CAS endonuclease and guide RNA variants with improved efficiency

Abstract

Compositions and methods for genome modification of a target sequence in the genome of a cell using novel engineered Cas polypeptides are provided. The engineered Cas polypeptides and related methods, systems, and compositions disclosed herein can be used in guidance of polynucleotide / endonuclease systems to modify or alter target sequences within the genome of a cell or organism. Also provided are novel effector and endonucleases systems, as well as elements comprising such systems, such systems, for example, guide polynucleotide / endonucleases systems comprising endonucleases. Also provided are compositions and methods for directing polynucleotide / endonucleases systems comprising at least one endonucleases optionally covalently or non-covalently linked to or assembled with at least one further protein subunit; and compositions and methods for direct delivery of endonucleases that are ribonucleotide proteins are provided.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional No. 63 / 352,100, filed on June 14, 2022; U.S. Provisional No. 63 / 378,897, filed on October 10, 2022; U.S. Provisional No. 63 / 481,458, filed on January 25, 2023; and U.S. Provisional No. 63 / 497,314, filed on April 20, 2023, the disclosures of each of which are incorporated herein by reference in their entirety.

[0003] References to electronically submitted sequence listings

[0004] An official copy of the sequence listing is submitted electronically here as an xml file named 9213-WO-PCT_ST26v2, created on June 13, 2023, and having a size of 422,257 bytes. The sequence listing contained in this xml file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

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

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

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

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

[0009] Described herein are novel engineered Cas polypeptides and endonucleases and methods and compositions for their use. Summary of the invention

[0010] Disclosed herein are compositions of novel engineered Cas polypeptides and methods of using the same. These Cas polypeptides are capable of targeting double-stranded DNA in a PAM-dependent manner under the guidance of a guide polynucleotide. In some embodiments, the engineered Cas polypeptide is an active endonuclease capable of introducing a break at a target site of a target double-stranded DNA. In some embodiments, the Cas polypeptide comprises one or more mutations that render it incapable of double-stranded cleavage but allow single-stranded cleavage. In some embodiments, the Cas polypeptide comprises one or more mutations that render it incapable of cleaving one or both strands of a double-stranded polynucleotide, but retains the ability to bind to a target polynucleotide sequence.

[0011] On the one hand, novel engineered Cas polypeptides are provided, comprising at least one zinc finger-like domain, at least one bridge-helix-like domain, a trisection RuvC domain (comprising discontinuous RuvC-I domain, RuvC-II domain and RuvC-III domain), optionally comprising heterologous polynucleotides. Also provided are compositions comprising the synthesis of novel engineered Cas polypeptides or endonucleases. In many aspects of the disclosed compositions or methods, at least one component optimized for expression in eukaryotic cells, particularly plant cells, fungal cells or animal cells is provided. In a particular example, the Cas polypeptide is less than about 500 amino acids.

[0012] In all aspects of the novel Cas polypeptides disclosed herein, the polypeptide has been engineered to include an amino acid sequence that is different from the native effector protein obtained or derived from Syntrophomonas palmitatica.

[0013] In a first aspect, an engineered Cas polypeptide or a polynucleotide encoding an engineered Cas polypeptide is provided, wherein when aligned with SEQ ID NO: 14 and numbered relative to the amino acid position of SEQ ID NO: 14, the engineered Cas polypeptide comprises one or more of the following amino acids: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamine at position 85. In some examples of this first aspect, when aligned with SEQ ID NO: 14 and numbered relative to the amino acid position of SEQ ID NO: 14, the engineered Cas polypeptide further comprises one or more of the following amino acids: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamine at position 85. At least one, at least two, at least three, at least four, at least five, at least six or seven of the following amino acid position numbers of NO: 14: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at 226, glycine at 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at position 376, cysteine ​​at position 379, cysteine ​​at position 395, cysteine ​​at position 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467 or proline at position 468.

[0014] In a second aspect, an engineered Cas polypeptide or a polynucleotide encoding the engineered Cas polypeptide is provided, wherein the engineered Cas polypeptide comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to SEQ ID NO: 14, and, when aligned with SEQ ID NO: 14 and numbered relative to the amino acid positions of SEQ ID NO: 14, comprises one or more of the following amino acids: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamine at position 85, and wherein the engineered Cas polypeptide is capable of site-specifically binding to a target site of a polynucleotide. In some examples of this second aspect, the engineered Cas polypeptide comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to one of SEQ ID NOs: 15-56, SEQ ID NO: 59, SEQ ID NO: 60, or SEQ ID NOs: 309-323, or SEQ ID NO: 362.

[0015] Further examples of each of the foregoing first aspect or second aspect are provided, wherein an engineered Cas polypeptide or a polynucleotide encoding the engineered Cas polypeptide is provided such that when aligned with SEQ ID NO: 14 and relative to SEQ When numbering the amino acid positions according to ID NO: 14, the engineered Cas polypeptide further comprises at least one, at least two, at least three, at least four, at least five, at least six or seven of the following amino acids: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at 226, glycine at 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at 376, cysteine ​​at 379, cysteine ​​at 395, cysteine ​​at 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

[0016] In yet further examples of each of the aforementioned first aspect or second aspect, when aligned with SEQ ID NO: 14 and relative to SEQ ID When the amino acid positions of NO: 14 are numbered, the engineered Cas polypeptide does not include at least one of the following: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at position 226, glycine at position 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at position 376, cysteine ​​at position 379, cysteine ​​at 395, cysteine ​​at position 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

[0017] In a specific embodiment of the aforementioned example of the first aspect or the second aspect, the engineered Cas polypeptide comprises less than 500 amino acids in length. In a specific embodiment of the aforementioned first aspect or the second aspect, the engineered Cas polypeptide is a Cas endonuclease.

[0018] Also provided are embodiments of each of the aforementioned first aspect or second aspect, wherein the engineered Cas polypeptide comprises at least one zinc finger-like domain, at least one bridge-helix-like domain, a trisection RuvC domain (comprising a discontinuous RuvC-I domain, a RuvC-II domain, and a RuvC-III domain), and optionally comprises a heterologous polynucleotide.

[0019] In a third aspect, a synthetic composition is provided comprising a CRISPR-Cas polypeptide, or a polynucleotide encoding the CRISPR-Cas polypeptide, wherein the CRISPR-Cas polypeptide comprises at least 50%, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, or more than 400 contiguous amino acids having at least 50%, between 50% and 55%, at least 55%, between 55% and 60%, at least 60%, between 60% and 65%, at least 65%, between 65% and 70%, at least 70%, 70% or more than 70% identical to a sequence selected from the group consisting of SEQ ID NOs: 15-47, SEQ ID NOs: 49-56, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NOs: 309-323, and SEQ ID NO: 362. %, at least 75%, between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, between 99% and 100%, or 100% sequence identity.

[0020] Any method or composition herein may further comprise a heterologous polynucleotide. The heterologous polynucleotide may be selected from the group consisting of: a non-coding regulatory expression element, such as a promoter, intron, enhancer or terminator; a donor polynucleotide; a polynucleotide modification template, which optionally comprises at least one nucleotide modification compared to a polynucleotide sequence in a cell; a transgene; a guide RNA; a guide DNA; a guide RNA-DNA hybrid; an endonuclease; a nuclear localization signal; and a cellular transit peptide.

[0021] On the other hand, a method for using any composition disclosed herein is provided. In some embodiments, a method is provided for combining a target sequence (e.g., in a cell genome or in vitro) of a polynucleotide comprising a disclosed Cas polypeptide or endonuclease (i.e., any Cas polypeptide or endonuclease in the first aspect or the second aspect or the third aspect embodiment). In some embodiments, the disclosed Cas polypeptide or endonuclease forms a complex with a guide polynucleotide (e.g., a guide RNA (gRNA), which can be a single guide RNA (sgRNA)). In some embodiments, the complex recognizes, binds to a polynucleotide at or near the target sequence and optionally produces a nick (one chain) or a break (two chains) in a polynucleotide at or near the target sequence. In some embodiments, the nick or break is repaired by non-homologous end joining (NHEJ). In some embodiments, the nick or break is repaired by homology directed repair (HDR) or by homologous recombination (HR) using a polynucleotide modified template or donor DNA molecule.

[0022] In any aspect, the engineered Cas polypeptide or endonuclease can be used to bind, cut and / or break a polynucleotide comprising a target sequence at a temperature below about 45 degrees Celsius, such as about 40 degrees Celsius or less, about 37 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 28 degrees Celsius or less, or about 25 degrees Celsius or less. Therefore, a method is also provided, which includes contacting a polynucleotide with any engineered Cas polypeptide or endonuclease disclosed herein (e.g., any Cas polypeptide described in the first, second or third aspects disclosed herein) at a temperature below about 45 degrees Celsius, such as about 40 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 28 degrees Celsius or less, or about 25 degrees Celsius or less, and generating a break in the polynucleotide. This break can be used to generate a target site (such as base editing, deletion or insertion) for targeted modification or alteration in a polynucleotide. Thus, in some examples, the engineered Cas polypeptide or endonuclease of the first, second or third aspects disclosed herein provides greater activity (binding, targeting efficiency and / or endonuclease activity) than the wild-type Cas-α10 endonuclease SEQ ID NO: 48 or its basic variant SEQ ID NO: 14 at a temperature below about 45 degrees Celsius, for example, about 40 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 28 degrees Celsius or less or about 25 degrees Celsius or less.

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

[0024] On the other hand, the engineered Cas polypeptides described herein include one or more mutations that provide nuclease inactivation or dead Cas polypeptides. For example, the engineered Cas polypeptides disclosed herein can be changed so that when aligned with SEQ ID NO: 14 or 48 and numbered relative to the amino acid positions of SEQ ID NO: 14 or 48, the engineered Cas polypeptides disclosed herein include alanine at position 228; alanine at relative position 327 or alanine at position 434. The disclosed inactivated engineered Cas polypeptides can be connected to effectors or effector proteins, which can be molecules that recognize, bind and / or cut or incise polynucleotide targets. The disclosed inactivated engineered Cas polypeptides can be connected to base editing molecules (e.g., deaminases) for targeted base editing. The disclosed inactivated engineered Cas polypeptides, optionally linked to an effector or effector protein, can be used for targeted delivery of effector molecules at temperatures of about 45 degrees Celsius or less, about 40 degrees Celsius or less, about 37 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 25 degrees Celsius or less.

[0025] In yet another aspect, a method of altering a target Cas polypeptide is provided; the method comprises modifying the target Cas polypeptide to include one or more amino acid changes, such that when aligned with SEQ ID NO: 14 or 48 and numbered relative to the amino acid positions of SEQ ID NO: 14 or 48, the target Cas polypeptide comprises one or more of the following amino acid changes: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, and glutamine at position 85, wherein the engineered Cas polypeptide is capable of site-specifically binding to a target site of a polynucleotide, and thereby producing an altered Cas polypeptide. Optionally, in some examples of this aspect, the target Cas polypeptide is further modified such that when aligned with SEQ ID NO: 14 or 48 and numbered relative to SEQ ID NO: 14 or 48, the target Cas polypeptide comprises one or more of the following amino acid changes: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, and glutamine at position 85. When the amino acid position is numbered as NO: 14 or 48, the target Cas polypeptide comprises one of the following amino acid changes: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at 226, glycine at 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at 376, cysteine ​​at 379, cysteine ​​at 395, cysteine ​​at 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

[0026] For example, in certain examples of the foregoing aspects, the method includes creating an alignment of a target Cas polypeptide (e.g., a Cas polypeptide of less than 500 amino acids in length) with SEQ ID NO: 14 or 48, identifying one or more target Cas polypeptide amino acid residues corresponding to one or more of amino acid positions 34, 36, 45, 54, 77, and 85 of SEQ ID NO: 14 or 48, and modifying the one or more target Cas polypeptide amino acid residues to one or more of the following: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, and glutamine at position 85, respectively. Optionally, the method further includes identifying a target Cas polypeptide corresponding to SEQ ID NO: 14 or 48. NO: 14 or 48 of amino acid positions 38, 79, 120, 149, 226, 230, 293, 298, 306, 329, 313, 325, 327, 338, 376, 379, 395, 398, 406, 409, 421, 430, 467 and 468 of one or more target Cas polypeptide amino acid residues, and modifying the one or more target Cas polypeptide amino acid residues to one or more of the following: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, aspartic acid at position 226, respectively. glycine at position 230, glycine at position 293, histidine at position 298, phenylalanine at position 306, glutamic acid at position 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at position 376, cysteine ​​at 379, cysteine ​​at 395, cysteine ​​at 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468. In these methods, the target Cas polypeptide can (i) have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 14, (ii) less than 500 amino acids in length, or (iii) both of (i) and (ii). In a particular example, the modified target Cas polypeptide provides greater activity (binding, targeting efficiency, and / or endonuclease activity) than an unmodified target Cas polypeptide at a temperature below about 45 degrees Celsius, e.g., about 40 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 28 degrees Celsius or less, or about 25 degrees Celsius or less.

[0027] Such methods of altering a target Cas polypeptide may include, for example, modifying a nucleic acid sequence encoding the target Cas polypeptide to include one or more altered codons that, when aligned with SEQ ID NO: 14 or 48 and numbered relative to the amino acid positions of SEQ ID NO: 14 or 48, encode one or more of the following amino acid changes: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, and glutamine at position 85, thereby generating an altered coding sequence that encodes an altered Cas polypeptide. Optionally, the method includes altering a nucleic acid sequence encoding a target Cas polypeptide to further include one or more additional ... SEQ ID When numbering the amino acid positions of NO:14 or 48, the one or more additional altered codons encode one or more of the following amino acid changes: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at 226, glycine at 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at 376, cysteine ​​at 379, cysteine ​​at 395, cysteine ​​at 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

[0028] Also provided herein are systems comprising a Cas polypeptide and an engineered guide RNA (egRNA), wherein the egRNA is based on a template guide RNA sequence (sgRNA) comprising a Cas polypeptide recognition domain, and wherein the egRNA comprises (i) a deletion, substitution, or insertion at one or more of nucleotide positions 1-3, 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 147-149, 149-151, 151-153, 153-155, 157-159, 159-161, 163-165, or 167-169 of the sgRNA Cas polypeptide recognition domain, or any combination thereof; (ii) a sequence of one of SEQ ID NOs: 62-156, SEQ ID NOs: 192-207, SEQ ID NOs: 260-284, SEQ ID NOs: 324-327, or SEQ ID NOs: 346-355; or (iii) a deletion, substitution, or insertion at one of nucleotide positions 1-3, 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 147-149, 149-151, 151-153, 153-155, 157-159, 159-161, 163-165, or 167-169 of the sgRNA Cas polypeptide recognition domain, or any combination thereof; An aptamer inserted at position 1, 3, 4, 5, 6, 7, 9, 10 or 11 of the Cas polypeptide recognition domain. Thus, the disclosed egRNA may comprise a deletion, substitution or insertion at: (i) positions 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 149-151, 151-153, 153-155, 157-159, 163-165, or 167-169 or any combination thereof; (ii) positions 1-9, or 146-153, or (iii) positions 1-9, 6-63, 61-63, 146-153, 157-159, 158-170, 167-169 or any combination thereof. Such engineered guides (egRNA) and their functionality (effect on target DNA cleavage efficiency) are shown in Tables 6, 7, 8, 9, 10, 11 and 12 herein.

