick, or cleave a target site (introducing a single- or double-stranded break within the target site). This portion or subsequence of a Cas endonuclease can include 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 entire functional portion of the Cas3 helicase domain, or the entire functional portion of a protein (such as, but not limited to, Cas5, Cas5d, Cas7, and Cas8b1).

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

[0311] Cas endonucleases may also include multifunctional Cas endonucleases. The terms "multifunctional Cas endonuclease" and "multifunctional Cas endonuclease polypeptide" are used interchangeably herein and include reference to a single polypeptide having a Cas endonuclease function (comprising at least one protein domain capable of acting as a Cas endonuclease) and at least one other function, such as, but not limited to, a complexing function (comprising at least a second protein domain capable of forming a complex with another protein). In one embodiment, a multifunctional Cas endonuclease comprises at least one additional protein domain (internal, upstream (5'), downstream (3'), or both internal 5' and 3', or any combination thereof) relative to the domains typical of a Cas endonuclease.

[0312] The terms "cascade" and "cascade complex" are used interchangeably herein and include reference to a multisubunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP). A cascade is a PNP that relies on a polynucleotide for complex assembly and stability and for target nucleic acid sequence identification. A cascade functions as a surveillance complex that finds and optionally binds to target nucleic acids complementary to the variable targeting domain of a 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 interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP), where one of the Cascade proteins is a Cas endonuclease that can recognize, bind to, and optionally unwind, nick, or cleave all or part of a target sequence.

[0314] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. 7-methylguanylate residues are 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 extreme 5' phosphate group of an mRNA transcript is removed by an RNA terminal phosphatase, leaving two terminal phosphate groups. Guanosine monophosphate (GMP) is added to the terminal phosphate group of the transcript by a guanylyltransferase, leaving a 5'-5' triphosphate-linked guanine at the end of the transcript. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyltransferase.

[0315] The term "not having a 5'-cap" is used herein to refer to RNA that has, for example, a 5'-hydroxyl group instead of a 5'-cap. Such RNA can be referred to, for example, as "decapped RNA." Because 5'-capped RNA undergoes nuclear export, decapped RNA can accumulate more fully in the nucleus after transcription. One or more RNA components herein are decapped.

[0316] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease, such as a Cas endonuclease described herein, and enables the Cas endonuclease to recognize, optionally bind to, and optionally cleave a DNA target site. A guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (a combined RNA-DNA sequence).

[0317] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a guide RNA, crRNA, or tracrRNA are used interchangeably herein and refer to a portion or subsequence of a guide RNA, crRNA, or tracrRNA of the present disclosure that retains the ability to function as a guide RNA, crRNA, or tracrRNA, respectively.

[0318] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" of a guide RNA, crRNA, or tracrRNA (respectively) are used interchangeably herein and refer to a variant of a guide RNA, crRNA, or tracrRNA of the present disclosure that retains the ability to function as a guide RNA, crRNA, or tracrRNA, respectively.

[0319] The terms "single guide RNA" and "sgRNA" are used interchangeably herein and refer to a synthetic fusion of two RNA molecules: a crRNA (CRISPR RNA) containing a variable targeting domain (linked to a tracr mate sequence that hybridizes to the tracrRNA) fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA may comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of a Type II CRISPR / Cas system that can form a complex with a Type II Cas endonuclease, and the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, allowing the Cas endonuclease to recognize, optionally bind to, and optionally nick or cut (introduce a single- or double-strand break) the DNA target site.

[0320] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and comprise a nucleotide sequence that can hybridize to (be complementary to) one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be comprised of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0321] The terms "Cas endonuclease recognition domain" or "CER domain" (of a guide polynucleotide) are used interchangeably herein and include a nucleotide sequence that interacts with a Cas endonuclease polypeptide. A CER domain includes a (trans-acting) tracr nucleotide mate sequence followed by a tracr nucleotide sequence. A CER domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., U.S. Patent Application Publication No. 2015 / 0059010A1, published February 26, 2015), or any combination thereof.

[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," "PGEN," are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease that are capable of forming a complex, wherein said guide polynucleotide / Cas endonuclease complex is capable of directing the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single- or double-strand break) the DNA target site. The guide polynucleotide / Cas endonuclease complex herein may comprise a Cas protein and a suitable polynucleotide component from any known CRISPR system (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).

[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," and "RGEN" are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease that can form a complex, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, allowing the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single- or double-strand break) the DNA target site.

[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" are used interchangeably herein and refer to a polynucleotide sequence, such as, but not limited to, 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), that a guide polynucleotide / Cas endonuclease complex can recognize, bind to, and optionally nick or cleave. A target site can be an endogenous site in the genome of a cell, or alternatively, a target site can be heterologous to the cell, such that it naturally occurs in the genome of the cell, or the target site can be found in a genomic location heterologous to where it occurs in nature. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein and refer to a target sequence that is endogenous or natural to the genome of a cell and is present in the endogenous or natural location of that target sequence in the genome of the cell. "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that is introduced into the genome of a cell. Such an artificial target sequence may be identical in sequence to an endogenous or natural target sequence in the genome of the cell, but may be located at a different location in the genome of the cell (i.e., a non-endogenous or non-occurring location).

[0325] As used herein, the term "protospacer adjacent motif" (PAM) refers to a short nucleotide sequence adjacent to the target sequence (protospacer) recognized (targeted) by the guide polynucleotide / Cas endonuclease system described herein. The Cas endonuclease cannot properly recognize a target DNA sequence unless the target DNA sequence is followed by a PAM sequence. The sequence and length of the PAM herein can vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is generally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0326] The terms "altered target site," "altered target sequence," "modified target site," and "modified target sequence" are used interchangeably herein and refer to a target sequence disclosed herein that contains at least one alteration compared to the unaltered target sequence. Such an "alteration" can include, for example, (i) a substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, (iv) a chemical alteration of at least one nucleotide, or (v) any combination of (i)-(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) a substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, (iv) a chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

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

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

[0330] The term "polynucleotide modification template" includes a polynucleotide that contains at least one nucleotide modification compared to the nucleotide sequence to be edited. The nucleotide modification can be a substitution, addition, or deletion of at least one nucleotide. Optionally, the polynucleotide modification template can further include a homologous nucleotide sequence adjacent to the at least one nucleotide modification, wherein the flanking homologous nucleotide sequence provides sufficient homology to the desired nucleotide sequence to be edited.

[0331] The term "plant-optimized Cas endonuclease" herein refers to a Cas protein, including a multifunctional Cas protein, that is encoded by a nucleotide sequence that is optimized for expression in a plant cell or plant.

[0332] The terms "plant-optimized nucleotide sequence encoding a Cas endonuclease," "plant-optimized construct encoding a Cas endonuclease," and "plant-optimized polynucleotide encoding a Cas endonuclease" are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, that has been optimized for expression in a plant cell or plant. Plants comprising a plant-optimized Cas endonuclease include plants comprising a nucleotide sequence encoding a Cas sequence and / or plants comprising a Cas endonuclease protein. In one aspect, the plant-optimized Cas endonuclease nucleotide sequence is a maize-optimized, rice-optimized, wheat-optimized, soybean-optimized, cotton-optimized, or canola-optimized Cas endonuclease.

[0333] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and their progeny. Plants can be monocotyledons or dicotyledons. 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 the following: whole plants, seedlings, meristems, ground tissues, vascular tissue, epidermal tissue, seeds, leaves, roots, shoots, stems, flowers, fruits, stolons, bulbs, tubers, corms, stems, shoots, shoots, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, callus tissue). It should be noted that because protoplasts lack a cell wall, protoplasts are not technically "intact" plant cells (as found in nature with all their components). The term "plant organ" refers to a plant tissue or a group of tissues that constitute a morphologically and functionally independent part of a plant. As used herein, "plant element" is synonymous with plant "part" and refers to any part of a plant, may include separate tissues and / or organs, and may be used interchangeably with the term "tissue" throughout. Similarly, "plant reproductive element" is generally intended to refer to any part of a plant that is capable of initiating other plants by sexual or asexual reproduction of that plant, such as, but not limited to, a seed, seedling, root, shoot, cutting, scion, graft, stolon, bulb, tuber, corm, keiki, or sprout. The plant element may be in a plant or plant organ, tissue culture, or cell culture.

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

[0335] As used herein, the term "plant part" refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant mass, and intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, pods, stalks, roots, root tips, anthers, and parts thereof, as well as parts thereof. Grain is intended to mean mature seeds produced by growers for purposes other than the cultivation or propagation of the species. Progeny, variants, and mutants of the regenerated plants are also within the scope of the present invention, provided that the part comprises the introduced polynucleotide.

[0336] The terms "monocotyledonous" or "monocotyledonous plant" refer to a subclass of angiosperms, also known as "monocotyledoneae," whose seeds generally contain only one primary leaf or cotyledon. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0337] The terms "dicotyledonous" or "dicotyledonous plant" refer to a subclass of angiosperms, also known as "dicotyledoneae," whose seeds generally contain only two primary leaves or cotyledons. The term includes reference to whole plants, plant elements, plant organs (e.g., 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 sterile plants and female sterile plants can be female fertile and male fertile, respectively. It is further recognized that male fertile (other than female fertile) plants can produce viable offspring when crossed with female fertile plants, and female fertile (other than male fertile) plants can produce viable offspring when crossed with male fertile plants.

[0339] As used herein, the term "non-conventional yeast" refers to any yeast that is not a Saccharomyces yeast species (e.g., S. cerevisiae) or a Schizosaccharomyces yeast species (see "Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols," K. Wolf, KD Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003).

[0340] In the context of this disclosure, the terms "hybridized," or "crossing," or "crossing" refer to the fusion of gametes by pollination to produce offspring (i.e., cells, seeds, or plants). The term encompasses both sexual crossing (pollination of one plant by another) and selfing (self-pollination, i.e., where the pollen and ovules (microspores and megaspores) are from the same plant or from genetically identical plants).

[0341] The term "introgression" refers to the transfer of a desired allele at a genetic locus from one genetic background to another. For example, introgression of a desired allele at a particular genetic locus can be transferred to at least one progeny plant by sexual crossing between two parent plants, where at least one parent plant has the desired allele in its genome. Alternatively, for example, transfer of the allele can be achieved by recombination between two donor genomes, e.g., in fused protoplasts, where at least one donor protoplast has the desired allele in its genome. 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 line" is a comparative term, referring to genetically identical but differently treated reference organisms. In one example, two genetically identical maize plant embryos can be separated into two distinct groups: one that has undergone a treatment (such as the introduction of a CRISPR-Cas effector endonuclease) and the other that has not undergone such a treatment, a control. In this way, any phenotypic differences between the two groups can be attributed solely to the treatment and not to any inherent nature of the plant's endogenous genetic makeup.