[0029] In some examples of any of the foregoing systems comprising a Cas polypeptide and an engineered guide RNA (egRNA), the Cas polypeptide is one of the engineered Cas polypeptides disclosed herein, e.g., the engineered Cas polypeptide (i) when aligned with SEQ ID NO: 14, numbered relative to the amino acid positions of SEQ ID NO: 14, comprises one or more of the following amino acid changes: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamine at position 85 (ii) comprises a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to SEQ ID NO: 14 and, when aligned with SEQ ID NO: 14, numbered relative to the amino acid positions of SEQ ID NO: 14. NO: 14, when the amino acid position is numbered, comprising one or more of the following amino acids: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamine at position 85, and wherein the engineered Cas polypeptide is capable of site-specific binding to a target site of a polynucleotide, (iii) comprising a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity to one of SEQ ID NO: 15-56, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 309-323 or SEQ ID NO: 362. Specific examples of systems comprising engineered Cas polypeptides and engineered guide RNAs (egRNAs) are disclosed in each of Tables 6, 7, 8, 9, 10, 11 and 12 herein.

[0030] DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0031] The present disclosure can be more fully understood from the following detailed description and accompanying drawings and sequence listing, which form a part of this application.

[0032] Figure 1Cas endonuclease variant yeast expression vectors are depicted, wherein: 1 = ROX3 promoter (SEQ ID NO: 1); 2 = yeast optimized Cas endonuclease gene (SEQ ID NO: 2); 3 = sequence encoding SV40 NLS (SEQ ID NO: 3); 4 = CYC1 terminator (SEQ ID NO: 4); 5 = SNR52 promoter (SEQ ID NO: 5); 6 = sequence encoding Cas endonuclease sgRNA (Cas endonuclease recognition domain) (SEQ ID NO: 6); 7 = sequence encoding Cas-α10sgRNA ADE2 targeting domain (SEQ ID NO: 7); 8 = sequence encoding Cas-α10sgRNA CAN1 targeting domain (SEQ ID NO: 8); 9 = sequence encoding Cas-α10sgRNA targeting ADE2 (SEQ ID NO: 9); 10 = sequence encoding Cas-α10sgRNA targeting CAN1 (SEQ ID NO: 10).

[0033] Figure 2 Schematic diagram of target cleavage detection in S. cerevisiae, where target cleavage and cellular repair result in the formation of a non-functional ade2 gene, which results in adenine auxotrophy and a switch from a white to a red / pink cell phenotype. This color switch allows identification of phenotypic differences and can be used to select cells expressing Cas endonuclease variants and / or associated guide RNAs with improved targeted DSB activity. Colonies with functional ade2 genes appear white. Colonies with non-functional ade2 genes appear red. Colonies mottled with red and white represent multiple sectors containing non-functional ade2 genes. Colonies with a single sector containing a non-functional ade2 gene appear white with red sections.

[0034] Figure 3 Schematic diagram of target cleavage assay in Saccharomyces cerevisiae, where target cleavage of the CAN1 gene and cellular non-functional repair results in resistance to L-canavanine, allowing positive selection of Cas endonuclease variants with improved double-strand break activity.

[0035] Figure 4 is the polypeptide sequence of the Cas endonuclease (SEQ ID NO: 14), which includes several modifications that enable it to function at lower temperatures, and which is further altered in the examples described herein. The key catalytic residues are shown in bold underlined font. The zinc finger domain is indicated by a dotted underline.

[0036] Figure 5Is the polypeptide sequence of an engineered Cas polypeptide (SEQ ID NO: 60) with improved activity shown in the examples herein. Key catalytic residues are shown in bold underlined font. Zinc finger domains are underlined with dashed lines. Double underlined letters show residues that are further altered relative to the basic Cas endonuclease (SEQ ID NO: 14) used herein.

[0037] Figure 6 Two schematics of human optimized expression cassettes are shown: the top panel shows the cassette for Cas-α nuclease and the bottom panel shows the cassette for Cas-α sgRNA expression.

[0038] Figure 7 An engineered sgRNA containing an MS2 recognition sequence incorporated at the 5' end, at the tip of the stem-loop, and within the 5'-GAAA-3' linker connecting the tracrRNA and crRNA of the sgRNA is shown; Figure 7 Also shown are examples of positions 1-12 at which aptamer or Cas6 sequences are inserted as described in Table 9 herein.

[0039] Figure 8 Two schematics of optimized expression cassettes for Zea mays are shown: the top panel shows the cassette for Cas-α nuclease and the bottom panel shows the cassette for Cas-α sgRNA expression.

[0040] Fig. 9 The figure shows the difference in cleavage at different temperatures between the basic variant Cas endonuclease SEQ ID NO: 14 and the wild-type Cas endonuclease SEQ ID NO: 157.

[0041] Fig.10 The figure shows the difference in cleavage between the variant Cas endonucleases SEQ ID NO: 14 and SEQ ID NO: 48 at different temperatures.

[0042] Fig.11 The figure shows the difference in cleavage at different temperatures between the variant Cas endonucleases SEQ ID NO: 14 and SEQ ID NO: 362, which shows that SEQ ID NO: 362 has significantly improved cleavage activity at 25°C.

[0043] Table 1 provides the description of the sequences cited herein. The isolated polynucleotides, nucleic acids and nucleotide sequences disclosed herein additionally encompass all complementary forms (e.g., reverse complementary sequences) of each sequence disclosed for such constructs. The sequence description and sequence listing appended herein meet the rules for disclosure of nucleotide and amino acid sequences in the patent application specified in 37 CFR §§ 1.821-1.835.

[0044] Table 1

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] DETAILED DESCRIPTION

[0057] The optimum temperature of many natural Cas endonucleases is higher than the typical biological temperature of some organisms (including plants and yeast). Therefore, wild-type Cas endonucleases (e.g., SEQ ID NO: 48) require a heat shock of about 45 degrees Celsius to achieve optimal activity. For some applications, it may be beneficial to modify this property. Provided herein are methods and compositions for novel engineered CRISPR effectors, systems, and elements comprising such effectors, including but not limited to novel guide polynucleotide / endonuclease complexes, guide polynucleotides, guide RNA elements, Cas proteins and endonucleases, and proteins comprising endonuclease functions (domains). Also provided are compositions and methods for directly delivering endonucleases, cutting ready complexes, guide RNAs, and guide RNA / Cas endonuclease complexes. The present disclosure further includes compositions and methods for genome modification of target sequences in cell genomes, for gene editing, and for inserting target polynucleotides into cell genomes. The identified variants should improve the genome editing results of various cell types including humans, and contribute to the widespread adoption of Cas nucleases guided by such microRNAs.

[0058] Unless otherwise specified, the terms used in the claims and specification are defined as set forth below. It must be noted that, as used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0059] As used herein, "nucleic acid" means polynucleotides, and includes a single-stranded polymer or a double-stranded polymer of a deoxyribonucleotide base or a ribonucleotide base. Nucleic acids can also include fragments and modified nucleotides. Therefore, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence" and "nucleic acid fragment" are used interchangeably to represent single-stranded or double-stranded RNA and / or DNA and / or RNA-DNA polymers, optionally containing synthetic, non-natural or altered nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0060] The term "genome" when applied to a prokaryotic or eukaryotic cell or cell of an organism encompasses not only the chromosomal DNA found within the nucleus, but also organelle DNA found within subcellular components of the cell (eg, mitochondria, or plastids).

[0061] "Open reading frame" is abbreviated as ORF.

[0062] The term "selectively hybridizes" includes reference to hybridization of a nucleic acid sequence to a specific nucleic acid target sequence under stringent hybridization conditions to a detectably greater degree (e.g., at least 2 times background) than the nucleic acid sequence to non-target nucleic acid sequences and to substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have about at least 80% sequence identity, or 90% sequence identity, up to and including 100% sequence identity (i.e., fully complementary) to each other.

[0063] The term "stringent conditions" or "stringent hybridization conditions" includes reference to conditions under which a probe will selectively hybridize with its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different situations. By controlling the stringency of hybridization conditions and / or washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous detection). Alternatively, stringent conditions can be adjusted to allow some mismatches in the sequence so that a lower degree of similarity is detected (heterologous detection). Typically, the probe length is less than about 1000 nucleotides, optionally less than 500 nucleotides in length. Typically, stringent conditions will be conditions where the salt concentration is less than about 1.5 M Na ions, typically about 0.01 M to 1.0 M Na ion concentration (or other one or more salts) at pH 7.0 to 8.3, and 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). Adding a destabilizing agent such as formamide can also achieve stringent conditions. Exemplary low stringency conditions include hybridization at 37° C. with a buffer solution of 30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1X to 2X SSC (20X SSC=3.0 M NaCl / 0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary medium stringency conditions include hybridization at 37° C. in 40% to 45% formamide, 1 M NaCl, 1% SDS, and washing in 0.5X to 1X SSC at 55° C. to 60° C. Exemplary high stringency conditions include hybridization at 37° C. in 50% formamide, 1 M NaCl, 1% SDS, and washing in 0.1X SSC at 60° C. to 65° C.

[0064] "Homologous" means that the DNA sequences are similar. For example, a "region homologous to a genomic region" found on a donor DNA is a region of DNA having a similar sequence to a given "genomic sequence" in the genome of a cell or organism. A homologous region can have any length sufficient to promote homologous recombination at the target site of cleavage. For example, the length of a homologous region can include at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-2000 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-200, 1-210, 1-220, 1-230, 1-240, 1-250, 1-260, 1-270, 1-280, 1-290, 1-300, 1-310 or more bases, so that the homologous region has enough homology to carry out homologous recombination with the corresponding genomic region." enough homology" means that two polynucleotide sequences have enough structural similarities to serve as substrates for homologous recombination reactions. Structural similarity includes the total length of each polynucleotide fragment and the sequence similarity of the polynucleotides. Sequence similarity can be described by percent sequence identity over the entire length of the sequence and / or by conserved regions that include local similarity (eg, contiguous nucleotides with 100% sequence identity) as well as percent sequence identity over a portion of the length of the sequence.

[0065] As used herein, a "genomic region" is a chromosomal segment in the genome of a cell that is present on either side of a target site, or alternatively, also comprises a portion of a target site. A genomic region may comprise at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-120 0, 5-1300, 5-1400, 5-1500, 5-1600, 5-1700, 5-1800, 5-1900, 5-2000, 5-2100, 5-2200, 5-2300, 5-2400, 5-2500, 5-2600, 5-2700, 5-2800. 5-2900, 5-3000, 5-3100 or more bases, so that the genomic region has sufficient homology to undergo homologous recombination with the corresponding homologous region.

[0066] As used herein, "homologous recombination (HR)" includes the exchange of DNA fragments at homologous sites between two DNA molecules. The frequency of homologous recombination is affected by multiple factors. Different organisms vary in the amount of homologous recombination and the relative proportion of homology and non-homologous recombination. Usually, the length of the homologous region can affect the frequency of homologous recombination events: the longer the homologous region, the higher the frequency. The length of the homologous region required for observing homologous recombination also varies with species. In many cases, at least 5kb of homology has been utilized, but homologous recombination with a homology as low as 25bp-50bp has been observed. See, e.g., 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.

[0067] "Sequence identity" or "identity" in the context of nucleic acid or polypeptide sequences refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.

[0068] The term "percentage of sequence identity" refers to a value determined by comparing two optimally aligned sequences over a comparison window, wherein for the optimal alignment of the two sequences, compared to a reference sequence (which does not include additions or deletions), the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., spaces). The percentage is calculated in the following manner: the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window, and then these results are multiplied by 100 to produce the percentage of sequence identity. Useful examples of percentage sequence identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%, or any percentage from 50% to 100%. These identities can be determined using any program described herein.

[0069] Sequence alignments and percent identity or similarity calculations can be determined using a variety of comparison methods designed to detect homologous sequences, including but not limited to MegAlign® of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, Wisconsin). TM Program. In the context of this application, it will be understood that, where sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" will mean any set of values ​​or parameters that are initially loaded into the software when it is first initialized.

[0070] The "Clustal V method of alignment" corresponds to the alignment method labeled Clustal V (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci [Computer Applications in Biological Sciences] 8:189-191) and is found in MegAlign of the LASERGENE bioinformatics computing package (DNASTAR, Inc., Madison, Wisconsin). TMProgram. For multiple alignments, the default values ​​correspond to GAP PENALTY = 10 and GAP LENGTH PENALTY = 10. The default parameters for pairwise alignments and percentage identity calculations of protein sequences using the Clustal method are KTUPLE = 1, GAP PENALTY = 3, WINDOW = 5, and DIAGONALS SAVED = 5. For nucleic acids, these parameters are KTUPLE = 2, GAP PENALTY = 5, WINDOW = 4, and DIAGONALS SAVED = 4. After aligning sequences using the Clustal V program, it is possible to obtain "percent identity" by viewing the "sequence distance" table in the same program. The "Clustal W method of alignment" corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al., (1992) Comput Appl Biosci [Computer Applications in Biological Sciences] 8:189-191) and found in MegAlign of the LASERGENE bioinformatics computing package (DNASTAR, Inc., Madison, Wisconsin). TMv6.1 program. Default parameters for multiple alignment (Gap Penalty = 10, Gap Length Penalty = 0.2, Delay Divergen Seqs (%) = 30, DNA Transformation Weight = 0.5, Protein Weight Matrix = Gonnet Series, DNA Weight Matrix = IUB). After aligning sequences using the Clustal W program, it is possible to obtain "percent identity" by viewing the "Sequence Distance" table in the same program. Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the values ​​obtained using GAP version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: % identity and % similarity for nucleotide sequences using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for amino acid sequences using a gap creation penalty weight of 8 and a gap length extension penalty weight of 2 and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 89: 10915). GAP uses the algorithm of Needleman and Wunsch (1970) J Mol Biol [Journal of Molecular Biology] 48: 443-53 to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible comparisons and gap positions, and uses the gap generation penalty and gap extension penalty in the unit of matching bases to produce the comparison with the maximum number of matching bases and the minimum gap. " BLAST " is the search algorithm for finding the region of similarity between biological sequences provided by the National Center for Biotechnology Information (NCBI) of the United States. The program compares nucleotide or protein sequence with the sequence database, and calculates the statistical significance of the match to identify the sequence with enough similarities to the query sequence, so that similarity will not be predicted to have occurred randomly. The sequences identified by BLAST report and their local comparison with the query sequence. It is well understood by those skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or modified natural or synthetic polypeptides, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage from 50% to 100%.Indeed, any amino acid identity from 50% to 100% may be useful in describing the present disclosure, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0071] Polynucleotide and polypeptide sequences, variants thereof, and the structural relationships of these sequences can be described using the terms "homology", "homologous", "substantially identical", "substantially similar", and "substantially corresponding", which are used interchangeably herein. These refer to polypeptide or nucleic acid sequences in which changes in one or more amino acids or nucleotide bases do not affect the function of the molecule, such as the ability to mediate gene expression or produce a certain phenotype. These terms also refer to one or more modifications of a nucleic acid sequence that do not substantially change the functional properties of the resulting nucleic acid relative to the initial unmodified nucleic acid. These modifications include deletions, substitutions, and / or insertions of one or more nucleotides in a nucleic acid fragment. Substantially similar nucleic acid sequences encompassed can be defined by the ability of these nucleic acid sequences to hybridize with the sequences exemplified herein, or to hybridize with any portion of a nucleotide sequence disclosed herein and functionally equivalent to any nucleic acid sequence disclosed herein (under moderate stringency conditions, e.g., 0.5X SSC, 0.1% SDS, 60°C). Stringent conditions can be adjusted to screen for moderately similar fragments (e.g., homologous sequences from distantly related organisms) to highly similar fragments (e.g., genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions.

[0072] A "centimorgan" (cM) or "map distance unit" is the distance between two polynucleotide sequences, linked genes, markers, target sites, loci, or any pair thereof, at which 1% of the products of meiosis are recombinant. Thus, one centimorgan is equivalent to a distance equal to the 1% average recombination frequency between two linked genes, markers, target sites, loci, or any pair thereof.

[0073] "Isolated" or "purified" nucleic acid molecules, polynucleotides, polypeptides or proteins, or biologically active portions thereof, are substantially or essentially free of components that normally accompany or interact with polynucleotides or proteins as found in their naturally occurring environment. Thus, isolated or purified polynucleotides or polypeptides or proteins are substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, "isolated" polynucleotides do not contain sequences that naturally flank the polynucleotides (i.e., sequences at the 5' and 3' ends of the polynucleotides) in the genomic DNA of the organism from which the polynucleotides are derived (optimally protein coding sequences). For example, in various embodiments, the isolated polynucleotides may 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 polynucleotides in the genomic DNA of the cells from which the polynucleotides are derived. Isolated polynucleotides may be purified from cells in which they naturally occur. Conventional nucleic acid purification methods known to technicians can be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

[0074] The term "fragment" refers to a continuous set of nucleotides or amino acids. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 continuous nucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 continuous amino acids. A fragment may or may not exhibit the function of a sequence having a certain percentage of identity over the length of the fragment.

[0075] The terms "functionally equivalent fragments" and "functionally equivalent fragments" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment or polypeptide that exhibits the same activity or function as the longer sequence from which it is derived. In one example, whether or not the fragment encodes an active protein, the fragment retains the ability to alter gene expression or produce a certain phenotype. For example, fragments can be used to design genes to produce a desired phenotype in a modified plant. Genes can be designed for use in suppression by connecting nucleic acid fragments, whether or not they encode an active enzyme, in a sense or antisense orientation relative to a plant promoter sequence.

[0076] "Gene" includes a nucleic acid fragment that expresses a functional molecule (such as, but not limited to, a specific protein), including regulatory sequences preceding the coding sequence (5' non-coding sequences) and following the coding sequence (3' non-coding sequences). "Native gene" refers to a gene found in its natural endogenous location with its own regulatory sequences.

[0077] The term "endogenous" refers to a sequence or other molecule that is naturally present in a cell or organism. In one aspect, an endogenous polynucleotide is typically found in the genome of a cell; that is, not heterologous.

[0078] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are identical, the plant is homozygous at that locus. If the alleles present at a given locus on a chromosome are different, the plant is heterozygous at that locus.

[0079] "Coding sequence" refers to a polynucleotide sequence that encodes a specific amino acid sequence. "Regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), internal nucleotide sequence, or downstream nucleotide sequence (3' non-coding sequence) of a coding sequence, and it affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5' non-translated sequences, 3' non-translated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0080] A "mutant gene" is a gene that has been altered by human intervention. Such a "mutant gene" has a sequence that differs from the sequence of the corresponding non-mutant gene by at least one nucleotide addition, deletion or substitution. In certain embodiments of the present disclosure, the mutated gene comprises an alteration caused by a guide polynucleotide / Cas endonuclease system as disclosed herein. A mutated plant is a plant comprising a mutated gene.

[0081] As used herein, a "targeted mutation" is a mutation in a gene (referred to as a target gene), including a native gene, generated by altering a target sequence within the target gene using any method known to those skilled in the art, including methods involving a guided Cas endonuclease system as disclosed herein.

[0082] The terms "knockout," "gene knock-out," and genetic knock-out are used interchangeably herein. Knockout means that a DNA sequence of a cell has been partially or completely ineffective by targeting with a Cas protein; for example, the DNA sequence may have encoded an amino acid sequence before knockout, or may have had a regulatory function (e.g., a promoter).

[0083] The terms "knock-in", "gene knock-in", "gene insertion" and "gene knock-in" are used interchangeably herein. Knock-in represents the replacement or insertion of a DNA sequence at a specific DNA sequence in a cell by targeting with a Cas protein (e.g., by homologous recombination (HR), wherein a suitable donor DNA polynucleotide is also used). Examples of knock-ins are the specific insertion of a heterologous amino acid coding sequence in a gene coding region, or the specific insertion of a transcriptional regulatory element in a genetic locus.

[0084] "Domain" means a continuous stretch of nucleotides (which may be RNA, DNA, and / or RNA-DNA combination sequences) or amino acids.

[0085] The term "conserved domain" or "motif" refers to a group of polynucleotides or amino acids that are conserved at specific positions along the aligned sequences of evolutionarily related proteins. Although amino acids at other positions can vary between homologous proteins, amino acids that are highly conserved at specific positions indicate amino acids that are essential to the structure, stability, or activity of the protein. Because they are identified by their high conservation in the aligned sequences of a family of protein homologs, they can be used as identifiers or "signatures" to determine whether a protein with a newly determined sequence belongs to a previously identified protein family.

[0086] A "codon-modified gene" or "codon-biased gene" or "codon-optimized gene" is a gene whose frequency of codon usage has been designed to mimic the frequency of preferred codon usage of the host cell.

[0087] An "optimized" polynucleotide is a sequence that has been optimized for improved expression in a specific heterologous host cell.

[0088] A "plant optimized nucleotide sequence" is a nucleotide sequence optimized for expression in plants (particularly for increased expression in plants). Plant optimized nucleotide sequences include codon optimized genes. Plant optimized nucleotide sequences can be synthesized by modifying the nucleotide sequence encoding a protein (such as the Cas endonucleases disclosed herein) using one or more plant preferred codons to improve expression. For a discussion of host preferred codon usage, see, e.g., Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92: 1-11.

[0089] "Promoter" is a DNA region that participates in the recognition and combination of RNA polymerase and other proteins to initiate transcription. The promoter sequence is composed of a proximal upstream element and a distal upstream element, and the latter element is generally referred to as an enhancer. "Enhancer" is a DNA sequence that can stimulate promoter activity, and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. Promoter can be derived from natural genes in its entirety, or be composed of different elements derived from different promoters existing in nature, and / or comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may guide gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental conditions. It is further recognized that, since the exact boundaries of regulatory sequences are not yet fully defined in most cases, some DNA fragments of variations may have the same promoter activity.

[0090] Promoters that cause gene expression in most cell types under most circumstances are generally referred to as "constitutive promoters". The term "inducible promoter" refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, such as by a chemical compound (chemical inducer), or in response to environmental, hormone, chemical, and / or developmental signals. Inducible or regulated promoters include promoters that are induced or regulated, for example, by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounds, or chemicals such as ethanol, abscisic acid (ABA), jasmonate, salicylic acid, or safeners.

[0091] "Translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence and the coding sequence of a gene. The translation leader sequence is present upstream of the translation initiation sequence in the mRNA. The translation leader sequence can affect the processing of the primary transcript to the mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (e.g., Turner and Foster, (1995) Mol Biotechnol [Molecular Biotechnology] 3: 225-236).

[0092] "3' non-coding sequence", "transcription terminator" or "termination sequence" refers to a DNA sequence located downstream of a coding sequence and includes polyadenylation recognition sequences and other sequences encoding regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is generally characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor. The use of various 3' non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.

[0093] "RNA transcript" refers to the product produced by the transcription catalyzed by RNA polymerase of a DNA sequence. When the RNA transcript is a fully complementary copy of a DNA sequence, the RNA transcript is referred to as a primary transcript or pre-mRNA. When the RNA transcript is an RNA sequence derived from the post-transcriptional processing of the pre-mRNA of the primary transcript, the RNA transcript is referred to as a mature RNA or mRNA. "Messenger RNA" or "mRNA" refers to an RNA that does not contain introns and can be translated into protein by a cell. "cDNA" refers to a DNA that is complementary to an mRNA template and synthesized from an mRNA template using a reverse transcriptase. The cDNA can be single-stranded or can be converted into a double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to an RNA transcript that includes mRNA and can be translated into protein in a cell or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and blocks the expression of a target gene (see, e.g., U.S. Patent No. 5,107,065). Antisense RNA can be complementary to any part of a particular gene transcript, i.e., 5' non-coding sequence, 3' non-coding sequence, intron or coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but still has an effect on cellular processes. The terms "complementary sequence" and "reverse complementary sequence" are used interchangeably herein with respect to mRNA transcripts, and are intended to define the antisense RNA of the messenger.

[0094] The term "genome" refers to the entire complementary sequence of genetic material (genes and non-coding sequences) present in every cell of an organism or virus or organelle; and / or the complete set of chromosomes inherited as a (haploid) unit from one parent.

[0095] The term "operably connected" refers to the association of a nucleic acid sequence on a single nucleic acid fragment so that the function of one of the nucleic acid sequences is regulated by another nucleic acid sequence. For example, when a promoter is capable of regulating the expression of a coding sequence (that is, the coding sequence is under the transcriptional control of a promoter), the promoter is operably connected to the coding sequence. The coding sequence can be operably connected to a regulatory sequence in a sense or antisense orientation. In another example, a complementary RNA region can be directly or indirectly operably connected to or within a target mRNA at 5' or 3' of a target mRNA, or the first complementary region is at 5' of the target mRNA and its complementary sequence is at 3' of the target mRNA.

[0096] Typically, "host" refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced. As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell) in vivo or in vitro, or a cell (e.g., a cell line) from a multicellular organism cultured as a unicellular entity into which a heterologous polynucleotide or polypeptide has been introduced. In some embodiments, the cell is selected from the group consisting of: a primitive cell, a bacterial cell, a eukaryotic cell, a eukaryotic unicellular organism, a somatic cell, a germ cell, a stem cell, a plant cell, an algae cell, an animal cell, an invertebrate cell, a vertebrate cell, a fish cell, a frog cell, a bird cell, an insect cell, a mammalian cell, a pig cell, a cattle cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell. In some cases, the cell is an in vitro cell. In some cases, the cell is an in vivo cell.

[0097] The term "recombinant" refers to the artificial combination of two originally separate sequence segments, for example, by chemical synthesis or by the manipulation of separate nucleic acid segments through genetic engineering techniques.

[0098] The terms "plasmid", "vector" and "box" refer to linear or circular extrachromosomal elements, which usually carry genes that are not part of the central metabolism of the cell, and are usually in the form of double-stranded DNA. Such elements can be autonomous replication sequences, genome integration sequences, phages, or nucleotide sequences derived from any source, single-stranded or double-stranded DNA or RNA, in linear or circular form, many of which have been connected or recombined into unique structures that can introduce the polynucleotide of interest into cells." transformation box" refers to a specific vector that contains genes and has elements other than genes that promote transformation of specific host cells. "expression cassette" refers to a specific vector that contains genes and has elements other than genes that allow expression of the gene in a host.

[0099] The terms "recombinant DNA molecule", "recombinant DNA construct", "expression construct", "construct", and "recombinant construct" are used interchangeably herein. A recombinant DNA construct comprises an artificial combination of nucleic acid sequences, such as regulatory sequences and coding sequences that are not all found together in nature. For example, a recombinant DNA construct may comprise regulatory sequences and coding sequences derived from different sources, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from that of natural occurrence. Such constructs may be used alone or in combination with a vector. If a vector is used, the selection of the vector depends on the method that will be used to introduce the vector into a host cell as is well known to those skilled in the art. For example, a plasmid vector may be used. The skilled person is fully aware of the genetic elements that must be present on the vector in order to successfully transform, select, and propagate the host cell. The skilled artisan will also recognize that different independent transformation events may result in different expression levels and patterns (Jones et al., (1985) EMBO J [Journal of the European Molecular Biology Association] 4: 2411-2418; De Almeida et al., (1989) MolGen Genetics [Molecular and General Genetics] 218: 78-86), and therefore typically multiple events are screened to obtain lines that display the desired expression level and pattern. Such screening can be accomplished by standard molecular biology assays, biochemical assays, and other assays, including Southern blot analysis, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzyme assays or activity assays, and / or phenotypic analysis.

[0100] The term "heterologous" refers to the difference between the original environment, position, or composition of a particular polynucleotide or polypeptide sequence and its current environment, position, or composition. Non-limiting examples include taxonomic derivation (e.g., if a polynucleotide sequence obtained from Zea mays is inserted into the genome of a different variety or cultivar of a rice (Oryza sativa) plant or maize, the polynucleotide sequence will be heterologous; or a polynucleotide obtained from a bacterium is introduced into a plant's cell) or sequence (e.g., a polynucleotide sequence is obtained from maize, separated, modified, and reintroduced into a maize plant). As used herein, "heterologous" with respect to a sequence may refer to a sequence that is derived from a different species, variant, exotic species, or, if derived from the same species, is substantially modified from its native form in a composition and / or genomic locus by deliberate human intervention. For example, the promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide is derived, or, if from the same / similar species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not a native promoter of the polynucleotide operably linked. Alternatively, one or more regulatory regions and / or polynucleotides provided herein can be synthesized as a whole. In another example, the target polynucleotide for being cut by the Cas endonuclease can belong to an organism different from the Cas endonuclease. In another example, the Cas endonuclease and the guide RNA can be introduced into the target polynucleotide together with another polynucleotide used as a template or donor for insertion into the target polynucleotide, wherein the other polynucleotide is heterologous to the target polynucleotide and / or the Cas endonuclease.

[0101] As used herein, the term "expression" refers to the production of a functional end-product (eg, mRNA, guide RNA, or protein) in either precursor or mature form.

[0102] A "mature" protein refers to a post-translationally processed polypeptide (ie, a polypeptide from which any pre-peptide or propeptide present in the primary translation product has been removed).

[0103] "Precursor" protein refers to the primary product of translation of mRNA (ie, pre- or propeptides are still present). Pre- or propeptides may be, but are not limited to, intracellular localization signals.

[0104] “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats ( C lustered R egularly I Interspaced S hort Palindromic R CRISPR loci refer to certain genetic loci encoding components of DNA cleavage systems, such as those used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327: 167-170; WO 2007025097 published March 1, 2007). CRISPR loci can consist of a CRISPR array that can be flanked by different Cas (CRISPR associated) genes, comprising short direct repeat sequences (CRISPR repeats) separated by short variable DNA sequences (called 'spacers').

[0105] As used herein, an "effector" or "effector protein" is a protein having an activity that includes recognition, binding, and / or cleavage or nicking of a polynucleotide target. An effector or effector protein may also be an endonuclease. The "effector complex" of a CRISPR system includes Cas proteins that participate in crRNA and target recognition and binding. Some component Cas proteins may additionally include domains that participate in target polynucleotide cleavage.

[0106] The term "Cas protein" refers to a protein that is produced by Cas ( CThe polypeptides encoded by genes (associated with RISPR). Cas proteins include proteins encoded by genes in the cas locus, and include adaptation molecules and interfering molecules. Interfering molecules of bacterial adaptive immune complexes include endonucleases. The Cas endonucleases described herein include one or more nuclease domains. Cas endonucleases include, but are not limited to, novel Cas endonucleases 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. When compounded with suitable polynucleotide components, Cas proteins can be "Cas endonucleases" or "Cas effector proteins" that can recognize, bind, and optionally nick or cut all or part of a specific polynucleotide target sequence. Cas endonucleases disclosed herein include those endonucleases having one or more RuvC nuclease domains. The Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a functional fragment or functional variant of a native Cas protein, or a functional fragment or functional variant of a native Cas protein having at least 50%, between 50 and 100%, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, between 400 and 450, at least 500 or more consecutive amino acids. %, between 55% and 60%, at least 60%, between 60% and 65%, at least 65%, between 65% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, 96% and 97%, at least 97%, between 97% and 98%, at least 98%, 98% and 99%, at least 99%, 99% and 100%, or 100% sequence identity and retains at least some of the activity of the native sequence.