[0343] "Introducing" is intended to mean providing a polynucleotide or polypeptide or polynucleotide-protein complex to a target, such as a cell or organism, in such a way that the component enters the interior of a cell of the organism or into the cell itself.

[0344] A "polynucleotide of interest" includes a nucleotide sequence that encodes a protein or polypeptide that improves the desirability, i.e., agronomically beneficial, trait of a crop. Polynucleotides of interest include, but are not limited to, polynucleotides that encode important traits for agronomically important traits, herbicide resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, crop characteristics, commercial products, phenotypic markers, or other traits of agricultural or commercial importance. Polynucleotides of interest may further be utilized in 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 that has multiple transgenes genetically linked to each other.

[0346] The compositions and methods herein may provide plants with improved "agronomic traits" or "agronomically important traits" or "agronomically beneficial traits", which may include, but are not limited to, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, increased yield, enhanced health, improved vigor, improved growth, improved photosynthetic capacity, nutritional enhancement, modified protein content, modified oil content, increased biomass, increased root length, improved root architecture, modulation of metabolites, modulation of the proteome, increased seed weight, modified seed carbohydrate composition, modified seed oil composition, modified seed protein composition, modified seed nutrient composition, compared to an isogenic plant not modified by the methods or compositions described herein.

[0347] "Agronomic trait potential" is intended to mean the ability of a plant element, at a certain point in its life cycle, to exhibit a phenotype, preferably an improved agronomic trait, or to transmit said phenotype to another related plant element of the same plant.

[0348] As used herein, the terms "reduced," "less," "slower," and "increased," "faster," "enhanced," and "greater" refer to a decrease or increase in a property of a modified plant element or resulting plant compared to an unmodified plant element or resulting plant. For example, the decrease in the property may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5% to 10%, at least 10%, 10% to 20%, at least 15%, at least 20%, 20% to 30%, at least 25%, at least 30%, 30% to 40%, at least 35%, at least 40%, 40% to 50%, at least 45%, at least 50%, 50% to 60%, at least about 60%, 60% to 70%, 70% to 80%, at least 75%, at least about 80%, 80% to 90%, at least about 90%, 90% to 100%, at least 100%, 100% to 200%, at least 200%, at least about 300%, at least about 400%) or more less than an untreated control. and the increase in the property may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5% to 10%, at least 10%, 10% to 20%, at least 15%, at least 20%, 20% to 30%, at least 25%, at least 30%, 30% to 40%, at least 35%, at least 40%, 40% to 50%, at least 45%, at least 50%, 50% to 60%, at least about 60%, 60% to 70%, 70% to 80%, at least 75%, at least about 80%, 80% to 90%, at least about 90%, 90% to 100%, at least 100%, 100% to 200%, at least 200%, at least about 300%, at least about 400% or more over an untreated control.

[0349] As used herein, the term "before," in reference to sequence position, refers to the presence of one sequence upstream or 5' of another sequence.

[0350] The abbreviations have the following meanings: "sec" means second, "min" means minute, "h" means hour, "d" means day, "μL" means microliter, "mL" means milliliter, "L" means liter, "μM" means micromolar, "mM" means millimolar, "M" means mole, "mmol" means millimole, "μmole" or "umole" means micromole, "g" means gram, "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 the sequence and structural analysis of their components. Several CRISPR / Cas systems have been described, including class 1 systems with multisubunit effector complexes (including types I, III, and IV) and class 2 systems with single-protein effectors (including types II, V, and VI) (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] The CRISPR-Cas system comprises, at a minimum, a CRISPR RNA (crRNA) molecule and at least one CRISPR-associated (Cas) protein, which form a crRNA-ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus contains an array of identical repeats interspersed with DNA targeting spacers encoding the crRNA components and an operon-like unit of cas genes encoding 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)). The crRNA functions as a guide RNA, directing the sequence-specific binding of the effector (protein or complex) to double-stranded DNA sequences by base-pairing with the complementary DNA strand and forming a so-called R-loop, while displacing the non-complementary strand (Jore et al., 2011, Nature Structural & Molecular Biology 18, 529-536).

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

[0354] Different cas genes, encoding proteins with distinct domains, exist in different CRISPR systems. The cas operon contains genes encoding one or more effector endonucleases and other Cas proteins. Protein subunits include those described in Makarova et al. 2011, Nat Rev Microbiol. 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. Domain types include those involved in expression (pre-crRNA processing, e.g., Cas6 or RNase III), buffering (including effector modules for crRNA and target binding and domains for target cleavage), adaptation (spacer insertion, e.g., Cas1 or Cas2), and assistance (regulatory, helper, or unknown function). Some domains may serve more than one purpose; for example, Cas9 contains a domain for endonuclease function and a domain for target cleavage.

[0355] Cas endonucleases are guided by a single CRISPR RNA (crRNA) through direct RNA-DNA base pairing and recognize DNA target sites adjacent to a protospacer adjacent motif (PAM) (Jore, MM et al., 2011, Nat. Struct. Mol. Biol. 18:529-536; Westra, ER et al., 2012, Molecular Cell 46:595-605; and Sinkunas, T. et al., 2013, EMBO J. 32:385-394).

[0356] Class I CRISPR-Cas systems Class I CRISPR-Cas systems include types I, III, and IV. A distinctive feature of Class I systems is the presence of an effector endonuclease complex instead of a single protein. The Cascade complex contains an RNA recognition motif (RRM) and a nucleic acid-binding domain, which is the core fold of the diverse RAMP (repeat-associated 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 scaffold of the crRNA-effector complex), where the Cas5 subunit binds to the 5' handle of the crRNA and interacts with the large subunit, and Cas6, which is often loosely associated with the effector complex 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] Type I CRISPR-Cas systems contain a complex of effector proteins called Cascade (CRISPR-associated complex for antiviral defense), which includes at least Cas5 and Cas7. The effector complex functions with a single CRISPR RNA (crRNA) and Cas3 to defend against invading viral DNA (Brouns, SJJ et al. Science 321:960-964; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type I CRISPR-Cas loci contain the signature gene cas3 (or variants cas3' or cas3"), which encodes a metal-dependent nuclease with a single-stranded DNA (ssDNA)-stimulating superfamily 2 helicase that has demonstrated the ability to unwind double-stranded DNA (dsDNA) and RNA-DNA duplexes (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Following target recognition, the Cas3 endonuclease is recruited to the Cascade-crRNA-target DNA complex to cleave and degrade the DNA target (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, and Cas5 and Cas7 function as non-catalytic RNA-binding proteins, whereas in type I-C systems, crRNA processing may be catalyzed by Cas5 (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type I systems have been 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).A modified type I CRISPR-associated complex (Cascade) for adaptive antiviral defense comprising at least the protein subunits Cas7, Cas5, and Cas6, one of which is synthetically fused to the Cas3 endonuclease or the modified restriction endonuclease FokI, has been described (WO 2013098244, published July 4, 2013).

[0358] Type III CRISPR-Cas systems, containing multiple cas7 genes, target either ssRNA or ssDNA and function as either an RNase or a target RNA-activated DNA nuclease (Tamulaitis et al., Trends in Microbiology 25(10)49-61, 2017). The 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)-guided binding of the Csm or Cmr complex to nascent transcripts recruits the Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and the phage DNA, but not the host DNA. The Cas10 HD domain is responsible for the ssDNase activity, and the Csm3 / Cmr4 subunits are responsible for the endoribonuclease activity of the Csm / Cmr complex. The 3' flanking sequence of the target RNA is important for the ssDNase activity of Csm / Cmr: base pairing with the 5' handle of the crRNA protects the host DNA from degradation.

[0359] Type IV systems contain typical type I cas5 and cas7 domains in addition to a cas8-like domain, but can lack the CRISPR array that is 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 distinctive feature of class II systems is the presence of a single Cas effector protein instead of an effector complex. Type II and V Cas proteins contain a RuvC endonuclease domain that adopts an RNase H fold.

[0361] Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNA contains a spacer region that is complementary to one strand of the double-stranded DNA target and a region that base-pairs with the tracrRNA (trans-activating CRISPR RNA) to form an RNA duplex that allows the Cas endonuclease to cleave the DNA target, leaving a blunt end. The spacer is obtained through a poorly understood process involving the Cas1 and Cas2 proteins. Type II CRISPR / Cas loci typically 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 within each CRISPR array and can contain other proteins such as Csn1 and Csn2. The presence of cas9 adjacent to the cas1 and cas2 genes is a hallmark of type II loci (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).

[0362] Type V CRISPR / Cas systems contain a single Cas endonuclease, including Cpf1 (Cas12) (Koonin et al., Curr Opinion Microbiology 37:67-78, 2017), which, unlike Cas9, is an active RNA-guided endonuclease that does not necessarily require an additional trans-activating CRISPR (tracr) RNA for target cleavage.

[0363] Type VI CRISPR-Cas systems contain two HEPN (higher eukaryotic and prokaryotic nucleotide-binding) domains, but lack the HNH or RuvC domains and contain the cas13 gene, which is independent of tracrRNA activity. The majority of the HEPN domain contains a conserved motif that constitutes a metal-independent endo-RNase active site (Anantharam et al., Biol Direct 8:15, 2013). This feature suggests that type VI systems act on RNA targets instead of the DNA targets common to other CRISPR-Cas systems.

[0364] Novel CRISPR-Cas system Disclosed herein is a novel CRISPR-Cas system, its components, and methods of using said components. The system includes a novel Cas effector protein, Cas-beta.

[0365] The novel CRISPR-Cas system components described herein may include one or more subunits from different Cas systems, subunits derived from or modified from two or more different bacterial or archaeal prokaryotes, and / or synthetic or engineered components.

[0366] Described herein is a newly identified CRISPR-Cas system that includes novel sequences for the cas genes. Additionally, novel cas genes and proteins are described.