[0107] "Functional fragments", "functionally equivalent fragments" and "functionally equivalent fragments" of Cas endonucleases are used interchangeably herein and refer to a portion or subsequence of a Cas endonuclease of the present disclosure, wherein the ability to recognize, bind and optionally unwind, nick or cut (introduce single-strand or double-strand breaks) a target site is retained. A portion or subsequence of a Cas endonuclease may comprise a complete peptide or a partial (functional) peptide of any one of its domains, such as, but not limited to, a complete functional portion of a Cas3 HD domain, a complete functional portion of a Cas3 helicase domain, a complete functional portion of a protein (such as, but not limited to, Cas5, Cas5d, Cas7 and Cas8b1).

[0108] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" of a Cas endonuclease or Cas effector protein (including the Cas endonucleases described herein) are used interchangeably herein and refer to variants of the Cas effector proteins disclosed herein in which the ability to recognize, bind and optionally unwind, nick or cleave all or part of a target sequence is retained.

[0109] Cas endonucleases may also include multifunctional Cas endonucleases. The terms "multifunctional Cas endonucleases" and "multifunctional Cas endonuclease polypeptides" are used interchangeably herein and include reference to a single polypeptide having a Cas endonuclease function (comprising at least one protein domain that can be used as a Cas endonuclease) and at least one other function, such as, but not limited to, a function of forming a complex (which comprises at least a second protein domain that can form a complex with other proteins). On the one hand, the multifunctional Cas endonuclease comprises at least one additional protein domain relative to those typical domains of (inside, upstream (5') or downstream (3'), or at both 5' and 3' inside, or any combination thereof) Cas endonucleases.

[0110] The terms "cascade" and "cascade complex" are used interchangeably herein and include reference to a multi-subunit protein complex that can be assembled with a polynucleotide to form a polynucleotide-protein complex (PNP). A cascade is a polynucleotide-dependent PNP to achieve complex assembly and stability and to identify target nucleic acid sequences. The cascade is used as a monitoring complex that finds and optionally binds to a target nucleic acid that is complementary to the variable targeting domain of a guide polynucleotide.

[0111] The terms "cleavage-ready Cascade", "crCascade", "cleavage-ready Cascade complex", "crCascade complex", "cleavage-ready Cascade system", "CRC" and "crCascade system" are used interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP), wherein one of the cascade proteins is a Cas endonuclease that is capable of recognizing, binding, and optionally unwinding, nicking or cleaving all or part of a target sequence.

[0112] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. The 7-methylguanylate residue is located at the 5' end of messenger RNA (mRNA) in eukaryotes. In eukaryotes, RNA polymerase II (Pol II) transcribes mRNA. Messenger RNA capping typically occurs as follows: the very terminal 5' phosphate group of the mRNA transcript is removed with an RNA terminal phosphatase, leaving two terminal phosphates. Guanosine monophosphate (GMP) is added to the terminal phosphate of the transcript with a guanylyltransferase, leaving a 5'-5' triphosphate-linked guanine at the end of the transcript. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyltransferase.

[0113] The term "without a 5'-cap" herein is used to refer to an RNA having, for example, a 5'-hydroxyl group instead of a 5'-cap. For example, such an RNA may be referred to as an "uncapped RNA". Because 5'-capped RNA has a tendency to be exported from the nucleus, uncapped RNA may accumulate better in the nucleus after transcription. One or more RNA components herein are uncapped.

[0114] As used herein, the term "guide polynucleotide" refers to a polynucleotide sequence that can form a complex with a Cas endonuclease (including a Cas endonuclease described herein) and enable the Cas endonuclease to recognize, optionally bind, and optionally cleave a DNA target site. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence).

[0115] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a guide RNA, crRNA, or tracrRNA are used interchangeably herein and refer to a portion or subsequence of a guide RNA, crRNA, or tracrRNA of the present disclosure, respectively, in which the ability to function as a guide RNA, crRNA, or tracrRNA, respectively, is retained.

[0116] The terms "functional variant," "functionally equivalent variant," and "functionally equivalent variant" of a guide RNA, crRNA, or tracrRNA (respectively) are used interchangeably herein and refer to variants of a guide RNA, crRNA, or tracrRNA of the present disclosure, respectively, in which the ability to function as a guide RNA, crRNA, or tracrRNA, respectively, is retained.

[0117] 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 pairing sequence that hybridizes to the tracrRNA) and a tracrRNA (transactivating RNA). CR 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, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, such that the Cas endonuclease can recognize, optionally bind, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0118] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and include a nucleotide sequence that can hybridize (be complementary) to one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a continuous stretch of 12 to 30 nucleotides. The variable targeting domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0119] The terms "Cas endonuclease recognition domain" or "CER domain" (of a guide polynucleotide) are used interchangeably herein and include nucleotide sequences that interact with a Cas endonuclease polypeptide. A CER domain comprises a (trans-acting) tracr nucleotide chaperone sequence followed by a tracr nucleotide sequence. A CER domain may be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., US20150059010A1, published on February 26, 2015), or any combination thereof.

[0120] As used herein, the terms "guide polynucleotide / Cas endonuclease complex", "guide polynucleotide / Cas endonuclease system", "guide polynucleotide / Cas complex", "guide polynucleotide / Cas system" and "guided Cas system", "polynucleotide-guided endonuclease", "PGEN" are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease capable of forming a complex, wherein the guide polynucleotide / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site. The guide polynucleotide / Cas endonuclease complex herein may comprise one or more Cas proteins of known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170; Makarova et al. 2015, Nature Reviews Microbiology 13: 1-15; Zetsche et al. 2015, Cell 163, 1-13; Shmakov et al. 2015, Molecular Cell 60, 1-13) and one or more suitable polynucleotide components.

[0121] The terms "guide RNA / Cas endonuclease complex", "guide RNA / Cas endonuclease system", "guide RNA / Cas complex", "guide RNA / Cas system", "gRNA / Cas complex", "gRNA / Cas system", "RNA-guided endonuclease", "RGEN" are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease capable of forming a complex, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind and optionally nick or cut (introduce single-strand or double-strand breaks) the DNA target site.

[0122] 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, for example, but not limited to, a nucleotide sequence on a chromosome, episome, locus or any other DNA molecule in the genome of a cell (including chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA), at which a guide polynucleotide / Cas endonuclease complex can recognize, bind and optionally nick or cut. The target site can be an endogenous site in the genome of the cell, or alternatively, the target site can be heterologous to the cell and thus not naturally present in the genome of the cell, or the target site can be found in a heterologous genomic location compared to the location where it occurs in nature. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of the cell and is located at the endogenous or native location of the target sequence in the genome of the cell. "Artificial target site" or "artificial target sequence" are used interchangeably herein and refer to a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence may be identical in sequence to an endogenous or native target sequence in the genome of the cell, but located at a different location in the genome of the cell (i.e., a non-endogenous or non-native location).

[0123] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to the (targeted) target sequence (pre-spacer) recognized by the guide polynucleotide / Cas endonuclease system described herein. If the target DNA sequence is not followed by a PAM sequence, the Cas endonuclease may not successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0124] "Altered target site", "altered target sequence", "modified target site", "modified target sequence" are used interchangeably herein and refer to a target sequence as disclosed herein, which comprises at least one change when compared to a non-altered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0125] "Modified nucleotides" or "edited nucleotides" refer to a nucleotide sequence of interest that contains at least one change when compared to its unmodified nucleotide sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, (iv) chemical change of at least one nucleotide, or (v) any combination of (i)-(iv).

[0126] Methods for "modifying a target site" and "altering a target site" are used interchangeably herein and refer to methods for generating an altered target site.

[0127] As used herein, "donor DNA" is a DNA construct comprising a polynucleotide of interest to be inserted into the target site of a Cas endonuclease.

[0128] The term "polynucleotide modification template" includes a polynucleotide comprising at least one nucleotide modification when compared to a nucleotide sequence to be edited. The nucleotide modification may be at least one nucleotide substitution, addition or deletion. Optionally, the polynucleotide modification template may further comprise a homologous nucleotide sequence flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequence provides sufficient homology to the desired nucleotide sequence to be edited.

[0129] The term "plant-optimized Cas endonuclease" herein refers to a Cas protein encoded by a nucleotide sequence that has been optimized for expression in plant cells or plants, including multifunctional Cas proteins.

[0130] "Plant optimized nucleotide sequence encoding a Cas endonuclease," "plant optimized construct encoding a Cas endonuclease," and "plant optimized polynucleotide encoding a Cas endonuclease" are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, which has been optimized for expression in a plant cell or plant. Plants comprising a plant optimized Cas endonuclease include: plants comprising a nucleotide sequence encoding a Cas sequence, and / or plants comprising a Cas endonuclease protein. In one aspect, the plant optimized Cas endonuclease nucleotide sequence is a maize optimized, rice optimized, wheat optimized, soybean optimized, cotton optimized, or canola optimized Cas endonuclease.

[0131] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, progeny of seeds and plants. Plants are monocots or dicots. Plant cells include but are not limited to cells derived from: seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen and microspores." Plant elements" are intended to refer to whole plants or plant components, which may include differentiation and / or undifferentiated tissues, such as but not limited to plant tissues, parts and cell types. In one embodiment, plant elements are one of: whole plants, seedlings, meristematic tissues, basic tissues, vascular tissues, epithelial tissues, seeds, leaves, roots, buds, stems, flowers, fruits, runners, bulbs, tubers, corms, asexual tip branches, buds, young shoots, tumor tissues, and various forms of cells and cultures (e.g., unicellular, protoplasts, embryos and callus). It should be noted that protoplasts are not technically "complete" plant cells (all components naturally occurring) because protoplasts do not have cell walls. The term "plant organ" refers to a plant tissue or a group of tissues that constitute the morphological and functionally different parts of a plant. As used herein, "plant element" is a synonym for "part" of a plant, refers to any part of a plant, and may include different tissues and / or organs, and may be used interchangeably with the term "tissue" in the full text. Similarly, "plant propagation element" is intended to generally refer to any plant part that can create other plants through the sexual or asexual propagation of the plant, such as but not limited to: seed, seedling, root, bud, cutting, scion, grafted seedling, runner, bulb, tuber, corm, asexual terminal branch or young shoot. Plant elements may be present in a plant or in a plant organ, tissue culture or cell culture.

[0132] "Progeny" includes any subsequent generation of a plant.

[0133] As used herein, the term "plant part" refers to plant cells, plant protoplasts, plant cell tissue cultures that can regenerate plants, plant calli, plant pieces, and intact plant cells in plants or plant parts (such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, spikes, cobs, shells, stems, roots, root tips, anthers, etc.), together with these parts themselves. Grains mean mature seeds produced by commercial growers for purposes other than cultivation or propagation of species. Progeny, variants, and mutants of these regenerated plants are also included within the scope of the present invention, provided that these parts contain introduced polynucleotides.

[0134] The term "monocotyledonous" or "monocotyledonous plant" refers to a subclass of angiosperms, also known as the "monocots," whose seeds typically contain only one embryonic leaf or cotyledon. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0135] The term "dicotyledonous" or "dicots" refers to a subclass of angiosperms, also known as the "dicots," whose seeds typically contain two embryonic leaves or cotyledons. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny thereof.

[0136] As used herein, a "male sterile plant" is a plant that does not produce viable or otherwise fertilized male gametes. As used herein, a "female sterile plant" is a plant that does not produce viable or otherwise fertilized female gametes. It should be recognized that male sterile plants and female sterile plants can be female fertile and male fertile, respectively. It should be further recognized that male fertile (but female sterile) plants can produce viable progeny when crossed with female fertile plants, and female fertile (but male sterile) plants can produce viable progeny when crossed with male fertile plants.

[0137] The term "non-conventional yeast" herein refers to any yeast that is not a yeast species of the genus Saccharomyces (e.g., Saccharomyces cerevisiae) or the genus Schizosaccharomyces. (See "Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols", K. Wolf, KD Breunig, G. Barth, eds., Springer-Verlag, Berlin, Germany, 2003).

[0138] In the context of the present disclosure, the term "hybridized" or "cross" refers to the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses sexual crosses (one plant being pollinated by another plant) and selfing (self-pollination, i.e., when the pollen and ovules (or microspores and megaspores) are from the same plant or genetically identical plants).

[0139] The term "infiltration" refers to the phenomenon that the allele of the expectation of the genetic locus is transferred to another genetic background from a genetic background. For example, the allele infiltration of the expectation at the specified locus can be passed to at least one progeny plant via the sexual hybridization between two parental plants, wherein at least one of the parental plants has the allele of expectation in its genome. Alternatively, for example, the transmission of alleles can occur by the recombination between two donor genomes, such as in fusion protoplasts, wherein at least one of the donor protoplasts has the allele of expectation in its genome. The allele of expectation can be, for example, the allele of the selection of transgenic, modified (mutated or edited) natural allele or marker or QTL.

[0140] The term "isoline" is a comparative term referring to organisms that are genetically identical but different in treatment. In one example, two genetically identical maize plant embryos can be divided into two different groups, one group receiving treatment (such as introduction of CRISPR-Cas effector endonucleases), and one group not receiving such treatment as a control. Therefore, any phenotypic differences between the two groups may be attributed only to the treatment, rather than to any inherent nature of the endogenous gene composition of the plant.

[0141] "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 or components gain access to the interior of a cell of the organism or to the cell itself.

[0142] "Purpose polynucleotides" include any nucleotide sequence encoding proteins or polypeptides that improve the desirability of crops (i.e., traits of agronomic significance). Purpose polynucleotides include, but are not limited to, polynucleotides encoding important traits for agronomy, herbicide-resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial products, phenotypic markers, or any other traits of agronomic or commercial importance. Purpose polynucleotides may additionally be utilized in sense or antisense orientations. In addition, more than one purpose polynucleotide may be utilized together or "stacked" to provide additional benefits.

[0143] "Complex trait loci" include genomic loci with multiple transgenes that are genetically linked to each other.

[0144] The compositions and methods herein can provide plants with improved "agronomic traits" or "traits of agronomic importance" or "traits of agronomic significance", which traits can include, but are not limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, improved yield, enhanced health, improved vigor, improved growth, improved photosynthetic capacity, enhanced nutrition, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root structure, modulation of metabolites, modulation of the proteome, increase in seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition compared to a homologous plant not comprising the modifications derived from the methods and compositions herein.

[0145] "Agronomic potential" is intended to mean the ability of a plant element to express a phenotype, preferably an improved agronomic trait, at some point in its life cycle, or to transmit said phenotype to another plant element with which it is associated in the same plant.

[0146] As used herein, the terms "reduced," "less," "slower," and "increased," "faster," "enhanced," "greater" refer to a decrease or increase in a characteristic of a modified plant element or resulting plant as compared to an unmodified plant element or resulting plant. For example, the reduction in a characteristic can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, between 5% and 10%, at least 10%, between 10% and 20%, at least 15%, at least 20%, between 20% and 30%, at least 25%, at least 30%, between 30% and 40%, at least 35%, at least 40%, between 40% and 50%, at least 45%, at least 50%, between 50% and 60%, at least about 60%, between 60% and 70%, between 70% and 80%, at least 75%, at least about 80%, between 80% and 90%, at least about 90%, between 90% and 100%, at least 100%, between 100% and 200%, at least 200%, at least about 300%, at least about 400%, or more than an untreated control. More, and the increase can be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5% to 10%, at least 10%, 10% to 20%, at least 15%, at least 20%, 20% to 30%, at least 25%, at least 30%, 30% to 40%, at least 35%, at least 40%, 40% to 50%, at least 45%, at least 50%, 50% to 60%, at least about 60%, 60% to 70%, 70% to 80%, at least 75%, at least about 80%, 80% to 90%, at least about 90%, 90% to 100%, at least 100%, 100% and 200%, at least 200%, at least about 300%, at least about 400% or more over an untreated control.