[0367] One distinguishing feature of the novel Cas-beta system is its locus structure, as depicted in Figure 1. The order of polynucleotides on the complementary strand is as follows: the cas-beta gene, the cas1 gene, the cas2 gene, and an unclassified cas gene that shares low sequence identity with cas4. Following the unclassified cas gene is a CRISPR array containing 37 repeats of a 37-nucleotide sequence, 33 of which are identical, three of which are 97.3% identical, and one at the end is present with 78.4% identity. The spacer polynucleotide between each array repeat is 35 or 36 nucleotides in length.

[0368] CRISPR-Cas system components Cas proteins Numerous proteins can be encoded in the CRISPR cas operon, including proteins involved in adaptation (spacer insertion), interference (effector module target binding, target nicking or cleavage—e.g., endonuclease activity), expression (pre-crRNA processing), regulation, and more.

[0369] Two proteins, Cas1 and Cas2, are conserved among many CRISPR systems (see, for example, Koonin et al., Curr Opinion Microbiology 37:67-78, 2017). Cas1 is a metal-dependent DNA-specific endonuclease that generates double-stranded DNA fragments. In some systems, Cas1 forms a stable complex with Cas2, which is essential for spacer acquisition and insertion for CRISPR systems (Nunez et al., Nature Str Mol Biol 21:528-534, 2014).

[0370] Many other proteins have been identified in various systems, including Cas4 (which may have similarity to RecB nuclease), 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, Cas9, the signature protein of class 2 type II systems, 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 endonucleases and effectors Endonucleases are enzymes that cleave phosphodiester bonds in polynucleotide chains, including restriction endonucleases, which cleave DNA at specific sites without damaging bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-associated) effector endonucleases.

[0374] Cas endonucleases, either as single effector proteins or forming effector complexes with other components, unwind DNA duplexes at target sequences and, optionally, cleave at least one DNA strand, mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) complexed with the Cas effector protein. Generally, such recognition and cleavage of a target sequence by a Cas endonuclease typically occurs when a precise protospacer adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, the Cas endonucleases herein, when complexed with a suitable RNA component, may lack DNA cleavage or nicking activity but still be capable of specifically binding to a DNA target sequence. (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] Described Cas endonucleases include, but are not limited to, 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).

[0377] Cas3 (and its variants Cas3' and Cas3") functions as a single-stranded DNA nuclease (HD domain) and an ATP-dependent helicase. Variants of the Cas3 endonuclease can be obtained by abolishing the functional activity of one or both domains of the Cas3 endonuclease polypeptide. ATPase-dependent helicase activity can be abolished (by deletion, knockout of the Cas3-helicase domain, or 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), disabling HD endonuclease activity can convert a cleavage-ready cascade containing a modified Cas3 endonuclease into a nickase (since the HD domain is still functional). Disabling HD endonuclease activity can be achieved by methods known in the art, including but not limited to, mutagenesis of key residues in the HD domain, and converting a cleavage-ready cascade containing a modified Cas3 endonuclease into a helicase. Disabling both Cas helicase and Cas3 HD endonuclease activity can be achieved by methods known in the art, including but not limited to, mutagenesis of key residues in both the helicase and HD domains, and converting a cleavage-ready cascade containing a modified Cas3 endonuclease into a binding protein that binds to a target sequence.

[0378] Cas9 (previously called Cas5, Csn1, or Csx12) is a Cas endonuclease that specifically recognizes and cleaves all or part of a DNA target sequence by complexing with cr and tracr nucleotides or a single guide polynucleotide. Cas9 recognizes the 3' GC-rich PAM sequence of target dsDNA. The Cas9 protein contains a RuvC nuclease with an HNH (HNH) nuclease adjacent to the RuvC-II domain. The RuvC nuclease and HNH nuclease can each cleave one DNA strand at the target sequence (the concerted action of both domains results in a DNA double-strand break, while the activity of one domain results in a nick). Generally, the RuvC domain contains subdomains I, II, and III; domain I is located near the N-terminus of Cas9, and subdomains II and III are located in the center of the protein and adjacent to the HNH domain (Hsu et al., Cell 157:1262-1278). Cas9 endonuclease is typically derived from a type II CRISPR system, which includes a DNA cleavage system that utilizes Cas9 endonuclease complexed with at least one polynucleotide component. For example, Cas9 can be complexed with CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In another example, Cas9 can 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) contains a RuvC nuclease domain and generates staggered 5' overhangs on dsDNA targets. Some variants do not require tracrRNA for their functionality, unlike Cas9. Cas12 and its variants recognize 5' AT-rich PAM sequences on target dsDNA. The insertion domain of the Cas12a protein, called Nuc, has been demonstrated to be involved in target strand cleavage (Yamano et al., Cell 2016, 165:949-962). Further mutational studies in other Cas12 proteins have demonstrated that the Nuc domain, along with the RuvC domain involved in cleavage, contributes to guidance and target binding (Swarts et al., Mol Cell 2017, 66:221-233 e224).

[0380] Cas endonucleases and effector proteins can be used for targeted genome editing (through single and multiple double-strand breaks and nicks) and targeted genome regulation (by attaching epigenetic effector domains to Cas proteins or sgRNAs). Cas endonucleases can also be engineered to function as RNA-guided recombinases, and, through RNA tethers, can serve as scaffolds for the assembly of multiprotein and nucleic acid complexes (Mali et al., 2013, Nature Methods Vol. 10:957-963).

[0381] Cas-beta endonuclease Cas-beta endonucleases are defined as functional RNA-guided, PAM-dependent dsDNA cleavage proteins of less than 900 amino acids and contain at least one of the motifs listed in Table 1B.

[0382] The RuvC domain has been documented to contain endonuclease functionality. Cas-beta endonucleases can be isolated or characterized from loci containing cas-beta genes encoding effector proteins and arrays containing multiple repeats. In some embodiments, the cas-beta locus can further contain part or all of the cas1 gene, the cas2 gene, and / or the cas4 gene.

[0383] Zinc finger motifs are domains that coordinate one or more zinc ions, usually by cysteine ​​and histidine side chains, to stabilize their fold. Zinc fingers are named for the pattern of cysteine ​​and histidine residues that coordinate the zinc ion (e.g., C4 means that the zinc ion is coordinated by four cysteine ​​residues, while CH means that the zinc ion is coordinated by three cysteine ​​residues and one histidine residue).

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

[0385] Cas-beta endonucleases function as double-strand break inducers and may also be nickases or single-strand break inducers. In some embodiments, catalytically inactive Cas-beta endonucleases may be used to target or recruit to a target DNA sequence but do not induce cleavage. In some embodiments, catalytically inactive Cas-beta proteins may be used in conjunction with functional endonucleases that cleave the target sequence. In some embodiments, catalytically inactive Cas-beta proteins may be combined with base-editing molecules such as deaminase. In some embodiments, the deaminase may be cytidine deaminase. In some embodiments, the deaminase may be adenine deaminase. In some embodiments, the deaminase may be ADAR-2.

[0386] The Cas-beta endonuclease may comprise 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 consecutive amino acids of any of SEQ ID NOs: 13, 14, 15 or 57, or a functional fragment or functional variant thereof that retains at least some activity. A "functional fragment" of a Cas-beta endonuclease is further defined as an RNA-guided double-stranded DNA cleavage protein that shares 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 with the RNA-guided double-stranded DNA cleavage protein.

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

[0388] The Cas-beta endonuclease may be a nucleotide sequence of 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, ... It may be encoded by a polynucleotide sharing at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity with 50 to 1000, at least 1000, or more than 1000 contiguous nucleotides.

[0389] Cas endonucleases, effector proteins, or functional fragments thereof for use in the disclosed methods can be isolated from natural sources or from recombinant sources in which genetically engineered host cells have been modified to express nucleic acid sequences encoding the proteins. Alternatively, Cas proteins can be produced using cell-free protein expression systems or produced synthetically. Effector Cas nucleases can be isolated and introduced into heterologous cells, or modified from their native form to exhibit a different type or magnitude of activity than their native source. Such modifications include, but are not limited to, fragments, variants, substitutions, deletions, and insertions.

[0390] Fragments and variants of Cas endonucleases and Cas effector proteins can be obtained by methods such as site-directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art and include, but are not limited to, those described in WO2013166113, published November 7, 2013, WO2016186953, published November 24, 2016, and WO2016186946, published November 24, 2016.

[0391] Cas endonucleases can include modified forms of Cas polypeptides. Modified forms of Cas polypeptides can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the naturally occurring nuclease activity of the Cas protein. For example, in some instances, modified forms of Cas proteins have less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nuclease activity of the corresponding wild-type Cas polypeptide (U.S. Patent Application Publication No. 20140068797, published March 6, 2014). In some instances, modified forms of Cas polypeptides have substantially no nuclease activity and are referred to as catalytically "inactivated Cas" or "deactivated Cas (dCas)." Inactivated Cas / deactivated Cas includes deactivated Cas endonuclease (dCas). Catalytically inactive Cas effector proteins can be fused to heterologous sequences to induce or modify activity.

[0392] The Cas endonuclease may be part of a fusion protein that includes one or more heterologous protein domains (e.g., one, two, three or more domains in addition to the Cas protein). Such a fusion protein may include any additional protein sequences and, optionally, a linker sequence between any two domains, e.g., between the Cas and the first heterologous domain. Examples of protein domains that can be fused to the Cas proteins herein include, but are not limited to, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], 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 having one or more of the following activities: methylase activity, demethylase activity, transcriptional activator activity (e.g., VP16 or VP64), transcriptional repressor activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also be fused to proteins that bind to DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

[0393] Catalytically active and / or inactive Cas endonucleases can be fused to heterologous sequences (U.S. Patent Application Publication No. 20140068797, published March 6, 2014). Suitable fusion partners include, but are not limited to, polypeptides that provide 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). Additional suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylating activity, deadenylating activity, sumoylating activity, desumoylating activity, ribosylation activity, deribosylation activity, myristoylating activity, or demyristoylating activity. Further suitable fusion partners include, but are not limited to, polypeptides that directly cause increased transcription of a target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that induce transcriptional activators, small molecule / drug-responsive transcriptional regulators, etc.) Partially active or catalytically inactive Cas-beta endonucleases can also be fused to another protein or domain, such as Clo51 nuclease or FokI nuclease, to generate a double-stranded break (Guilinger et al. Nature biotechnology, volume 32, number 6, June 2014).