[0147] As used herein, the term "preceded by" when referring to sequence position means that one sequence occurs upstream or 5' to another sequence.

[0148] The meanings of the abbreviations are as follows: “sec” means second, “min” means minute, “h” means hour, “d” means day, “μL” means microliter, “mL” means milliliter, “L” means liter, “μM” means micromole, “mM” means millimolar, “M” means molar, “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.

[0149] Classification of CRISPR-Cas systems

[0150] CRISPR-Cas systems have been classified based on sequence and structural analysis of the components. Multiple CRISPR / Cas systems have been described, including class 1 systems with multi-subunit effector complexes (including types I, III, and IV) and class 2 systems with single protein effectors (including types II, V, and VI) (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).

[0151] The CRISPR-Cas system includes at least CRISPR RNA (crRNA) molecules and at least one CRISPR-related (Cas) protein to form a crRNA ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus includes a series of identical repetitive sequences, which are interspersed with DNA targeting spacers encoding crRNA components and operon-like units of cas genes encoding Cas protein components. The generated ribonucleoprotein complex recognizes polynucleotides in a sequence-specific manner (Jore et al. 2011, Nature Structural & Molecular Biology [Natural Structure and Molecular Biology] 18, 529-536). CrRNA forms a base pair with a complementary DNA strand, while displacing a non-complementary strand to form a so-called R ring, thereby acting as a guide RNA for sequence-specific binding of an effector (protein or complex) to a double-stranded DNA sequence. (Jore et al. 2011).

[0152] The RNA transcripts (pre-crRNA) of the CRISPR loci are cleaved specifically in repetitive sequences by CRISPR-associated (Cas) endonucleases in type I and III systems or by RNase III in type II systems. The number of CRISPR-associated genes at a given CRISPR locus can vary between species.

[0153] There are different cas genes in different CRISPR systems, which encode proteins with different domains. The cas operon contains genes encoding one or more effector endonucleases and other Cas proteins. Protein subunits include those described in: Makarova et al. 2011, Nat Rev Microbiol. [Natural Review Microbiology] 20119 (6): 467-477; Makarova et al. 2015, Nature Reviews Microbiology [Natural Review Microbiology] Vol. 13: 1-15; and Koonin et al. 2017, Current Opinion Microbiology [Microbiology New View] 37: 67-78). The types of domains include those involved in expression (pre-crRNA processing, such as Cas 6 or RNase III), interference (including effector modules for crRNA and target binding, and one or more domains for target cutting), adaptation (spacer insertion, such as Cas1 or Cas2) and auxiliary (regulation or auxiliary or unknown function). Some domains can serve more than one role, for example Cas9 includes domains for endonuclease function as well as for target cleavage, etc.

[0154] Cas endonucleases are guided by a single CRISPR RNA (crRNA) through direct RNA-DNA base pairing to recognize a DNA target site immediately adjacent to a protospacer adjacent motif (PAM) (Jore et al. 2011, supra, Westra et al. 2012, Molecular Cell 46:595-605, and Sinkunas et al. 2013, EMBO J. 32:385-394).

[0155] Class I CRISPR-Cas systems

[0156] Class I CRISPR-Cas systems include Type I, Type III, and Type IV. Class I systems are characterized by the presence of effector endonuclease complexes rather than individual proteins. The Cascade complex contains an RNA recognition motif (RRM) and a nucleic acid binding domain, which is the core fold of a diverse superfamily of RAMP (repeat-associated mysterious protein) proteins (Makarova et al. 2013, Biochem Soc Trans [Biochemical Society Bulletin] 41, 1392-1400; Makarova et al. 2015, Nature Reviews Microbiology [Natural Review Microbiology] Vol. 13: 1-15). RAMP protein subunits include Cas5 and Cas7, which comprise the backbone of the crRNA-effector complex, where the Cas5 subunit binds to the 5' handle of the crRNA and interacts with the large subunit, and typically include Cas6, which is loosely associated with the effector complex and typically functions as a repeat-specific RNase in pre-crRNA processing (Charpentier et al., FEMS Microbiol Rev [FEMS Microbiology Review] 2015, 39:428-441; Niewoehner et al. 2016 RNA, 22:318-329).

[0157] Type I CRISPR-Cas systems contain an effector protein complex, called Cascade (CRISPR-associated complex for antiviral defense), which contains 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 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)-stimulated superfamily 2 helicase that has the ability to unwind double-stranded DNA (dsDNA) and RNA-DNA duplexes (Makarova et al. 2015, Nature Reviews; Microbiology 13: 1-15). Upon 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. [Journal of the European Molecular Biology Association] 32: 385-394). In some type I systems, Cas6 is the active endonuclease responsible for crRNA processing, and Cas5 and Cas7 act as non-catalytic RNA binding proteins; although in type IC systems, crRNA processing can be catalyzed by Cas5 (Makarova et al. 2015, Nature Reviews Microbiology [Nature Review Microbiology] Vol. 13: 1-15). Type I systems are divided into seven subtypes (Makarova et al. 2011, Nat Rev Microbiol. [Nature Review Microbiology] 2011 9 (6): 467-477; Koonin et al. 2017, Curr Opinion Microbiology [Microbiology New View] 37: 67-78).A modified type I CRISPR-associated complex (Cascade) for adaptive antiviral defense has been described, comprising at least the protein subunits Cas7, Cas5 and Cas6, wherein one of these subunits is synthetically fused to the Cas3 endonuclease or the modified restriction endonuclease FokI (WO 2013098244, published on July 4, 2013).

[0158] Type III CRISPR-Cas systems (including multiple cas7 genes) target ssRNA or ssDNA and function as RNases and targeted RNA-activated DNA nucleases (Tamulaitis et al. 2017, Trends in Microbiology 25(10)49-61). Csm (Type III-A) and Cmr (Type III-B) complexes function as RNA-activated single-stranded (ss) DNA enzymes (coupling target RNA binding / cleavage with ssDNA degradation). Upon infection with foreign DNA, CRISPR RNA (crRNA)-guided Csm or Cmr complexes bind to emerging transcripts and recruit Cas10 DNA enzymes to actively transcribed phage DNA, resulting in degradation of transcripts and phage DNA rather than host DNA. The Cas10 HD-domain is responsible for ssDNA enzyme activity, and the Csm3 / Cmr4 subunits are responsible for the endoribonuclease activity of the Csm / Cmr complex. The 3' flanking sequence of the target RNA is crucial for the ssDNA enzyme activity of Csm / Cmr: base pairing with the 5'-handle of crRNA protects the host DNA from degradation.

[0159] Type IV systems, although including the typical type I Cas5 and Cas7 domains and a Cas8-like domain, may lack the CRISPR array that is characteristic of most other CRISPR-Cas systems.

[0160] Class II CRISPR-Cas systems

[0161] Class II CRISPR-Cas systems include Type II, Type V, and Type VI. Class II systems are characterized by the presence of a single Cas effector protein rather than an effector complex. Type II and Type V Cas proteins contain a RuvC endonuclease domain that adopts an RNase H fold.

[0162] Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to guide Cas endonucleases to their DNA targets. CrRNA includes a spacer region complementary to one strand of a double-stranded DNA target and a region that forms an RNA duplex with tracrRNA (trans-activating CRISPR RNA) base pairing, which guides Cas endonucleases to cut DNA targets, leaving flat ends. Spacers are obtained by processes involving Cas1 and Cas2 proteins that are not fully understood. Type II CRISPR / Cas loci typically include cas1 and cas2 genes and cas9 genes (Chylinski et al., 2013, RNA Biology [RNA Biology] 10: 726-737; Makarova et al. 2015, Nature Reviews Microbiology [Natural Review Microbiology] Vol. 13: 1-15). Type II CRISR-Cas loci can encode tracrRNA, which is partially complementary to the repetitive sequences in the corresponding CRISPR array and can include other proteins (such as Csn1 and Csn2). The presence of cas9 near cas1 and cas2 genes is a hallmark of type II loci (Makarova et al. 2015, Nature Reviews Microbiology [microorganism natural review] Vol. 13: 1-15).

[0163] Type V CRISPR / Cas systems contain a single Cas endonuclease, including Cpf1 (Cas12) (Koonin et al. 2017 Curr Opinion Microbiology 37:67-78), which is an active RNA-guided endonuclease that does not necessarily require additional trans-activating CRISPR (tracr) RNA for target cleavage, unlike Cas9.

[0164] Type VI CRISPR-Cas systems contain cas13 genes encoding nucleases with two HEPN (higher eukaryotic and prokaryotic nucleotide binding) domains but without HNH or RuvC domains, and are independent of tracrRNA activity. Most HEPN domains contain conserved motifs that constitute metal-independent endonuclease active sites (Anantharam et al. 2013 Biol Direct [Biology Guide] 8:15). Due to this feature, type VI systems are believed to act on RNA targets rather than DNA targets common to other CRISPR-Cas systems.

[0165] Novel Cas endonuclease CRISPR-Cas system

[0166] Disclosed herein are novel CRISPR-Cas systems, components thereof, and methods of using the components. The system comprises novel Cas effector proteins, Cas endonucleases.

[0167] The novel CRISPR-Cas system components described herein may comprise one or more subunits from different Cas systems, subunits derived or modified from more than one different bacterial or archaeal prokaryotes, and / or synthetic or engineered components.

[0168] Described herein is a newly identified CRISPR-Cas system comprising a novel arrangement of cas genes. Further described are novel cas genes and proteins.

[0169] One feature of the novel Cas endonuclease system is the locus structure, which comprises an endonuclease gene upstream of the CRISPR array, but does not comprise a cas1 gene, a cas2 gene, or a cas4 gene.

[0170] Components of the CRISPR-Cas system

[0171] Cas proteins

[0172] Many proteins can be encoded in a CRISPR cas operon, including those involved in adaptation (spacer insertion), interference (effector module target binding, target nicking or cleavage - e.g., endonuclease activity), expression (pre-crRNA processing), regulation, or others.

[0173] The two proteins, Cas1 and Cas2, are conserved in many CRISPR systems (e.g., as described in Koonin et al. 2017, Curr Opinion Microbiology 37:67-78). Cas1 is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments. In some systems, Cas1 forms a stable complex with Cas2, which is essential for spacer acquisition and insertion in CRISPR systems ( et al. 2014, Nature Str Mol Biol [Nature Structural Molecular Biology] 21: 528-534).

[0174] Many other proteins have been identified in different systems, including Cas4, which may have similarities to the RecB nuclease and is thought to play a role in capturing new viral DNA sequences for incorporation into the CRISPR array (Zhang et al. 2012, PLOS One 7(10):e47232).

[0175] Some proteins may have multiple functions. For example, Cas9, the signature protein of the class II system, has been shown to be involved in pre-crRNA processing, target binding, and target cleavage.

[0176] In some natural systems, such as the Cas endonuclease CRISPR system from Synechocystis palmi, no genes encoding Cas1, Cas2, or Cas4 proteins were detected near the endonuclease gene.

[0177] Cas endonucleases and effectors

[0178] Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains and include restriction endonucleases that cleave DNA at specific sites without damaging bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas ( C RISPR- as societal) effector endonucleases.

[0179] Cas endonucleases (as single effector proteins or effector complexes with other components) unwind DNA duplexes at target sequences and optionally cut at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, crRNA or guide RNA) complexed with the Cas effector protein. If the correct protoplast adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence, such recognition and cutting of the target sequence by the Cas endonuclease typically occurs. Alternatively, the Cas endonucleases herein may lack DNA cutting or nicking activity, but when complexed with a suitable RNA component, can still specifically bind to the DNA target sequence. (See also U.S. Patent Application US20150082478, published on March 19, 2015, and US20150059010, published on February 26, 2015).

[0180] Cas endonucleases can occur as single effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).

[0181] Cas endonucleases that have been described include, but are not limited to, for example: Cas3 (characteristic of 1 class, type I systems), Cas9 (characteristic of 2 class, type II systems), and Cas12 (Cpf1) (characteristic of 2 class, type V systems).

[0182] Cas3 (and its variants Cas3' and Cas3") function as a single-stranded DNA nuclease (HD domain) and an ATP-dependent helicase. Variants of Cas3 endonucleases can be obtained by disabling the functional activity of one or both domains of the Cas3 endonuclease polypeptide. Disabling the ATPase-dependent helicase activity (by deleting, knocking out the Cas3-helicase domain, or by mutating key residues, or by assembling the reaction in the absence of ATP, as previously described (Sinkunas, T. et al., 2013, EMBO J. [Journal of the European Molecular Biology Association] 32:385-394)) can convert a cleavage-ready Cascade comprising a modified Cas3 endonuclease into a nickase (because the HD domain is still functional). Disabling the HD endonuclease activity can be accomplished by any method known in the art, such as, but not limited to, mutation of key residues of the HD domain, which can convert a cleavage-ready Cascade comprising a modified Cas3 endonuclease into a helicase. Disabling the Cas helicase and Cas3 Disabling the HD endonuclease activity can be accomplished by any method known in the art, such as, but not limited to, mutation of key residues in both the helicase and HD domains, which can convert a cleavage-ready Cascade comprising a modified Cas3 endonuclease into a binding protein that binds to the target sequence.

[0183] Cas9 (formerly known as Cas5, Csn1, or Csx12) is a Cas endonuclease that forms a complex with cr nucleotides and tracr nucleotides or with a single guide polynucleotide, which is used to specifically recognize and cut all or part of a DNA target sequence. Cas9 recognizes 3'GC-rich PAM sequences on target dsDNA. The Cas9 protein includes a RuvC nuclease and an HNH (HNH) nuclease adjacent to the RuvC-II domain. RuvC nuclease and HNH nuclease can each cut a single DNA chain at the target sequence (the synergistic action of the two domains leads to DNA double-stranded cutting, and the activity of one domain leads to a nick). Typically, the RuvC domain includes subdomains I, II, and III, wherein subdomain I is located near the N-terminus of Cas9, and subdomains II and III are located in the middle of the protein, i.e., on the flanks of the HNH domain (Hsu et al. 2013, Cell [Cell] 157: 1262-1278). Cas9 endonucleases are typically derived from type II CRISPR systems, which include DNA cleavage systems utilizing Cas9 endonucleases compounded with at least one polynucleotide component. For example, Cas9 can be compounded with CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In another example, Cas9 can be compounded with a single guide RNA (Makarova et al., 2015, Nature Reviews Microbiology [Natural Review Microbiology] Vol. 13: 1-15).

[0184] Cas12 (formerly known as Cpf1, and variants c2c1, c2c3, CasX and CasY) contains a RuvC nuclease domain and produces staggered 5' overhangs on dsDNA targets. Unlike the function of Cas9, some variants do not require tracrRNA. Cas12 and its variants recognize 5'AT-rich PAM sequences on target dsDNA. An insertion domain of Cas12a protein called Nuc has been shown to be responsible for the cutting of the target chain (Yamano et al., Cell [Cell] 2016, 165: 949-962). Other mutation studies in other Cas12 proteins show that the Nuc domain helps guide the binding of the object and the target, while the RuvC domain is responsible for cutting (Swarts et al., Mol Cell [Molecular Cell] 2017, 66: 221-233e224).

[0185] Cas endonucleases and effector proteins can be used for targeted genome editing (via single and multiple double-strand breaks and nicks) and targeted genome regulation (via tethering epigenetic effector domains to Cas proteins or sgRNAs). Cas endonucleases can also be engineered to function as RNA-guided recombinases and can serve as scaffolds for assembling polyprotein and nucleic acid complexes via RNA tethers (Mali et al. 2013, Nature Methods [Natural Methods] Vol. 10: 957-963).

[0186] Cas endonucleases and their variants

[0187] A Cas endonuclease or a functional variant thereof is defined as a functional RNA-guided, PAM-dependent dsDNA cleavage protein having less than 500 amino acids, comprising: a C-terminal RuvC catalytic domain (split into three subdomains) and further comprising a bridge-helix and one or more zinc finger motifs; and an N-terminal Rec subunit having a helical bundle, a WED wedge-like (or "oligonucleotide binding domain", OBD) domain, and optionally a zinc finger motif.

[0188] The novel Cas endonuclease variant proteins disclosed herein include effector proteins (endonucleases). The wild-type (WT) Cas endonuclease protein requires a PAM sequence of N(T>W>C)TTC at or near the target site of the target double-stranded polydeoxyribonucleotide.

[0189] Certain functional variants of the Cas endonucleases disclosed herein can have double-strand break or single-strand nick activity that can be lower than, about the same as, or even higher than the activity of the WT Cas endonuclease. Different levels of activity can have different uses depending on the practitioner's expectations. When aligned with SEQ ID NO: 14 or SEQ ID NO: 48, the Cas endonuclease or functional variant comprises (i) glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamine at position 85, and (II) the following amino acid motifs: GxxxG starting at amino acid position 226; ExL starting at amino acid position 327, Cx starting at amino acid position 376. n C; Cx starting at amino acid position 395 n(C, H); wherein X is any nucleotide and n is any number. In some aspects, the functional variant is a variant without: phenylalanine at position 38, alanine (A) at position 40, histidine at position 79, glutamic acid at relative position 81, alanine at relative position 87, threonine at relative position 335, cysteine ​​at position 409, glutamic acid at position 421, lysine at position 467, or glutamic acid at position 468, wherein each of these refers to a position relative to SEQ ID NO: 14 or 48 in an alignment with SEQ ID NO: 14 or 48. In some aspects, when aligned with SEQ ID NO: 14 or SEQ ID NO: 48, relative to SEQ ID NO: 14 or SEQ ID NO: 48. When the amino acid positions of NO:48 are numbered, a functional variant is a variant having aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at 226, glycine at 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at 376, cysteine ​​at 379, cysteine ​​at 395, cysteine ​​at 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

[0190] A "functional fragment" of a Cas endonuclease variant endonuclease refers to a polynucleotide of less than 497 amino acids that has at least 50%, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, between 400 and 450, at least 450 or more consecutive amino acids of any one of SEQ ID NOs: 15-47. 99%, at least 99%, 99% and 100%, or 100% sequence identity; and comprises the ability to recognize, or bind to, or nick a single strand of a double stranded polynucleotide, or cleave both strands of a double stranded polynucleotide, or any combination of the foregoing.

[0191] It has been documented that the RuvC domain comprises an endonuclease function. Cas endonucleases can be isolated or identified from a locus comprising a Cas endonuclease gene encoding an effector protein and an array comprising multiple repeat sequences.

[0192] Zinc finger motifs are domains that coordinate one or more zinc ions, usually through cysteine ​​and histidine side chains, to stabilize their folding. Zinc fingers are named after the pattern of cysteine ​​and histidine residues that coordinate the zinc ion (e.g., C4 indicates that the zinc ion is coordinated with four cysteine ​​residues; C3H indicates that the zinc ion is coordinated with three cysteine ​​residues and one histidine residue).

[0193] Cas endonucleases contain one or more zinc finger (ZFN) coordination motifs that can form zinc binding domains. Zinc finger-like motifs can help separate target and non-target strands and load guide RNA into DNA targets. Cas endonucleases containing one or more zinc finger motifs can provide additional stability for the ribonucleoprotein complex on the target polynucleotide. Cas endonucleases contain C4 or C3H zinc binding domains.

[0194] As used herein, "domain" is synonymous with "motif". For example, zinc finger domain and zinc finger motif are used synonymously. Similarly, zinc binding domain and zinc binding motif are used synonymously.

[0195] Cas endonucleases are RNA-guided endonucleases that are capable of binding and cleaving a double-stranded DNA target comprising: (1) a sequence with homology to the nucleotide sequence of the guide RNA, and (2) a PAM sequence.

[0196] Cas endonucleases have the function of double-strand break inducers, and may also be nickases or single-strand break inducers. In some aspects, catalytically inactivated Cas endonucleases can be used for targeting or recruitment to target DNA sequences but do not induce cutting. In some aspects, catalytically inactivated Cas endonucleases proteins can be used together with functional endonucleases to cut target sequences. In some aspects, catalytically inactivated Cas endonucleases proteins can be combined with base editing molecules such as deaminases. In some aspects, the deaminase can be a cytidine deaminase. In some aspects, the deaminase can be an adenine deaminase. In some aspects, the deaminase can be ADAR-2.

[0197] The Cas endonuclease is further defined as an RNA-guided double-stranded DNA cleavage protein having at least 50%, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, between 400 and 450, at least 450, or more than 450 contiguous amino acids of any one of SEQ ID NOs: 15-47. %, at least 97%, 97% and 98%, at least 98%, 98% and 99%, at least 99%, 99% and 100%, or 100% sequence identity and retains at least partial endonuclease activity.

[0198] The disclosed engineered Cas endonucleases or inactivated engineered Cas polypeptides may be encoded by a polynucleotide that is at least 50, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, between 400 and 500, or more. at least 400, between 400 and 450, at least 500, between 500 and 550, at least 600, between 600 and 650, at least 650, between 650 and 700, at least 700, between 700 and 750, at least 750, between 750 and 800, at least 800, between 800 and 850, at least 850, between 850 and 900, %, at least 70%, 70% to 75%, at least 75% to 80%, at least 80 ... , between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, between 99% and 100%, or 100% sequence identity.

[0199] The Cas endonucleases, effector proteins or functional fragments thereof used in the disclosed methods can be isolated from natural sources or recombinant sources, in which genetically modified host cells are modified to express nucleic acid sequences encoding proteins. Alternatively, a cell-free protein expression system can be used to produce Cas proteins or to produce Cas proteins synthetically. The effector Cas nuclease can be isolated and introduced into heterologous cells, or it can be modified from its native form to exhibit an activity type or activity size different from its natural source. Such modifications include, but are not limited to, fragments, variants, substitutions, deletions and insertions. The Cas endonuclease WT composition is described in WO 2020123887 published on July 16, 2020.

[0200] Fragments and variants of Cas endonucleases and Cas endonuclease effector proteins can be obtained via methods such as site-directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art, such as, but not limited to, WO 2013166113 published on November 7, 2013, WO 2016186953 published on November 24, 2016, and WO 2016186946 published on November 24, 2016.

[0201] Cas endonucleases may include modified forms of Cas polypeptides. Modified forms of Cas polypeptides may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the naturally occurring nuclease activity of Cas proteins. For example, in some cases, a modified form of a Cas protein has a nuclease activity of less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the corresponding wild-type Cas polypeptide (SEQ ID NO: 48; see also US20140068797 published on March 6, 2014). In some cases, the modified form of the Cas polypeptide has no substantial nuclease activity and is referred to as catalytically "inactivated Cas" or "inactivated Cas (dCas)". Inactivated Cas / inactivated Cas includes inactivated Cas endonucleases (dCas). Catalytically inactivated Cas effector proteins may be fused to heterologous sequences to induce or modify activity.

[0202] The Cas endonuclease can be part of a fusion protein that also comprises one or more heterologous protein domains (e.g., 1, 2, 3 or more domains other than the Cas protein). Such a fusion protein can comprise any additional protein sequence, and optionally a linker sequence between any two domains (e.g., between the Cas and the first heterologous domain). Examples of protein domains that can be fused to the Cas proteins herein include, but are not limited to, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]); reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], β-galactosidase, β-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]); and domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity (e.g., VP16 or VP64), transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also be fused to proteins that bind to DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

[0203] The catalytically active and / or catalytically inactivated Cas endonucleases can be fused to heterologous sequences (US20140068797 disclosed on March 6, 2014). Suitable fusion partners include, but are not limited to providing active polypeptides, which increase transcription indirectly by acting directly on the target DNA or on a polypeptide (e.g., histone or other DNA-binding proteins) related to the target DNA. Other suitable fusion partners include, but are not limited to providing methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinase activity, adenylation activity, deadenylation activity, SUMOylation (SUMOylating) activity, deSUMOylation (deSUMOylating) activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity polypeptide. Further suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription of the target nucleic acid (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecule / drug-responsive transcriptional regulators, etc.). Partially active or catalytically inactive Cas endonucleases can also be fused to another protein or domain (e.g., Clo51 or FokI nucleases) to generate double-strand breaks (Guilinger et al. Nature Biotechnology, Vol. 32, No. 6, June 2014).

[0204] A catalytically active or catalytically inactive Cas protein, such as a Cas endonuclease protein described herein, can also be fused to a molecule that directs the editing of single or multiple bases in a polynucleotide sequence, such as a site-specific deaminase that can change nucleotide identity, such as from C·G to T·A or from A·T to G·C (Gaudelli et al. 2017, "Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage." Nature 551(7681):464-471; Nishida et al. 2016, "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353(6305):aaf8729; Komor et al., "Programmable editing of a target base in genomic DNA without DNA cleavage." Nature 551(7681):464-471; Nishida et al. 2016, "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems." Science 353(6305):aaf8729; Komor et al., "Programmable editing of a target base in genomic DNA without DNA cleavage." Double-stranded DNA cleavage. [Programmable editing of target bases in genomic DNA without double-stranded DNA cleavage]" Nature [Nature] 533(7603) (2016): 420-4). The base editing fusion protein can include, for example, active (double-strand break generation), partially active (nickase) or inactivated (catalytically inactive) Cas endonucleases and deaminases (such as but not limited to cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). In some embodiments, base editing repair inhibitors and glycosylase inhibitors (e.g., uracil glycosylase inhibitors (to prevent uracil removal)) are considered as other components of the base editing system.

[0205] The Cas endonucleases described herein can be expressed and purified by methods known in the art, for example as described in WO / 2016 / 186953 published on November 24, 2016.

[0206] To date, many Cas endonucleases have been described that can recognize specific PAM sequences (WO 2016186953 published on November 24, 2016, WO 2016186946 published on November 24, 2016, and Zetsche B et al. 2015. Cell [Cell] 163, 1013) and cut target DNA at a specific position. It should be understood that based on the methods and embodiments described herein using the novel guided Cas system, those skilled in the art can now customize these methods so that they can utilize any guided endonuclease system.

[0207] Cas effector protein may include a heterologous nuclear localization sequence (NLS). For example, the heterologous NLS amino acid sequence herein may have sufficient strength to drive the accumulation of detectable amounts of Cas proteins in the nucleus of yeast cells herein. NLS may include one (monotype) or multiple (e.g., ditype) short sequences (e.g., 2 to 20 residues) of alkaline, positively charged residues (e.g., lysine and / or arginine), and may be located anywhere in the Cas amino acid sequence, but such that it is exposed on the protein surface. For example, NLS may be operably connected to the N-terminus or C-terminus of the Cas protein herein. Two or more NLS sequences may be connected to the Cas protein, such as at both the N-terminus and the C-terminus of the Cas protein. The Cas endonuclease gene may be operably connected to the SV40 nuclear targeting signal upstream of the Cas codon region and the ditype VirD2 nuclear localization signal downstream of the Cas codon region (Tinland et al., (1992) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 89: 7442-6). Non-limiting examples of suitable NLS sequences herein include those disclosed in US Pat. Nos. 6,660,830 and 7,309,576.

[0208] Guide polynucleotide

[0209] Instruction polynucleotide enables Cas endonuclease to target recognition, binding and optionally cutting, and can be unimolecular or bimolecular.Instruction polynucleotide sequence can be RNA sequence, DNA sequence or its combination (RNA-DNA combination sequence). Optionally, instructing polynucleotide can include at least one nucleotide, phosphodiester bond or connection modification, such as but not limited to locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, thiophosphate bond, connection with cholesterol molecule, connection with polyethylene glycol molecule, connection with spacer 18 (hexaethylene glycol chain) molecule or cause cyclization 5' to 3' covalent connection. Instruction polynucleotides comprising only ribonucleic acid are also referred to as "guidance RNA" or "gRNA" (US20150082478 published on March 19, 2015 and US20150059010 published on February 26, 2015). Instruction polynucleotide can be engineered or synthesized.

[0210] The guide polynucleotide includes a chimeric non-naturally occurring guide RNA that includes regions that are not found together in nature (i.e., they are heterologous to each other). For example, the chimeric non-naturally occurring guide RNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA, and the first nucleotide sequence domain is connected to a second nucleotide sequence that can recognize a Cas endonuclease, such that the first and second nucleotide sequences are not found connected together in nature.

[0211] The guiding polynucleotide can be a double molecule (also referred to as a duplex guiding polynucleotide) comprising a cr nucleotide sequence (e.g., crRNA) and a tracr nucleotide (e.g., tracrRNA) sequence. In some cases, there is a linker polynucleotide connecting crRNA and tracrRNA to form a single guiding object (e.g., sgRNA).

[0212] The tracr nucleotide includes a first nucleotide sequence domain (called the variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in the target DNA and a second nucleotide sequence (also called the tracr mate sequence) that is part of the Cas endonuclease recognition (CER) domain. The tracr mate sequence can hybridize with the tracr nucleotide along the complementary region and together form CAs endonuclease recognition domain or CER domain. The CER domain can interact with the Cas endonuclease polypeptide. The cr nucleotides and tracr nucleotides of the duplex guidance polynucleotide can be RNA, DNA and / or RNA-DNA combination sequences. In some embodiments, the cr nucleotide molecules of the duplex guidance polynucleotide are referred to as "crDNA" (when composed of continuous extensions of DNA nucleotides) or "crRNA" (when composed of continuous extensions of RNA nucleotides) or "crDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). The cr nucleotides may be included in fragments of crRNA naturally occurring in bacteria and archaea. The size of the crRNA fragments naturally occurring in bacteria and archaea that may be present in the cr nucleotides disclosed herein may be, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides.

[0213] In some embodiments, the tracr nucleotide is referred to as "tracrRNA" (when composed of a continuous extension of RNA nucleotides) or "tracrDNA" (when composed of a continuous extension of DNA nucleotides) or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In one embodiment, the RNA of the guide RNA / Cas9 endonuclease complex is a duplexed RNA comprising a duplex crRNA-tracrRNA. In the 5'- to -3' direction, the tracrRNA (trans-activating CRISPR RNA) comprises (i) a sequence annealed to the repeat region of the CRISPR II type crRNA and (ii) a portion containing a stem loop (Deltcheva et al. Nature [Nature] 471: 602-607). The duplex guide polynucleotide can form a complex with the Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind to the target, and optionally nick or cut (introduce single-stranded or double-stranded breaks) the target site. (US 20150082478 published on March 19, 2015 and US 20150059010 published on February 26, 2015).

[0214] In one aspect, the guide polynucleotide is a guide polynucleotide capable of forming a PGEN as described herein, wherein the guide polynucleotide comprises a first nucleotide sequence domain complementary to a nucleotide sequence in a target DNA and a second nucleotide sequence domain that interacts with a Cas polypeptide disclosed herein.

[0215] In one aspect, the guide polynucleotide is a guide polynucleotide described herein, wherein the first nucleotide sequence domain and the second nucleotide sequence domain are selected from the group consisting of a DNA sequence, an RNA sequence, and a combination thereof.