[0394] Catalytically active or inactive Cas proteins, such as the Cas-beta proteins described herein, can also be fused to molecules that direct the editing of single or multiple bases in a polynucleotide sequence, such as site-specific deaminases that can change the identity of a nucleotide, for example, from C·G to T·A or from A·T to G·C (Gaudelli et al., "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al., "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353(6305) (2016); Komor et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage." Nature 533(7603)(2016):420-4. Base editing fusion proteins can include, for example, active (double-strand breaking), 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 (e.g., uracil glycosylase inhibitors (which prevent the removal of uracil)) are contemplated as other components of the base editing system in some embodiments.

[0395] The Cas endonucleases described herein can be expressed and purified by methods known in the art, for example, as described in WO 2016 / 186953, published November 24, 2016.

[0396] Many Cas endonucleases have been described that can recognize specific PAM sequences (WO2016186953 published November 24, 2016; WO2016186946 published November 24, 2016; and Zetsche B et al. 2015. Cell 163, 1013) and cleave target DNA at specific locations. Of course, those skilled in the art will understand that based on the methods and embodiments described herein that use the novel inducible Cas system, these methods can be adapted to use any inducible endonuclease system.

[0397] The Cas effector protein may comprise a heterologous nuclear localization sequence (NLS). The heterologous NLS amino acid sequence herein may be strong enough to drive the accumulation of detectable amounts of the Cas protein in the nucleus of, for example, a yeast cell herein. The NLS may comprise a short sequence (e.g., 2-20 residues) of one (monoclavicular) or multiple (e.g., biclavicular) basic, positively charged residues (e.g., lysine and / or arginine) and may be positioned anywhere within the Cas amino acid sequence so that it is exposed on the protein surface. The NLS may be operably linked, for example, to the N-terminus or C-terminus of the Cas protein herein. For example, two or more NLS sequences may be linked to the Cas protein, for example, to the N-terminus and C-terminus of the Cas protein. The Cas endonuclease gene is operably linked to an SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) of the Cas codon region. Non-limiting examples of suitable NLS sequences herein include those disclosed in U.S. Patent Nos. 6,660,830 and 7,309,576.

[0398] Guide polynucleotide The guide polynucleotide enables the Cas endonuclease to recognize, bind to, and optionally cleave the target, and can be a single molecule or a double molecule. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide can include at least one nucleotide, a phosphodiester bond, or a linkage modification, including, but not limited to, locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, phosphorothioate linkage, a linkage to a cholesterol molecule, a linkage to a polyethylene glycol molecule, a linkage to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent linkage resulting in cyclization. A guide polynucleotide that contains only ribonucleic acid is also called a "guide RNA" or "gRNA" (U.S. Patent Application Publication No. 20150082478, published March 19, 2015, and U.S. Patent Application Publication No. 20150059010, published February 26, 2015). Guide polynucleotides can be engineered or synthesized.

[0399] Guide polynucleotides include chimeric non-natural guide RNAs (i.e., they are heterologous to each other) that contain regions that are not found together in nature, such as a chimeric non-natural guide RNA that contains a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA linked to a second nucleotide sequence that can recognize a Cas endonuclease, where the first and second nucleotide sequences are not found linked together in nature.

[0400] The guide polynucleotide can be a duplex molecule (also called a double-stranded guide polynucleotide) that includes a cr nucleotide sequence (e.g., crRNA) and a tracr nucleotide sequence (e.g., tracrRNA). Optionally, a linker polynucleotide is present that connects the crRNA and tracrRNA to form a single guide, e.g., sgRNA.

[0401] The cr nucleotide comprises a first nucleotide sequence domain (called a variable targeting domain or VT domain) capable of hybridizing to a nucleotide sequence in the target DNA and a second nucleotide sequence (also called a tracr mate sequence) that is part of the Cas endonuclease recognition (CER) domain. The tracr mate sequence hybridizes to the tracr nucleotide along a complementary region, and together they form a Cas endonuclease recognition domain, i.e., a CER domain. The CER domain can interact with a Cas endonuclease polypeptide. The cr nucleotide and tracr nucleotide of the double-stranded guide polynucleotide can be RNA, DNA, and / or RNA-DNA combination sequences. In some embodiments, the cr nucleotide molecule of the double-stranded guide polynucleotide is referred to as "crDNA" (when composed of a continuous stretch of DNA nucleotides), "crRNA" (when composed of a continuous stretch of RNA nucleotides), or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The cr nucleotide can comprise a fragment of the crRNA naturally occurring in Bacteria and Archaea. The size of the fragments of naturally occurring crRNA in Bacteria and Archaea that can be present in the cr nucleotides disclosed herein can vary from, but are not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. In some embodiments, the crRNA molecule is selected from the group consisting of SEQ ID NOs: 22, 23, and 24.

[0402] In some embodiments, the tracr nucleotide sequence is referred to as "tracrRNA" (when composed of a continuous stretch of RNA nucleotides), or "tracrDNA" (when composed of a continuous stretch of DNA nucleotides), or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In one embodiment, the RNA that guides the RNA / Cas9 endonuclease complex is a double-stranded RNA comprising a double-stranded crRNA-tracrRNA. tracrRNA (trans-activating CRISPR RNA) contains, from 5' to 3', (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 the guide polynucleotide / Cas endonuclease complex (also called a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, allowing the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single- or double-strand break) the target site (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, wherein said guide polynucleotide comprises a first nucleotide sequence domain that is complementary to a nucleotide sequence of a target DNA and a second nucleotide sequence domain that interacts with said Cas endonuclease polypeptide.

[0405] In one aspect, the guide polynucleotide is a guide polynucleotide described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain are guide polynucleotides selected from the group consisting of DNA sequences, RNA sequences, and combinations thereof.

[0406] In one aspect, the guide polynucleotide is a guide polynucleotide described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain are selected from the group consisting of stability-enhancing RNA backbone modifications, stability-enhancing DNA backbone modifications, and combinations thereof (see Kanasty et al., 2013, Common RNA-backbone modifications, Nature Materials 12:976-977; U.S. Patent Application Publication No. 20150082478 published March 19, 2015; U.S. Patent Application Publication No. 20150059010 published February 26, 2015).

[0407] The guide RNA includes a duplex molecule containing a chimeric non-natural crRNA linked to at least one tracrRNA. The chimeric non-natural crRNA includes a crRNA that contains regions that are not found together in nature (i.e., they are heterologous to each other). For example, a crRNA that contains a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in target DNA linked to a second nucleotide sequence (also called a tracrmate sequence), where the first and second sequences are not found linked together in nature.

[0408] The guide polynucleotide can also be a single molecule (also referred to as a single guide polynucleotide) that includes a cr nucleotide sequence linked to a tracr nucleotide sequence. A single guide polynucleotide includes a first nucleotide sequence domain (called a 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 a Cas endonuclease polypeptide. In some embodiments, the sgRNA molecule is selected from the group consisting of SEQ ID NOs: 31-36.

[0409] The VT and / or CER domains of a single guide polynucleotide can comprise RNA, DNA, or a combined RNA-DNA sequence. A single guide polynucleotide composed of a sequence derived from cr and tracr nucleotides can be referred to as "single guide RNA" (when composed of a continuous stretch of RNA nucleotides), "single guide DNA" (when composed of a continuous stretch of DNA nucleotides), or "single guide RNA-DNA" (when composed of a combination of RNA and DNA nucleotides). A single guide polynucleotide can form a complex with a Cas endonuclease, and the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, allowing the Cas endonuclease to recognize, bind, and optionally nick or cleave (introduce a single- or double-strand break) the target site. (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-naturally occurring single guide RNAs (sgRNAs) include sgRNAs that contain regions that are not found together in nature (i.e., they are heterologous to each other), such as an sgRNA that contains a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in target DNA linked to a second nucleotide sequence (also called a target mate sequence) that is not found linked together in nature.

[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 linking the cr and tracr nucleotides of the single guide polynucleotide (also referred to as the "loop") can comprise 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, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, It can be 3, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the cr and tracr nucleotides of the single guide polynucleotide can comprise a tetraloop sequence, such as, but not limited to, a GAAA tetraloop sequence.

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

[0413] Protospacer adjacent motif (PAM) As used herein, the term "protospacer adjacent motif" (PAM) refers to a short nucleotide sequence adjacent to the target sequence (protospacer) recognized (targeted) by the guide polynucleotide / Cas endonuclease system. The Cas endonuclease cannot correctly recognize a target DNA sequence unless the target DNA sequence is followed by a PAM sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence may be of any length but is generally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.

[0414] The terms "randomized PAM" and "randomized protospacer adjacent motif" are used interchangeably herein and refer to a random DNA sequence adjacent to a target sequence (protospacer) recognized (targeted) by a guide polynucleotide / Cas endonuclease system. The randomized PAM sequence can be of any length, but is generally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. The randomized nucleotides include any of the 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 typically comprises a Cas protein having all of its endonuclease domains functionally (e.g., a wild-type endonuclease domain or a variant thereof that retains the activity of part or all of each endonuclease domain). Thus, a wild-type Cas protein (e.g., a Cas protein disclosed herein) or a variant thereof that retains the activity of part or all of each endonuclease domain of the Cas protein are suitable examples of Cas proteins that can cleave both strands of a DNA target sequence.

[0417] A guide polynucleotide / Cas endonuclease complex capable of cleaving a single strand of a DNA target sequence may be characterized herein as having nickase activity (e.g., partial cleavage ability). Cas nickases typically contain a single functional endonuclease domain that enables Cas to cleave (i.e., nick) only one strand of a DNA target sequence. For example, a Cas9 nickase may contain (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, a Cas9 nickase may contain (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a mutated, dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Application Publication No. 20140189896, published July 3, 2014. To increase the specificity of DNA targeting, a pair of Cas nickases can be used. Generally, this can be accomplished by providing two Cas nickases that are linked to RNA components with different guide sequences to target and nick DNA sequences on opposite strands in the region in close proximity for the desired targeting. Such nearby cleavage of each DNA strand creates a double-strand break (i.e., a DSB with a single-strand overhang), which is then recognized as a substrate for non-homologous end-joining (NHEJ) (prone to imperfect repair leading to mutations) or homologous recombination (HR). In these embodiments, the nicks can be separated from one another by, for example, at least about 5, 5-10, at least 10, 10-15, at least 15, 15-20, at least 20, 20-30, at least 30, 30-40, at least 40, 40-50, at least 50, 50-60, at least 60, 60-70, at least 70, 70-80, at least 80, 80-90, at least 90, 90-100, or 100 or more (or any integer between 5 and 100) bases. One or two Cas nickase proteins herein can be used in a Cas nickase pair.For example, a Cas9 nickase with a mutated RuvC domain but a functional HNH domain (i.e., Cas9 HNH / RuvC) can be used (e.g., Streptococcus pyogenes Cas9 HNH / RuvC). Each Cas9 nickase (e.g., Cas9 HNH / RuvC) can be directed to specific DNA sites close together (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences that target each nickase to its specific DNA site.

[0418] In certain embodiments, the guide polynucleotide / Cas endonuclease complex can bind to a DNA target site sequence but does not cleave any strand at the target site sequence. Such a complex can include a Cas protein in which all of its nuclease domains are 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 include both a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. Cas proteins herein that bind to but do not cleave a target DNA sequence can be used to regulate gene expression; for example, the Cas protein can be fused to a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein).

[0419] In one aspect, a guide polynucleotide / Cas endonuclease complex (PGEN) described herein is a PGEN in which said Cas endonuclease is optionally covalently or non-covalently linked or assembled to at least one protein subunit or functional fragment thereof.

[0420] In one embodiment of the present disclosure, the guide polynucleotide / Cas endonuclease complex is a guide polynucleotide / Cas endonuclease complex (PGEN) comprising at least one guide polynucleotide and at least one Cas endonuclease polypeptide, wherein said Cas endonuclease polypeptide comprises at least one protein subunit or functional fragment thereof, said guide polynucleotide is a chimeric non-natural guide polynucleotide, and said guide polynucleotide / Cas endonuclease complex is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or a portion of a target sequence.

[0421] The Cas effector protein may be a 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 the guide polynucleotide / Cas effector protein complex is capable of recognizing, binding to, and optionally nicking, unwinding, or cleaving all or a portion of a target sequence.

[0423] The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises one or more copies of at least one protein subunit or functional fragment thereof. In some embodiments, the protein subunit is selected from the group consisting of a Cas1 protein subunit, a Cas2 protein subunit, a Cas4 protein subunit, and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises at least two different protein subunits selected from the group consisting of Cas1, Cas2, and Cas4.

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

[0425] In one aspect, the guide polynucleotide / Cas effector protein complex (PGEN) described herein is a PGEN in which the Cas effector protein is covalently or non-covalently linked to at least one protein subunit or functional fragment thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex in which the Cas effector protein polypeptide is covalently or non-covalently linked to or assembled with one copy or multiple copies of at least one protein subunit or functional fragment thereof selected from the group consisting of a Cas1 protein subunit, a Cas2 protein subunit, optionally one additional Cas protein subunit including a Cas4 protein, and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex in which the Cas effector protein is covalently or non-covalently linked to or assembled with at least two different protein subunits selected from the group consisting of a Cas1 protein subunit, a Cas2 protein subunit, and optionally one additional Cas protein including a Cas4 protein. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein is covalently or non-covalently linked to at least three different protein subunits selected from the group consisting of Cas1, Cas2, and optionally one additional Cas protein, including Cas4, and combinations thereof.

[0426] Any component of the guide polynucleotide / Cas effector protein complex, the guide polynucleotide / Cas effector protein complex itself, and the polynucleotide modification template and / or donor DNA can be introduced into a heterologous cell or organism by any method known in the art.

[0427] Recombinant constructs for cell transformation The disclosed guide polynucleotides, Cas endonucleases, polynucleotide modification templates, donor DNA, guide polynucleotide / Cas endonuclease systems disclosed herein, and any combination thereof, optionally further comprising one or more polynucleotides of interest, can be introduced into cells, including but 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.

[0428] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in more detail in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described below.

[0429] Vectors and constructs include circular plasmids and linear polynucleotides, which contain a polynucleotide of interest and optionally other components, including linkers, adapters, regulatory elements, or analytical elements. In some embodiments, recognition 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 further provides expression constructs for expressing guide RNA / Cas systems in prokaryotic or eukaryotic cells / organisms that are capable of recognizing, binding to, and optionally nicking, unnicking, or cleaving all or part of a target sequence.

[0431] In one embodiment, an expression construct of the present disclosure comprises a promoter operably linked to a nucleotide sequence encoding a Cas gene (or an optimized plant comprising a Cas endonuclease gene as described herein) and a promoter operably linked to a guide RNA of the present disclosure. The promoter is capable of driving expression of the operably linked nucleotide sequence in a prokaryotic or eukaryotic cell / organism.

[0432] Nucleotide sequence modifications of the guide polynucleotide, VT domain, and / or CER domain can be selected from the group consisting of, but are not limited to, a 5' cap, a 3' polyadenylation tail, a riboswitch sequence, a stable regulatory sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a binding site for a protein, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2'-fluoro A nucleotides, 2'-fluoro U nucleotides; 2'-O-methyl RNA nucleotides, phosphorothioate linkages, linkages to cholesterol molecules, linkages to polyethylene glycol molecules, linkages to spacer 18 molecules, 5' to 3' covalent linkages, or any combination thereof. These modifications can result in at least one additional advantageous characteristic selected from the group of modified or modulated stability, intracellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity for complementary target sequences, modified resistance to cellular degradation, and increased cell permeability.

[0433] A method for expressing RNA components, such as gRNAs, in eukaryotic cells for Cas9-mediated DNA targeting is to use an RNA polymerase III (Pol III) promoter, which allows transcription of RNAs with precisely defined, unmodified 5' and 3' ends (DiCarlo et al., Nucleic Acids Res. 41:4336-4343; Ma et al., Mol. Ther. Nucleic Acids 3:e161). This strategy has been successfully applied to cells of several different species, including corn and soybean (U.S. Patent Application Publication No. 20150082478, 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] Various methods and compositions can be used to obtain cells or organisms with a polynucleotide of interest inserted into a target site for a Cas endonuclease. Such methods can use homologous recombination (HR) to integrate the polynucleotide of interest into the target site. In one method described herein, the polynucleotide of interest is introduced into the cells of an organism via a donor DNA construct.

[0435] The donor DNA construct further comprises first and second regions of homology flanking the polynucleotide of interest, the first and second regions of homology of the donor DNA having homology to first and second genomic regions, respectively, present in or flanking the target site in the genome of the cell or organism.

[0436] The donor DNA can be linked to a guide polynucleotide. Tethered donor DNA may allow for co-localization of target and donor DNA, which is useful for genome editing, gene insertion, and target genome regulation, and may be useful for targeting postmitotic cells, which are thought to have greatly reduced function of the endogenous HR machinery (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, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-600 bp, 550-750 bp, 600-800 bp, 700-800 bp, 800-900 bp, 900-1000 bp, 1000-1200 bp, 1500-1600 bp, 1700-1800 bp The term "target region" includes the entire length and / or entire region having an integer value within the range of 0 bp, 500-1,000 bp, 600-1,250 bp, 700-1,500 bp, 800-1,750 bp, 900-2,000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to the entire length of the target site. These ranges include all integers within this range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bp. The amount of homology can also be described in terms of percent sequence identity over the fully aligned length of two polynucleotides, including percent sequence identities of at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98% to 99%, 99%, 99% to 100% or 100%. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity; for example, sufficient homology can be described as a 75-150 bp region having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be demonstrated by the predictive ability of two polynucleotides to specifically hybridize under high stringency conditions; see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds. (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[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 a "homologous region" of the donor DNA and a "genomic region" of the organism's genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination.

[0439] The homologous region on the donor DNA may have homology to any sequence adjacent to the target site. In some instances, the homologous region shares substantial sequence homology with the genomic sequence immediately adjacent to the target site, but it is recognized that the homologous region can be designed to have sufficient homology to a region that may be further 5' or 3' to the target site. The homologous region can also have homology to a fragment of the target site in addition to downstream genomic regions.

[0440] In one embodiment, the first homologous region further comprises a first fragment of the target site and the second homologous region comprises a second fragment of the target site, wherein the first fragment and the second fragment are different.

[0441] Polynucleotide of interest Polynucleotides of interest are described in detail herein, including polynucleotides that reflect commercial markets and commercial market interests related to crop development. Crops of interest and markets will change, and as developing countries expand global markets, new crops and technologies will emerge. Furthermore, as our understanding of agronomic traits and characteristics such as yield and heterosis increases, gene selection for genetic modification will change accordingly.

[0442] General categories of polynucleotides of interest include, for example, genes involved in signaling such as zinc fingers, genes involved in signal transduction such as kinases, and genes involved in housekeeping such as heat shock proteins. More specific polynucleotides of interest include, but are not limited to, genes involved in traits of agronomic interest such as those affecting crop yield, grain quality, grain nutrient content, starch and carbohydrate quality and quantity and kernel size, sucrose loading, protein quality and quantity, nitrogen fixation and / or utilization, fatty acid and oil composition, genes encoding proteins that confer resistance to abiotic stresses (such as those that confer resistance to drought, nitrogen, temperature, salinity, toxic metals or trace elements, or toxins such as pesticides and herbicides), genes encoding proteins that confer resistance to biotic stresses (such as attack by fungi, viruses, bacteria, insects and nematodes and the development of diseases associated with these organisms), and the like.

[0443] Agronomically important traits such as oil, starch, and protein content can be genetically modified in addition to using traditional breeding methods. Modifications include increasing oleic acid, saturated and unsaturated oil content, increasing lysine and sulfur concentrations, providing essential amino acids, and modifying starch. Modifications of the phoridothionin protein are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0444] The polynucleotide sequence of interest may encode a protein involved in conferring disease resistance or pest resistance. "Disease resistance" or "pest resistance" refers to the avoidance of harmful symptoms in a plant resulting from the interaction of the plant with a pathogen. Pest resistance genes may encode resistance to pests that inhibit high yields, such as cutworms, armyworms, or the European corn borer. Disease and insect resistance genes, such as lysozyme or cecropins for antibacterial protection, or proteins such as defensins, glucanases, or chitinases for antifungal protection, or Bacillus thuringiensis endotoxin for controlling nematodes or insects, protease inhibitors, collagenases, lectins, or glucosidases, are all examples of useful gene products. Genes encoding disease resistance traits include antidote genes such as those for fumonisins (U.S. Pat. No. 5,792,931), avirulence (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 cutworms, armyworms, and European corn borers. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109).