[0216] In one aspect, the guide polynucleotide is a guide polynucleotide as described herein, wherein the first nucleotide sequence domain and the second nucleotide sequence domain are selected from the group consisting of: RNA backbone modifications that enhance stability, DNA backbone modifications that enhance stability, and combinations thereof (see Kanasty et al. 2013, Common RNA-backbone modifications, Nature Materials 12:976-977; US20150082478 published on March 19, 2015 and US 20150059010 published on February 26, 2015)

[0217] The guide RNA includes a bimolecule comprising a chimeric non-naturally occurring crRNA connected to at least one tracrRNA. The chimeric non-naturally occurring crRNA includes a crRNA that is contained in a region that is not found together in nature (i.e., they are heterologous to each other). For example, the crRNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize with a nucleotide sequence in a target DNA, and the first nucleotide sequence domain is connected to a second nucleotide sequence (also referred to as a tracr pairing sequence) so that the first and second sequences are not found to be connected together in nature.

[0218] The guide polynucleotide can also be a single molecule comprising a cr nucleotide sequence linked to a tracr nucleotide sequence (also referred to as a single guide polynucleotide). The single guide polynucleotide comprises a first nucleotide sequence domain (referred to as a variable targeting ( V ariable T argeting) domain or VT domain) and interacting with the Cas endonuclease polypeptide C as endonuclease recognition domain (CER domain).

[0219] The VT domain and / or CER domain of a single guide polynucleotide may comprise an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. A single guide polynucleotide consisting of a sequence from a cr nucleotide and a tracr nucleotide may be referred to as a "single guide RNA" (when consisting of a continuous extension of RNA nucleotides) or a "single guide DNA" (when consisting of a continuous extension of DNA nucleotides) or a "single guide RNA-DNA" (when consisting of a combination of RNA and DNA nucleotides). A single guide polynucleotide may form a complex with a Cas endonuclease, wherein the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) may guide the Cas endonuclease to a genomic target site, enabling the Cas endonuclease to recognize, bind, and optionally nick or cut (introduce single-strand or double-strand breaks) the target site. (US 20150082478, published on March 19, 2015, and US20150059010, published on February 26, 2015).

[0220] Chimeric non-naturally occurring single guide RNAs (sgRNAs) include sgRNAs that include regions that are not found together in nature (i.e., they are heterologous to each other). For example, an sgRNA includes a first nucleotide sequence domain (referred to as a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA, linked to a second nucleotide sequence (also referred to as a tracr mate sequence) that is not found linked together in nature.

[0221] The nucleotide sequence connecting the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or an RNA-DNA combination sequence. In an embodiment, the nucleotide sequence connecting the cr nucleotides and the tracr nucleotides of the single guide polynucleotide (also referred to as a "loop") can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 98, 99, or 100 nucleotides in length. In another embodiment, the nucleotide sequence linking the cr nucleotides and the tracr nucleotides of the single guide polynucleotide can include a tetraloop sequence, such as, but not limited to, a GAAA tetraloop sequence.

[0222] The guide polynucleotides can be produced by any method known in the art, including chemically synthesized guide polynucleotides (such as but not limited to Hendel et al. 2015, Nature Biotechnology 33, 985-989), in vitro generated guide polynucleotides, and / or self-splicing guide RNAs (such as but not limited to Xie et al. 2015, PNAS 112: 3570-3575).

[0223] The guide polypeptide can be used in combination with any engineered Cas polypeptide disclosed herein, including any of the engineered Cas polypeptides or endonucleases disclosed in paragraphs

[0007] to

[0027] of the Summary of the Invention herein. See also the discussion of guide polynucleotide / Cas endonuclease complexes herein.

[0224] Protospacer adjacent motif (PAM)

[0225] "Pre-spacer adjacent motif" (PAM) herein refers to a short nucleotide sequence adjacent to a (targeted) target sequence (pre-spacer) that can be recognized by a guide polynucleotide / Cas endonuclease system. If the target DNA sequence is not followed by a PAM sequence, the Cas endonuclease may not be able to successfully recognize the target DNA sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence can be any length, but typically is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0226] "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 typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides in length. The randomized nucleotides include any of the nucleotides A, C, G or T.

[0227] Guide polynucleotide / Cas endonuclease complex

[0228] The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding, and optionally nicking, unwinding, or cleaving all or part of a target sequence.

[0229] The guiding polynucleotide / Cas endonuclease complex that can cut both chains of the DNA target sequence typically comprises a Cas protein with all its endonuclease domains in a functional state (e.g., a wild-type endonuclease domain or a variant thereof that retains some or all activity in each endonuclease domain). Therefore, a wild-type Cas protein (e.g., a Cas protein disclosed herein) or a variant thereof that retains some or all activity in each endonuclease domain of the Cas protein is a suitable example of a Cas endonuclease that can cut both chains of the DNA target sequence.

[0230] The guiding polynucleotide / Cas endonuclease complex that can cut one strand of the DNA target sequence can be characterized herein as having nickase activity (e.g., partial cleavage ability). The Cas nickase typically comprises a functional endonuclease domain that allows Cas to cut only one strand of the DNA target sequence (i.e., form a nick). For example, the Cas9 nickase can comprise (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, the Cas9 nickase can comprise (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 US20140189896, published on July 3, 2014. A pair of Cas nickases can be used to increase the specificity of DNA targeting. In general, this can be performed by providing two Cas nickases that target and nick near a DNA sequence in a desired targeted region on opposite strands by associating with RNA components having different guide sequences. Such near cleavage of each DNA strand generates a double-strand break (i.e., a DSB with a single-stranded overhang), which is then recognized as a substrate for non-homologous end joining (NHEJ) (which tends to produce incomplete repairs that lead to mutations) or homologous recombination (HR). Each nick in these embodiments can be separated from each other by, for example, at least about 5, 5 to 10, at least 10, 10 to 15, at least 15, 15 to 20, at least 20, 20 to 30, at least 30, 30 to 40, at least 40, 40 to 50, at least 50, 50 to 60, at least 60, 60 to 70, at least 70, 70 to 80, at least 80, 80 to 90, at least 90, 90 to 100, or 100 or more (or any integer between 5 and 100) bases. One or two Cas nickase proteins herein can be used for Cas nickase pairs. For example, a Cas9 nickase with a mutated RuvC domain but with a functional HNH domain (i.e., Cas9HNH+ / RuvC-) (e.g., Streptococcus pyogenes Cas9 HNH+ / RuvC-) can be used. By using the appropriate RNA components herein (which have guide RNA sequences that target each nickase to each specific DNA site), each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) is guided to specific DNA sites that are adjacent to each other (separated by up to 100 base pairs).

[0231] In certain embodiments, the polynucleotide / Cas endonuclease complex can bind to the DNA target site sequence, but does not cut any chain at the target site sequence. Such a complex can include a Cas protein in which all nuclease domains are mutated and dysfunctional. For example, a Cas9 protein that can bind to a DNA target site sequence but does not cut any chain at the target site sequence can include a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. The Cas protein herein that binds but does not cut a target DNA sequence can be used to regulate gene expression, for example, in this case, the Cas protein can be fused to a transcription factor (or part thereof) (e.g., an inhibitor or activator, such as any of those disclosed herein).

[0232] In one aspect, the guide polynucleotide / Cas endonuclease complex (PGEN) described herein is a PGEN, wherein the Cas endonuclease is optionally covalently or non-covalently linked or assembled to at least one protein subunit or a functional fragment thereof.

[0233] In one embodiment of the present disclosure, the guide polynucleotide / Cas endonuclease complex is a guide polynucleotide / Cas endonuclease complex (PGEN) comprising at least one guide polynucleotide and at least one Cas endonuclease polypeptide, wherein the Cas endonuclease polypeptide comprises at least one protein subunit or a functional fragment thereof, wherein the guide polynucleotide is a chimeric non-naturally occurring guide polynucleotide, wherein the guide polynucleotide / Cas endonuclease complex is capable of recognizing, binding, and optionally nicking, unwinding or cutting all or part of a target sequence.

[0234] The Cas effector protein may be a Cas endonuclease effector protein as disclosed herein.

[0235] 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 endonuclease effector protein, wherein the guide polynucleotide / Cas effector protein complex is capable of recognizing, binding, and optionally nicking, unwinding or cutting all or part of a target sequence.

[0236] The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein is a Cas polypeptide disclosed herein, further comprising one or more copies of at least one protein subunit or a functional fragment thereof. In some embodiments, the protein subunit is selected from the group consisting of Cas1 protein subunits, Cas2 protein subunits, Cas4 protein subunits, and any combination thereof. The PGEN may 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.

[0237] The PGEN may be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises at least three different protein subunits or functional fragments thereof selected from the group consisting of Cas1, Cas2 and optionally an additional Cas protein comprising Cas4.

[0238] In one aspect, the guide polynucleotide / Cas effector protein complex (PGEN) described herein is a PGEN, wherein the Cas effector protein is a Cas polypeptide disclosed herein, which is covalently or non-covalently linked to at least one protein subunit or a functional fragment thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein polypeptide is covalently or non-covalently linked or assembled to one or more copies of at least one protein subunit or a functional fragment thereof, the at least one protein subunit being selected from the group consisting of: Cas1 protein subunit, Cas2 protein subunit, optionally comprising a Cas4 protein subunit, and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein is covalently or non-covalently linked or assembled to at least two different protein subunits, the at least two different protein subunits being selected from the group consisting of: Cas1, Cas2, and optionally comprising a Cas4. A further Cas protein. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein is covalently or non-covalently linked to at least three different protein subunits or functional fragments thereof, the at least three different protein subunits being selected from the group consisting of Cas1, Cas2, and optionally an additional Cas protein comprising Cas4, and any combination thereof.

[0239] Any components of the guide polynucleotide / Cas effector protein complex, the guide polynucleotide / Cas effector protein complex itself, as well as one or more polynucleotide modification templates and / or one or more donor DNAs can be introduced into a heterologous cell or organism by any method known in the art.

[0240] Recombinant constructs for cell transformation

[0241] 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. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein.

[0242] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are more fully described in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, New York (1989). Transformation methods are well known to those skilled in the art and are described below.

[0243] Vectors and constructs include circular plasmids and linear polynucleotides comprising a polynucleotide of interest and optionally other components, including linkers, adapters, regulatory components, or analytical components. In some examples, the recognition site and / or target site may be contained within an intron, coding sequence, 5'UTR, 3'UTR, and / or regulatory region.

[0244] Components for expressing and utilizing novel CRISPR-Cas systems in prokaryotic and eukaryotic cells

[0245] The present invention also provides expression constructs for expressing a guide RNA / Cas system in a prokaryotic or eukaryotic cell / organism that is capable of recognizing, binding, and optionally nicking, unwinding or cleaving all or part of a target sequence.

[0246] In one embodiment, the expression construct of the present disclosure comprises a promoter operably connected to a nucleotide sequence encoding a Cas gene (or plant-optimized, including a Cas endonuclease gene as described herein) and a promoter operably connected to a guide RNA of the present disclosure. The promoter is capable of driving expression of an operably connected nucleotide sequence in a prokaryotic or eukaryotic cell / organism.

[0247] The nucleotide sequence modifications of the guide polynucleotide, VT domain and / or CER domain may be selected from, but not limited to, the group consisting of: a 5' cap, a 3' poly-A tail, a riboswitch sequence, a stability control sequence, a sequence forming a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a protein binding site, a locked nucleic acid (LNA), a 5-methyl dC nucleotide, a 2,6-diaminopurine nucleotide, a 2'-fluoro A nucleotide, a 2'-fluoro U nucleotide; a 2'-O-methyl RNA nucleotide, a phosphorothioate bond, a connection to a cholesterol molecule, a connection to a polyethylene glycol molecule, a connection to a spacer 18 molecule, a 5' to 3' covalent connection, or any combination thereof. These modifications may produce at least one additional beneficial feature, wherein the additional beneficial feature is selected from the group consisting of: modified or modulated stability, subcellular targeting, tracking, fluorescent labeling, a binding site for a protein or protein complex, modified binding affinity to a complementary target sequence, modified resistance to cellular degradation, and increased cellular permeability.

[0248] Methods for expressing RNA components (e.g., gRNA) in eukaryotic cells for Cas9-mediated DNA targeting have used RNA polymerase III (Pol III) promoters, which allow RNA transcription with precisely defined unmodified 5'- and 3'-ends (DiCarlo et al. 2013, Nucleic Acids Res. 41(7):4336-4343; Ma et al. 2014, Mol. Ther. Nucleic Acids 3:el61). This strategy has been successfully applied in cells of several different species, including maize and soybean (US20150082478 published on March 19, 2015). Methods for expressing RNA components that do not have a 5' cap have been described (WO 2016 / 025131 published on February 18, 2016).

[0249] Different methods and compositions can be used to obtain a cell or organism having a target polynucleotide inserted into the target site of a Cas endonuclease. Such a method can use homologous recombination (HR) to provide integration of the target polynucleotide at the target site. In a method described herein, the target polynucleotide is introduced into an organism cell via a donor DNA construct.

[0250] The donor DNA construct further comprises a first homology region and a second homology region flanking the target polynucleotide. The first homology region and the second homology region of the donor DNA have homology with the first and second genomic regions present in the target site of the cell or organism genome or flanking the target site, respectively.

[0251] Donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, can be used for genome editing, gene insertion and targeted genome regulation, and can also be used to target late mitotic cells, in which the function of endogenous HR machinery is expected to be greatly reduced (Mali et al. 2013, Nature Methods [Natural Methods] Vol. 10: 957-963).

[0252] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or having a length between about 1 bp-20 bp, 20 bp-50 bp, 50 bp-100 bp, 75 bp-150 bp, 100 bp-250 bp, 150 bp-300 bp, 200 bp-400 bp, 250 bp-500 bp, 300 bp-600 bp, 350 bp-750 bp, 400 bp-800 bp, 450 bp- The target site can be a target site having a length of 1-20 bp, 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, and 20 bp. The amount of homology can also be described by the percent sequence identity over the entire compared length of the two polynucleotides, including at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98% to 99%, 99%, 99% to 100% or 100% percent sequence identity. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of contiguous nucleotides or local percent sequence identity, for example, sufficient homology can be described as a region of 75 bp-150 bp having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be described by the predicted ability of two polynucleotides to hybridize specifically under high stringency conditions, see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, New York); 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).

[0253] 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 possessed by a "homologous region" of the donor DNA and a "genomic region" of the genome of the organism 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, allowing the sequences to undergo homologous recombination.

[0254] The homology region on the donor DNA can have homology with any sequence flanking the target site. Although in some cases, the homology region has significant sequence homology with the genomic sequence immediately flanking the target site, it should be recognized that the homology region can be designed to have sufficient homology with regions that may be further 5' or 3' of the target site. The homology region can also have homology with fragments of the target site and downstream genomic regions.

[0255] In one embodiment, the first region of homology further comprises a first segment in the target site, and the second region of homology comprises a second segment in the target site, wherein the first segment and the second segment are different.

[0256] Target polynucleotide

[0257] Target polynucleotides are further described herein, and include polynucleotides that reflect those commercial markets and commercial interests related to crop development. Target crops and markets change, and as developing countries open international markets, new crops and technologies will also appear. In addition, as our understanding of agronomic traits and characteristics (such as increase in yield and heterosis) deepens gradually, the selection of genes for genetic engineering will change accordingly.