[0445] Proteins obtained by expression of "herbicide tolerance proteins" or "nucleic acid molecules encoding herbicide tolerance" include proteins that confer the ability of cells to tolerate higher concentrations of herbicide than cells that do not express the protein, or to tolerate a particular concentration of herbicide for a longer period of time than cells that do not express the protein. Herbicide tolerance traits can be introduced into plants by genes encoding tolerance to herbicides that act by inhibiting the activity of acetolactate synthase (ALS, also known as acetohydroxyacid synthase, AHAS), particularly sulfonylurea (UK: sulfonylurea)-type herbicides, genes encoding tolerance to herbicides that act by inhibiting the activity of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), genes encoding tolerance to glyphosate (e.g., the EPSP synthase gene and the GAT gene), genes encoding tolerance to HPPD inhibitors (e.g., the HPPD gene), or other such genes known in the art. See, e.g., U.S. Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and 9,187,762. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene mutant encodes resistance to the herbicide chlorsulfuron.

[0446] Furthermore, the polynucleotide of interest may also contain an antisense sequence complementary to at least a portion of the messenger RNA (mRNA) for the target gene sequence of interest. The antisense nucleotide is constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequence can be made as long as the sequence hybridizes with the corresponding mRNA and disrupts the expression of the corresponding mRNA. In this method, antisense constructs having 70%, 80%, or 85% sequence identity to the corresponding antisense sequence can be used. Furthermore, a portion of the antisense nucleotide can be used to disrupt the expression of the target gene. Generally, a sequence of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or more can be used.

[0447] Additionally, polynucleotides of interest can be used in a sense orientation to silence the expression of endogenous genes in plants. Methods for silencing gene expression in plants with polynucleotides in a sense orientation are known in the art. These methods generally involve transforming a plant with a DNA construct comprising a promoter that drives expression in the plant, operably linked to at least a portion of a nucleotide sequence corresponding to the transcription of the endogenous gene. Typically, such nucleotide sequences have substantial sequence identity to the transcribed sequence of the endogenous gene, generally greater than about 65% sequence identity, greater than about 85% sequence identity, or greater than about 95% sequence identity. See U.S. Pat. Nos. 5,283,184 and 5,034,323.

[0448] The polynucleotide of interest can also be a phenotypic marker. Phenotypic markers are screening or selection markers, including visual markers and selection markers, regardless of whether they are positive or negative selection markers. Any phenotypic marker can be used. In particular, selection or screening markers often contain a DNA segment that allows for the identification of a molecule or cells containing this molecule, or the favorable or unfavorable selection of such molecules or cells, under specific conditions. These markers can encode an activity, such as, but not limited to, the production of RNA, peptides, or proteins, or can provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, etc.

[0449] Examples of selectable markers include, but are not limited to, DNA segments containing restriction enzyme sites; DNA segments encoding products that provide resistance to otherwise toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT); DNA segments encoding products that are otherwise deficient in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that are easily identifiable (e.g., phenotypic markers such as β-galactosidase, GUS, fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), and red (RFP) fluorescent protein, and cell surface proteins); generation of novel primer sites for PCR (e.g., juxtaposition of two DNA sequences not previously juxtaposed); inclusion of DNA sequences that are unaffected or have been acted upon by restriction endonucleases or other DNA-modifying enzymes, chemicals, etc., and 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). See, for example, acetolactate synthase (ALS) for resistance to sulfonylureas, imidazolinones, triazolopyrimidine sulfonamides, pyrimidinyl salicylates, and sulfonylaminocarbonyltriazolinones (Shaner and Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69-110); 5-enolpyruvylshikimate-3-phosphate (EPSPS) for glyphosate resistance (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 can be stacked together into composite trait loci, as described in U.S. Patent Application Publication No. 20130263324, published October 3, 2013, and WO 2013 / 112686, published August 1, 2013.

[0452] A polypeptide of interest includes any protein or polypeptide encoded by a polynucleotide of interest described herein.

[0453] Additionally, methods are provided for identifying at least one plant cell containing in its genome a polynucleotide of interest integrated at a target site. Various methods can be used to identify plant cells with insertion at or near the target site in the genome. Such methods can be considered direct analysis of the target sequence to detect alterations in the target sequence, including, but not limited to, PCR, sequencing, nuclease digestion, Southern blotting, and any combination thereof. See, for example, U.S. Patent Application Publication No. 20090133152, published May 21, 2009. The method also includes recovering a plant from the plant cell containing the polynucleotide of interest integrated into its genome. The plant can be sterile or fertile. Any polynucleotide of interest can be provided and confirmed to be capable of being integrated into the target site in the plant's genome and expressed in the plant.

[0454] Optimization of sequences for expression in plants Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent 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, the removal of one or more sequences encoding spurious polyadenylation signals, the removal of one or more exon-intron splice site signals, the removal of one or more transposon-like repeats, and the removal of other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence can be adjusted to an average level for a given plant host (calculated with reference to known genes expressed in that host plant cell). When possible, the sequence is modified to avoid predicted hairpin secondary structures in one or more mRNAs. Thus, the "plant-optimized nucleotide sequence" of the present disclosure includes one or more such sequence modifications.

[0455] Expression elements 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, and 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 the polynucleotide sequence has been modified from its natural state to function with more desirable characteristics in a particular host cell (e.g., but not limited to, a bacterial promoter can be "corn-optimized" to improve its expression in corn plants). Alternatively, an expression element can be "synthetic," meaning that the expression element is designed in silico and synthesized for use in a host cell. Synthetic expression elements can be entirely synthetic or partially synthetic (comprising 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 RNA synthesis at high levels only in particular cell or tissue types; when a promoter induces RNA synthesis preferentially in certain tissues and at lower levels in other tissues, it is often referred to as a "tissue-specific promoter" or "tissue-preferred promoter."

[0457] Plant promoters include promoters that can initiate 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); and the ALS promoter (U.S. Pat. No. 5,659,026).

[0459] Tissue-preferred promoters can be used to target enhanced expression in specific plant tissues. Examples of tissue-preferred promoters include those described in International Publication No. 2013103367 published on 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 Probabilistic Cell Differentiation 20:181-96; and Guevara-Garcia et al., (1993) Plant J 4:495-505. Leaf-preferential promoters include, for example, Yamamoto et al., (1997) Plant J 12:255-65; Kwon et al., (1994) Plant Physiol 105:357-67; Yamamoto et al., (1994) Plant Cell Physiol 35:773-8; Gotor et al., (1993) Plant J 3:509-18;Orozco et al.,(1993)Plant Mol Biol 23:1129-38;Matsuoka et al.,(1993)Proc.Natl.Acad.Sci.USA90:9586-90;Simpson et al.,(1958)EMBO J 4:2723-9;Timko et al. 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 synthetase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific regulatory element in the snap 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 andersenii) andersonii and Trema tomentosa; Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A. rhizogenes rolC and rolD root-inducible genes); Teeri et al., (1989) EMBO J 8:343-50 (Agrobacterium wound-inducible TR1' and TR2' genes); VfENOD-GRP3 gene promoter (Kuster et al., (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana et al., (1994) Plant Mol Biol 25:681-91); phaseolin gene (Murai et al., (1983) Science 23:476-82; Sengopta-Gopalen et al. al., (1988) Proc. Natl. Acad. Sci. USA 82:3320-4. See also U.S. Patent Nos. 5,837,876, 5,750,386, 5,633,363, 5,459,252, 5,401,836, 5,110,732, and 5,023,179.

[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, but are not limited to, Cim1 (cytokinin-inducible message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase); as well as those disclosed, for example, in International Publication No. 2000011177 published March 2, 2000, and U.S. Patent No. 6,225,529. Seed-preferred promoters for dicotyledonous plants include, but are not limited to, bean-β-phaseolin, napin, β-conglycinin, soybean lectin, and cruciferin. Seed-preferred promoters for monocotyledons include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1. See also WO 2000012733, published March 9, 2000, which 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 regulators. The promoters can be chemically inducible promoters, in which application of chemicals induces gene expression, or chemically repressible promoters, in which application of chemicals represses gene expression. Chemically inducible promoters include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO 1993001294, published January 21, 1993), which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Other chemically regulated promoters include steroid-responsive promoters (e.g., glucocorticoid-inducible promoters (see 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 5,789,156).

[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] An example of a stress-inducible promoter is 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 evaluating the stress tolerance of plants exposed to simulated or naturally occurring stress conditions.

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

[0465] New promoters of various types useful in plant cells are constantly being discovered, and many examples can be found in Okamuro and Goldberg (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 induced Cas endonuclease can recognize and bind to DNA target sequences and introduce single-strand (nicks) or double-strand breaks. When single- or double-strand breaks are introduced into DNA, the cell's DNA repair machinery is activated to repair the break. Error-prone DNA repair mechanisms can create mutations at the double-strand break site. 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, but deletions, insertions, or other rearrangements (such as chromosomal translocations) are also 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 agents 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). Using DNA cleaving agents, a 2- to 9-fold increase in homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al., (1995) Plant Mol Biol 28:281-92). Experiments using 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 for repairing double-stranded and single-stranded DNA breaks in cells. Homologous recombination repair includes homologous recombination (HR) and single-stranded annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form of HDR is called homologous recombination (HR), which requires the longest sequence homology between the donor DNA and the acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and break-induced replication, which require shorter sequence homology than HR. Homologous recombination repair at nicks (single-stranded breaks) may occur via a different mechanism than HDR for double-stranded breaks (Davis and Maizels. PNAS (0027-8424), 111(10), p. E924-E932).

[0469] For example, modification of the genomes of prokaryotic and eukaryotic living cells or organisms by homologous recombination (HR) is a powerful tool in genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al., (1992) Mol Gen Genet 231:186-93) and insects (Dray and Gloor, 1997, Genetics 147:689-99). Homologous recombination has also been achieved in other organisms. For example, at least 150-200 bp of homology was required for homologous recombination in the protozoan parasite Leishmania (Papadopoulou and Dumas, (1997) Nucleic Acids Res 25:4278-86). In the filamentous fungus Aspergillus nidulans, gene replacement has been achieved using as little as 50 bp of contiguous 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 (ES) lines that can be grown in culture, transformed, selected, and introduced into mouse embryos (Watson et al., 1992, Recombinant DNA, 2nd Ed., Scientific American Books distributed by W.H. Freeman & Co.).

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

[0471] Generally, DNA targeting can be achieved by cleaving one or both strands at a specific polynucleotide sequence in a cell using a Cas protein in association with a suitable polynucleotide component. Upon induction of a single- or double-strand break in DNA, the cell's DNA repair machinery can be activated to repair the break by non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes, leading to modification of the target site.

[0472] The length of the DNA sequence of the target site can vary, including, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. The target site may be palindromic, i.e., a sequence on one strand can be read identically in the opposite direction on the complementary strand. The nick / cleavage site can be within the target sequence, or the nick / cleavage site can be outside the target sequence. In another variation, the cleavage can occur at nucleotide positions directly opposite each other, resulting in a blunt-end cleavage, or in other cases, the nicks can be offset, resulting in a single-stranded overhang (which may be a 5' overhang or a 3' overhang), also referred to as a "sticky end." Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a given target site, and the active variant retains biological activity and thus can be recognized and cleaved by a Cas endonuclease.

[0473] Assays for measuring single- or double-stranded cleavage of target sites by endonucleases are known in the art and generally measure the overall activity and specificity of the agent towards a DNA substrate containing the recognition site.

[0474] The targeting methods herein can be practiced, for example, such that two or more DNA target sites are targeted in the method. Such methods can optionally be characterized as multiplex methods. In certain embodiments, two, three, four, five, six, seven, eight, nine, ten, or more target sites can be simultaneously targeted. Multiplex methods are typically practiced by the targeting methods herein, in which multiple different RNA components are provided, each designed to guide the guide polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0475] Gene editing The process of editing a genome sequence by combining a DSB with a modified template generally involves introducing into a host cell a DSB inducer or a nucleic acid encoding the DSB inducer that can recognize a target sequence in a chromosomal sequence and introduce a DSB into the genome sequence, and at least one polynucleotide modified template containing at least one nucleotide modification compared to the nucleotide sequence to be edited. The polynucleotide modified template may further contain nucleotide sequences flanking the at least one nucleotide modification. The flanking sequences are substantially homologous to the chromosomal region adjacent to the DSB. Genome editing using a DSB inducer, such as a Cas-gRNA complex, is described, for example, in U.S. Patent Application Publication No. 20150082478, published March 19, 2015; International Publication No. 2015026886, published February 26, 2015; International Publication No. 2016007347, published January 14, 2016; and International Publication No. 2016 / 025131, published February 18, 2016.

[0476] Several uses of the guide RNA / Cas endonuclease system have been described (see, e.g., U.S. Patent Application Publication No. 20150082478A1, published March 19, 2015; WO 2015026886, published February 26, 2015; and U.S. Patent Application Publication No. 20150059010, published February 26, 2015), including, but not limited to, altering or substituting a nucleotide sequence of interest (such as a regulatory element), inserting a polynucleotide of interest, gene knockout, gene knockin, altering a splice site and / or introducing an alternative splice site, altering a nucleotide sequence encoding a protein of interest, fusion of amino acids and / or proteins, and gene silencing by expressing an inverted repeat in a gene of interest.

[0477] Proteins can be altered in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known. For example, amino acid sequence variants of proteins can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al., (1987) Meth Enzymol 154:367-82; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance on amino acid substitutions unlikely to affect the biological activity of a protein can be found, for example, in the model Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC). Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be preferred. Conservative deletions, insertions, and amino acid substitutions are not expected to cause radical changes in the properties of the protein, and the effects of substitutions, deletions, insertions, or combinations thereof can be evaluated in routine screening assays. 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 the target site.

[0478] Genome editing methods using Cas endonucleases and complexes comprising Cas endonucleases and guide polynucleotides are described herein. Following characterization of the guide RNA and PAM sequences, the endonucleases and associated 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), the genes comprising the complexes can be optimized as described in International Publication No. 2016186953, published November 24, 2016, and then delivered to cells as DNA expression cassettes using methods known in the art. The components necessary to comprise an active complex can be delivered 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-guided polynucleotide complex (WO 2017070032, 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 delivered as RNA, with or without modifications that protect the RNA from degradation, i.e., as capped or uncapped mRNA (Zhang et al. 2016 Nat. Commun. 7:12617), or as a Cas protein-guided polynucleotide complex (WO 2017070032, published April 27, 2017), or any combination thereof. To generate crRNA in vivo, tRNA-derived elements can be used to recruit endogenous RNAses to cleave the crRNA transcript into a mature form that can guide the complex to its DNA target site, as described, for example, in International Publication No. WO2017105991, published June 22, 2017. Nickase complexes can be used individually or in concert to generate single or multiple DNA nicks in one or both DNA strands.Furthermore, the cleavage activity of Cas endonucleases can be inactivated by modifying key catalytic residues in their cleavage domains (Sinkunas, T et al., 2013, EMBO J. 32:385-394), resulting in RNA-guided helicases that can be used to enhance homology-directed repair, induce transcriptional activation, or remodel DNA local structures. Furthermore, the activities of both the Cas cleavage and helicase domains can be knocked out and used in combination with other DNA cleavage, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancement, DNA integration, DNA inversion, and DNA repair agents.

[0479] The direction of transcription of the tracrRNA (if present) and other components of the CRISPR-Cas system (e.g., variable targeting domain, crRNA repeats, loops, anti-repeats) can be deduced as described in WO2016186946 published on November 24, 2016 and WO2016186953 published on November 24, 2016.

[0480] Once appropriate guide RNA requirements are established as described herein, the PAM preference for each new system disclosed herein can be investigated. If the cleavage complex results in degradation of the randomized PAM library, the complex can be converted into a nickase by disabling the ATPase-dependent helicase activity through 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). Two regions of PAM randomization separated by two protospacer targets can be used to generate double-stranded DNA breaks, which can be captured and sequenced to determine the PAM sequences that support cleavage by each complex.

[0481] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein into a cell and identifying at least one cell having a modification at the target, wherein the modification at the target site is selected from the group consisting of: (i) substitution of at least one nucleotide; (ii) deletion of at least one nucleotide; (iii) insertion of at least one nucleotide; chemical alteration of at least one nucleotide; and (v) any combination of (i) to (iv).

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

[0483] Knockouts can be generated by indels (insertion or deletion of nucleotide bases in a targeted 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-induced targeted mutations can occur within nucleotide sequences located within or outside the genomic target site recognized and cleaved by the Cas endonuclease.

[0485] The method for editing a nucleotide sequence in the genome of a cell can be a method that does not use an exogenous selectable marker by restoring function to a non-functional gene product.

[0486] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell.The method comprises: introducing at least one PGEN described herein and at least one donor DNA into a cell, wherein the donor DNA comprises a target polynucleotide; and optionally further comprising identifying at least one cell that has the target polynucleotide integrated at or near the target site.

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

[0488] Various methods and compositions can be used to generate cells or organisms having a polynucleotide of interest inserted into a target site through the activity of the CRISPR-Cas system components described herein. In one method described herein, the polynucleotide of interest is introduced into an organism cell via a donor DNA construct. As used herein, "donor DNA" refers to a DNA construct containing the polynucleotide of interest that is inserted into the target site of the Cas endonuclease. The donor DNA construct further comprises first and second homologous regions flanking the polynucleotide of interest. The first and second homologous regions of the donor DNA are homologous to first and second genomic regions, respectively, present in or flanking the target site in the genome of the cell or organism.

[0489] The donor DNA can be tethered to a guide polynucleotide. Tethered donor DNA may enable co-localization of target and donor DNA, which may be useful for genome editing, gene insertion, and target genome regulation, and may be useful for targeting postmitotic cells, which are thought to have significantly reduced function of the endogenous HR machinery (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, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-600 bp, 550-750 bp, 600-800 bp, 700-800 bp, 800-900 bp, 900-1000 bp, 1000-1200 bp, 1500-1600 bp, 1700-1800 bp The term "target region" includes the entire length and / or entire region having an integer value within the range of 0 bp, 500-1,000 bp, 600-1,250 bp, 700-1,500 bp, 800-1,750 bp, 900-2,000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or up to the entire length of the target site. These ranges include all integers within this range, for example, the range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bp. The amount of homology can also be described as percent sequence identity over the fully aligned length of two polynucleotides, including percent sequence identity of 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%. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity; for example, sufficient homology can be described as a 75-150 bp region having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be demonstrated by the predictive ability of two polynucleotides to specifically hybridize under high stringency conditions; see, e.g., Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds. (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0491] Episomal DNA molecules can also be ligated into double-strand breaks, for example, T-DNA can be integrated into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J 17:6086-95). When the sequence surrounding the double-strand break is modified, for example, by exonuclease activity involved in double-strand break maturation, the gene conversion pathway can regain its original structure if homologous sequences are available, such as in non-dividing somatic cells or sister chromatids after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA modification templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

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

[0493] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to enable editing (modification) of a genomic nucleotide sequence of interest (see also U.S. Patent Application Publication No. 20150082478, published March 19, 2015, and International Publication No. WO 2015026886, published February 26, 2015).

[0494] Polynucleotides and / or traits of interest can be stacked together at complex trait loci, as described in International Publication No. WO2012129373, published September 27, 2012, and International Publication No. WO2013112686, published August 1, 2013. The guide polynucleotide / Cas9 endonuclease system described herein provides a system for efficiently generating double-stranded breaks, allowing for the stacking of traits at complex trait loci.

[0495] The guide polynucleotide / Cas system described herein for mediating gene targeting can be used in methods for directing heterologous gene insertion and / or generating complex trait loci containing multiple heterologous genes, similar to those disclosed in International Publication No. WO2012129373, published September 27, 2012, where the guide polynucleotide / Cas system disclosed herein replaces the use of double-strand break-inducing agents to introduce genes of interest. Transgenes can be propagated as a single locus by inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or 5 centimorgans (cM) of each other (see, e.g., U.S. Patent Application Publication No. 20130263324, published October 3, 2013, or International Publication No. WO2012129373, published March 14, 2013). After plants containing the transgenes are selected, the plants containing (at least) one of the transgenes can be crossed to form an F1 containing both transgenes. Of the progeny from these F1 (F2 or BC1), 1 / 500 will have the two different transgenes recombined on the same chromosome. This composite locus can then be propagated as a single locus with both transgene traits. This process can be repeated to accumulate any number of desired traits.

[0496] Further uses of guide RNA / Cas endonuclease systems have been described (e.g., U.S. Patent Application Publication No. 20150082478 published March 19, 2015; International Publication No. WO 2015026886 published February 26, 2015; U.S. Patent Application Publication No. 20150059010 published February 26, 2015; International Publication No. WO 2016007347 published January 14, 2016; and PCT application No. WO 2016007347 published February 18, 2016). See Publication No. 2016025131), including, but not limited to, altering or substituting a nucleotide sequence of interest (such as a regulatory element), inserting a polynucleotide of interest, gene knockout, gene knockin, altering a splice site and / or introducing an alternative splice site, altering a nucleotide sequence encoding a protein of interest, fusion of amino acids and / or proteins, and gene silencing by expressing an inverted repeat in a gene of interest.

[0497] The characteristics obtained from the gene editing compositions and methods described herein can be evaluated. Chromosomal intervals correlated with the desired phenotype or trait can be identified. Various methods well known in the art can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers linked to genes that control the desired trait. In other words, chromosomal intervals are determined so that any marker present within the interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for a specific trait. In one embodiment, a chromosomal interval contains at least one QTL, and may actually contain more than one QTL. When multiple QTLs are very close to each other within the same interval, one marker is linked to more than one QTL, making the association of a specific marker with a specific QTL unclear. Conversely, for example, when two very close markers cosegregate with a desired phenotypic trait, it is often unclear whether these markers identify the same QTL or two different QTLs. 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, e.g., at least one breeding population. A QTL region encompasses or is closely linked to a gene that influences the trait in question. An "allele of a QTL" can include multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can refer to a haplotype within a specific window, where the window 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 the 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 a particular method for introducing a sequence into an organism or cell, but simply introduce a polynucleotide or polypeptide into at least one cell of the organism. Introduction includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient (direct) provision of a nucleic acid, protein, or polynucleotide-protein complex (PGEN, RGEN) to a cell.

[0499] Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are not limited to, microinjection, electroporation, stable transformation, transient transformation, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated transfer, transfection, transduction, cell-penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical application, sexual crossing, sexual propagation, and any combination thereof.

[0500] For example, guide polynucleotides (guide RNA, cr nucleotides + tracr nucleotides, guide DNA and / or guide RNA-DNA molecules) can be directly (transiently) introduced into cells as single-stranded or double-stranded polynucleotide molecules. Guide RNA (or crRNA + tracrRNA) can also be indirectly introduced into cells by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding guide RNA (or crRNA + tracrRNA) operably linked to a specific promoter capable of transcribing guide RNA (crRNA + tracrRNA molecule) in said cells. A particular promoter can be, but is not limited to, an RNA polymerase III promoter, which allows transcription of RNA with strictly defined, unmodified 5' and 3' ends (Ma et al., 2014, Mol. Ther. Nucleic Acids 3:e161; DiCarlo et al., 2013, Nucleic Acids Res. 41:4336-4343; WO 2015026887, published February 26, 2015). Any promoter capable of transcribing the guide RNA within 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, for example, in that plant cells contain a plant cell wall that can act as a barrier to the delivery of components.

[0502] Delivery of the Cas endonuclease, and / or guide RNA, and / or ribonucleoprotein complex, and / or polynucleotide encoding any one or more of the foregoing into plant cells can be achieved by methods known in the art, including, but not limited to, Rhizobiales-mediated transformation (e.g., Agrobacterium, Ochrobactrum), particle-mediated delivery (particle bombardment), polyethylene glycol (PEG)-mediated transfection (e.g., into protoplasts), electroporation, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery.

[0503] Cas endonucleases, such as those described herein, can be introduced into cells by directly introducing the Cas polypeptide itself (referred to as direct Cas endonuclease delivery), mRNA encoding the Cas protein, and / or the guide polynucleotide / Cas endonuclease complex itself using any method known in the art. Cas endonucleases can also be introduced into cells indirectly by introducing a recombinant DNA molecule encoding the Cas endonuclease. The endonuclease can be transiently introduced into cells or integrated into the genome of the host cell using any method known in the art. Cellular uptake of the endonuclease and / or guide polynucleotide can be facilitated using a cell-penetrating peptide (CPP), as described in International Publication No. WO2016073433, published May 12, 2016. Any promoter capable of expressing the Cas endonuclease in cells can be used, including heat-shock / heat-inducible promoters operably linked to a nucleotide sequence encoding the Cas endonuclease.

[0504] Direct delivery of polynucleotide-modified templates into plant cells can be achieved by particle-mediated delivery, and any other direct delivery method, including but not limited to polyethylene glycol (PEG)-mediated transfection into protoplasts, whisker-mediated transformation, electroporation, particle bombardment, cell-penetrating peptides or mesoporous silica nanoparticle (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. Donor DNA can be transiently present in the cell, or it can be introduced by a viral replicon. In the presence of the Cas endonuclease and the 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 be accompanied by direct delivery (co-delivery) of other mRNAs that can facilitate enrichment and / or visualization of cells that receive the guide polynucleotide / Cas endonuclease complex components. For example, direct co-delivery of the guide polynucleotide / Cas endonuclease components (and / or the guide polynucleotide / Cas endonuclease complex itself) with mRNAs encoding phenotypic markers (such as, but not limited to, transcriptional activators such as CRC (Bruce et al. 2000 The Plant Cell 12:65-79)) can restore function to the non-functional gene product, allowing for selection and enrichment of cells without the use of an exogenous selection marker, as described in International Publication No. WO 2017070032, published April 27, 2017.

[0507] Introduction of the guide RNA / Cas endonuclease complex (representing the cleavage-ready complex described herein) into a cell includes introducing the individual components of the complex into the cell, separately or collectively, directly (direct delivery of the guide RNA and the Cas endonuclease protein and protein subunit, or functional fragments thereof) or by recombinant constructs expressing the components (guide RNA, Cas endonuclease, protein subunit, or functional fragments thereof). Introduction of the guide RNA / Cas endonuclease complex (RGEN) into a cell includes introducing the guide RNA / Cas endonuclease complex into the cell as a ribonucleotide-protein. The ribonucleotide-protein can be assembled before introduction into the cell, 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) can be assembled by any means known in the art in vitro or before being introduced into a cell (which will be targeted for genome modification as described herein).

[0508] Direct delivery of RGEN ribonucleoprotein allows genome editing at the target site of the cell genome, with the complex then rapidly degrading, making its presence in the cell transient. This transient presence of the RGEN complex can lead to reduced off-target effects. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences results in sustained expression of RGEN from these plasmids, which can increase off-target effects (Cradick, TJ et al. (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. 31:822-826).

[0509] Direct delivery can be achieved by combining any one of the components (e.g., at least one guide RNA, at least one Cas protein, and optionally one additional protein) of the guide RNA / Cas endonuclease complex (RGEN) (representing the cleavage-ready complex described herein) with a delivery matrix comprising microparticles (such as, but not limited to, gold particles, tungsten particles, and silicon carbide whisker particles) (see also International Publication No. 2017070032, published April 27, 2017). The delivery matrix can include any one of the components, such as the Cas endonuclease, attached to a solid matrix (e.g., a bombardment particle).

[0510] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein that form the guide RNA / Cas endonuclease complex are introduced into a cell as RNA and protein, respectively.

[0511] In one aspect, a guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and Cas endonuclease protein that form the guide RNA / Cas endonuclease complex and at least one protein subunit of the complex are introduced into a cell as RNA and protein, respectively.

[0512] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein that form the guide RNA and Cas endonuclease complex (cleavage-ready complex) and at least one protein subunit of the complex are pre-assembled in vitro and introduced into a cell as a ribonucleotide-protein complex.

[0513] Protocols for introducing polynucleotides, polypeptides, or polynucleotide-protein complexes (PGENs, RGENs) into eukaryotic cells, such as plants or plant cells, are known, and include microinjection (Crossway et al., (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543), meristem transformation (U.S. Pat. No. 5,736,369), electroporation (Riggs et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-6, Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), whisker-mediated transformation (Ainley et al. 2013, Plant Biotechnology Journal 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); Singh 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. Acad. Sci. USA 85:4305-9 (corn); Klein et al.,(1988) Biotechnology 6:559-63 (maize); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol 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. 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.Li 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 (maize via Agrobacterium tumefaciens).

[0514] Alternatively, polynucleotides can be introduced into plants or plant cells by contacting the cells or organisms with a virus or viral nucleic acid. Generally, such methods involve incorporating the polynucleotide into a viral DNA or RNA molecule. In some embodiments, a polypeptide of interest may be first synthesized as part of a viral polyprotein, which may 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, e.g., U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931.

[0515] Polynucleotides or recombinant DNA constructs can be provided to or introduced into prokaryotic and eukaryotic cells or organisms using a variety of transient transformation methods, including, but not limited to, direct introduction of polynucleotide constructs into plants.

[0516] Nucleic acids and proteins can be provided to cells in any manner, including using molecules that facilitate uptake of any or all components (proteins and / or nucleic acids) of the inducible Cas system, such as cell-penetrating peptides and nanocarriers. See also U.S. Patent Application Publication No. 20110035836, published February 10, 2011, and European Patent Application Publication No. 2821486A1, published January 7, 2015.

[0517] Other methods for introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts can be used, such as plastid transformation and methods for 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 genome of the organism and can be inherited by its progeny. Transient transformation is intended to mean that a polynucleotide is introduced into an organism but is not integrated into the genome of this organism, or that a polypeptide is introduced into an organism. Transient transformation means that the introduced composition is only expressed or present temporarily in the organism.

[0519] A variety of methods are available for identifying those cells with modified genomes at or near the target site without using a screening marker phenotype. Such methods can be considered as 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 thereof.

[0520] Cells and plants The polynucleotides and polypeptides of the present disclosure can be introduced into cells, including but not limited to, human, non-human, animal, mammalian, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein. Any plant, including monocotyledonous and dicotyledonous plants and plant components, 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 millet (e.g., pearl millet, Pennisetum glaucum), common millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), wheat (Triticum species, e.g., Triticum aestivum), and the like. aestivum, Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palms, ornamentals, 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 napus, B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum), and the like. arboreum, Gossypium barbadense), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), and potato (Solanum tuberosum).

[0523] Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea plants (Camellia sinensis), bananas (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), and others. guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), vegetables, ornamental plants and conifers.

[0524] Vegetables that can be used include tomato (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis, such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and muskmelon (C. melo). Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanens...

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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