[0258] General categories of polynucleotides of interest include, for example, those genes of interest that are involved in information (e.g., zinc fingers), those genes that are involved in communication (e.g., kinases), and those genes that are involved in housekeeping (e.g., heat shock proteins). More specific polynucleotides of interest include, but are not limited to, genes that are involved in traits of agronomic interest (e.g., but not limited to: crop yield, grain quality, crop nutrient content, starch and carbohydrate quality and quantity), as well as genes that affect grain size, sucrose loading, protein amount and quantity, nitrogen fixation and / or nitrogen utilization, fatty acid and oil composition, genes encoding proteins that confer resistance to abiotic stresses (e.g., drought, nitrogen, temperature, salinity, toxic metals, or trace elements), or those that confer resistance to toxins (e.g., pesticides and herbicides), genes encoding proteins that confer resistance to biotic stresses (e.g., fungal, viral, bacterial, insect and nematode attack and the development of diseases associated with these organisms).

[0259] In addition to using traditional breeding methods, agronomically important traits (e.g., oil, starch, and protein content) can also be altered genetically. Modifications include increasing the content of oleic acid, saturated and unsaturated oils, increasing the levels of lysine and sulfur, providing essential amino acids, and also modifications to starch. Protein modifications of hordothionin are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0260] The polynucleotide sequence of interest can encode proteins that are involved in providing disease or pest resistance. "Disease resistance" or "pest resistance" is intended to be a plant that avoids harmful symptoms as a consequence of plant-pathogen interactions. Pest resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borers, etc. Disease resistance genes and insect resistance genes, such as lysozymes or cecropins for antibacterial protection, or proteins for antifungal protection, such as defensins, glucanases, or chitinases, or Bacillus thuringiensis endotoxins, protease inhibitors, collagenases, lectins, or glycosidases for controlling nematodes or insects, are examples of useful gene products. Genes encoding disease resistance traits include detoxification genes, such as for fumonisins (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089); etc. Insect resistance genes can encode resistance to pests that seriously affect yield, such as rootworms, cutworms, European corn borers, etc. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109); etc.

[0261] "Herbicide resistance proteins" or proteins expressed by "herbicide resistance encoding nucleic acid molecules" include proteins that confer to cells the ability to tolerate higher concentrations of herbicides compared to cells that do not express the protein, or confer to cells the ability to tolerate a certain concentration of herbicide for a longer period of time compared to cells that do not express the protein. The herbicide resistance trait can be introduced into plants by genes encoding resistance to herbicides that inhibit acetolactate synthase (ALS, also known as acetohydroxyacid synthase, AHAS) (particularly sulfonylurea (UK: sulfonylurea (sulphonylurea)) herbicides), genes encoding resistance to herbicides that inhibit glutamine synthase (such as glufosinate or basta) (e.g., bar genes), genes encoding resistance to glyphosate (e.g., EPSP synthase genes and GAT genes), genes encoding resistance to HPPD inhibitors (e.g., HPPD genes), or other such genes known in the art. See, e.g., U.S. Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and 9,187,762. The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutant encodes resistance to the herbicide chlorsulfuron.

[0262] In addition, it is recognized that the target polynucleotide can also include an antisense sequence complementary to at least a portion of the messenger RNA (mRNA) of the targeted target gene sequence. Antisense nucleotides are constructed to hybridize with the corresponding mRNA. The antisense sequence can be modified as long as the sequence hybridizes with the corresponding mRNA and interferes with the expression of the corresponding mRNA. In this way, an antisense construct with 70%, 80% or 85% sequence identity to the corresponding antisense sequence can be used. In addition, the part of the antisense nucleotide can be used to destroy the expression of the target gene. Generally, a sequence of at least 50 nucleotides, 100 nucleotides, 200 nucleotides or more nucleotides can be used.

[0263] In addition, the target polynucleotide can also be used in a sense orientation to suppress the expression of endogenous genes in plants. The method of using polynucleotides in a sense orientation to suppress gene expression in plants is known in the art. These methods generally involve transforming plants with a DNA construct comprising a promoter, which is operably connected to at least a portion of the nucleotide sequence of the transcript corresponding to the endogenous gene, driving the expression in the plant. Typically, such a nucleotide sequence has substantial sequence identity with the sequence of the transcript of the endogenous gene, usually greater than about 65% sequence identity, about 85% sequence identity or greater than about 95% sequence identity. Referring to U.S. Patent Nos. 5,283,184 and 5,034,323.

[0264] The purpose polynucleotide can also be a phenotypic marker. Phenotypic markers are screenable or selectable markers, which include visual markers and selectable markers, whether it is a positive selectable marker or a negative selectable marker. Any phenotypic marker can be used. In particular, selectable markers or screenable markers include DNA segments, which allow people to identify molecules or cells comprising it under specific conditions or for molecules or cells comprising it or for molecules or cells comprising it. These markers can encode activity, such as but not limited to the generation of RNA, peptides or proteins, or can provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, etc.

[0265] Examples of selectable markers include, but are not limited to, DNA segments comprising restriction endonuclease sites; DNA segments encoding products that provide resistance to otherwise toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT); DNA segments encoding products that are inherently deficient in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that are easily identified (e.g., phenotypic markers such as β-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (RFP), and cell surface proteins); generating new primer sites for PCR (e.g., juxtaposition of two DNA sequences that were not previously juxtaposed), comprising DNA sequences that are inactive or acted upon by restriction endonucleases or other DNA modifying enzymes, chemicals, etc.; and comprising DNA sequences required for specific modifications (e.g., methylation) that allow their identification.

[0266] Additional selectable markers include genes that confer resistance to herbicidal compounds such as sulfonylureas, glufosinate, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). See, for example, acetolactate synthase (ALS) for resistance to sulfonylureas, imidazolinones, triazolopyrimidinesulfonamides, pyrimidine salicylates, and sulfonylaminocarbonyl-triazolinones (Shaner and Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69-110); glyphosate resistance 5-enolpyruvylshikimate-3-phosphate (EPSPS) (Saroha et al., 1998, J. Plant Biochemistry & Biotechnology 7: 65-72);

[0267] Target polynucleotides include genes that are stacked or used in combination with other traits (such as, but not limited to, herbicide resistance or any other trait described herein). Target polynucleotides and / or traits can be stacked together in a complex trait locus, as described in US20130263324, published on October 3, 2013, and WO / 2013 / 112686, published on August 1, 2013.

[0268] A polypeptide of interest includes any protein or polypeptide encoded by a polynucleotide of interest described herein.

[0269] Further provided is a method for identifying at least one plant cell, the plant cell comprising the target polynucleotide integrated at the target site in its genome. Can use a variety of methods to identify those plant cells with insertion at or near the target site in the genome. Such method can be considered to be directly analyzing the target sequence to detect any change in the target sequence, including but not limited to PCR method, sequencing method, nuclease digestion, Southern blotting and any combination thereof. See, for example, US20090133152 disclosed on May 21, 2009. The method also includes reobtaining plants from the plant cell comprising the target polynucleotide integrated into its genome. The plant can be sterile or fertile. It should be appreciated that any target polynucleotide can be provided, the polynucleotide is integrated into the target site in the plant genome and expressed in plants.

[0270] Optimization of sequences for expression in plants

[0271] Methods for synthesizing plant-preferential 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. [Nucleic Acids Research] 17: 477-498. Additional sequence modifications are known to enhance gene expression in plant hosts. For example, these include elimination of: one or more sequences encoding false polyadenylation signals, one or more exon-intron splice site signals, one or more transposon-like repeats, and other such well-characterized sequences that may be harmful to gene expression. The GC content of the sequence can be adjusted to the average level of a given plant host, such as calculated by reference to known genes expressed in host plant cells. When possible, the sequence is modified to avoid the presence of one or more predicted hairpin secondary mRNA structures. Therefore, the "plant-optimized nucleotide sequence" of the present disclosure includes one or more such sequence modifications.

[0272] Expression elements

[0273] Any polynucleotide encoding a variant Cas polypeptide or other CRISPR system component disclosed herein may be functionally linked to a heterologous expression element to promote transcription or regulation in a host cell. Such expression elements include, but are not limited to, promoters, leader sequences, introns, and terminators. An expression element may be "minimal" - meaning a shorter sequence derived from a natural source that still acts as an expression regulator or modifier. Alternatively, an expression element may be "optimized" - meaning that its polynucleotide sequence has been changed from its native state so that it functions with more desirable characteristics in a specific host cell (for example, but not limited to, a bacterial promoter may be "maize optimized" to improve its expression in corn plants). Alternatively, an expression element may be "synthetic" - meaning that it is designed by computer and synthesized for use in a host cell. Synthetic expression elements may be fully synthetic or partially synthetic (fragments comprising naturally occurring polynucleotide sequences).

[0274] Certain promoters have been shown to be able to direct RNA synthesis at a higher rate than other promoters. These are referred to as "strong promoters". Certain other promoters have been shown to direct RNA synthesis at higher levels only in specific types of cells or tissues, and if the promoter preferentially directs RNA synthesis in certain tissues but also directs RNA synthesis in other tissues at reduced levels, it is often referred to as a "tissue-specific promoter" or "tissue-preferential promoter".

[0275] Plant promoters include promoters that are capable of initiating transcription in plant cells. For a review of plant promoters, see Potenza et al. 2004, In vitro Cell Dev Biol 40: 1-22; Porto et al. 2014, Molecular Biotechnology (2014), 56(1), 38-49.

[0276] Constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-2); rice actin (McElroy et al., (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al. (1989) Plant Mol Biol 12:619-32; ALS promoter (U.S. Pat. No. 5,659,026), etc.

[0277] Tissue-preferred promoters can be used to target enhanced expression within specific plant tissues. Tissue-preferred promoters include, for example, WO 2013103367, published July 11, 2013, Kawamata et al. (1997) Plant Cell Physiol 38:792-803; Hansen et al. (1997) Mol Gen Genet 254:337-43; Russell et al. (1997) Transgenic Res 6:157-68; Rinehart et al. (1996) Plant Physiol 112:1331-41; Van Camp et al. (1996) Plant Physiol. 112:525-35; Canevascini et al. (1996) Plant Physiol. 112:513-524; Lam, (1994) Results Probl. Cell Differ [Results and Problems in Cell Differentiation] 20:181-96; and Guevara-Garcia et al. (1993) Plant J. [Plant Journal] 4:495-505. Leaf-preferred promoters include, for example, Yamamoto et al. (1997) Plant J 12:255-65; Kwon et al. (1994) Plant Physiol 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol 35:773-8; Gotor et al. (1993) Plant J 3:509-18; Orozco et al. (1993) Plant Mol Biol 23:1129-38; Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90:9586-90; Simpson et al. (1958) EMBO J [Journal of the European Molecular Biology Association] 4: 2723-9; Timko et al. (1988) Nature [Nature] 318: 57-8.Root-preferred promoters include, for example, Hire et al. (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al. (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol Biol 14:433-43 (root-specific promoter of mannopine synthase (MAS) from Agrobacterium tumefaciens); Bogusz et al., (1990) Plant Cell 2:633-41 (root-specific promoter from Parasponia andersonii and Trema tomentosa); Leach and Aoyagi, (1991) Plant Sci 79:69-76 (rolC and rolD root-inducible genes of A. rhizogenes); Teeri et al. (1989) EMBO J 8:343-50 (TR1′ and TR2′ genes induced by wounding of Agrobacterium); VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana et al. (1994) Plant Mol Biol 30:757-76). Biol [Plant Molecular Biology] 25:681-91); phaseolin gene (Murai et al. (1983) Science [Science] 23:476-82; Sengopta-Gopalen et al. (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 82:3320-4). See also U.S. Patent Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179.

[0278] 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 [Biological Analysis] 10: 108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced messenger); cZ19B1 (maize 19 kDa zein); and milps (inositol-1-phosphate synthase); and, for example, those disclosed in WO 2000011177 (published on March 2, 2000) and U.S. Patent 6,225,529. For dicotyledons, seed-preferred promoters include, but are not limited to, bean β-phaseolin, rapeseed protein, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-preferred promoters 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 nucl. See also WO 2000012733 published on March 9, 2000, which discloses seed-preferred promoters from END1 and END2 genes.

[0279] Chemically inducible (regulatable) promoters can be used to regulate gene expression in prokaryotic and eukaryotic cells or organisms by application of exogenous chemical regulators. The promoter can be a chemically inducible promoter in the case where application of a chemical induces gene expression, or a chemically repressible promoter in the case where application of a chemical represses gene expression. Chemically inducible promoters include, but are not limited to, the maize In2-2 promoter activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter activated by hydrophobic electrophilic compounds used as pre-emergence herbicides (GST-II-27, WO 1993001294 published on January 21, 1993), and the tobacco PR-1a promoter activated by salicylic acid (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7). Other chemically-regulated promoters include steroid-responsive promoters (see, e.g., glucocorticoid-inducible promoters (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al. (1998) Plant J 14:247-257); tetracycline-inducible promoters and tetracycline-repressible promoters (Gatz et al. (1991) Mol Gen Genet 227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156)).

[0280] Pathogen-inducible promoters that are induced upon infection by pathogens include, but are not limited to, promoters that regulate the expression of PR proteins, SAR proteins, β-1,3-glucanase, chitinase, and the like.

[0281] Stress-inducible promoters include the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17: 287-91). Those skilled in the art are familiar with procedures for simulating stress conditions (such as drought, osmotic stress, salt stress, and temperature stress) and evaluating stress tolerance in plants that have been subjected to simulated or naturally occurring stress conditions.

[0282] Another example of an inducible promoter useful in plant cells is the ZmCAS1 promoter, described in US20130312137 published on November 21, 2013.

[0283] New promoters of different types useful in plant cells are continually being discovered; many examples can be found in the compilation by Okamuro and Goldberg, (1989) The Biochemistry of Plants, Vol. 115, ed. Stumpf and Conn (Academic Press, New York, NY), pp. 1-82.

[0284] Genome modification using novel CRISPR-Cas system components

[0285] As described herein, the guided Cas endonuclease can recognize, bind to the DNA target sequence, and introduce single-strand breaks (nicks) or double-strand breaks. Once single-strand breaks or double-strand breaks are induced in DNA, the DNA repair mechanism of the cell is activated to repair the break. Error-prone DNA repair mechanisms can produce mutations at double-strand break sites. The most common repair mechanism that combines the ends of the break is non-homologous end joining (NHEJ) pathway (Bleuyard et al. (2006) DNA Repair [DNA repair] 5: 1-12). The structural integrity of chromosomes is typically maintained by repair, but deletion, insertion or other rearrangements (such as chromosome translocation) are possible (Siebert and Puchta, 2002 Plant Cell [plant cells] 14: 1121-31; Pacher et al. 2007, Genetics [genetics] 175: 21-9).

[0286] DNA double-strand breaks appear to be potent factors in stimulating homologous recombination pathways (Puchta et al. (1995) Plant Mol Biol 28:281-92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot 56:1-14). Using DNA-breaking agents, a two- to nine-fold increase in homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al. (1995) Plant Mol Biol 28:281-92). In maize protoplasts, experiments with linear DNA molecules demonstrated enhanced homologous recombination between plasmids (Lyznik et al. (1991) Mol Gen Genet 230:209-18).

[0287] Homologous-directed repair (HDR) is a mechanism used to repair double-stranded DNA breaks and single-stranded DNA breaks in cells. Homologous directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber.2010Annu.Rev.Biochem.[Biological Chemistry Yearbook] 79:181-211). The most common form of HDR is called homologous recombination (HR), which has the longest sequence homology requirement between donor and recipient DNA. Other forms of HDR include single-strand annealing (SSA) and break-induced replication, and these require shorter sequence homology relative to HR. Homologous directed repair at the nick (single-strand break) can occur via a mechanism different from HDR at the double-strand break (Davis and Maizels.PNAS[Proceedings of the National Academy of Sciences of the United States] (0027-8424), 111 (10), pages E924-E932).

[0288] The alteration of the genome of prokaryotic and eukaryotic cells or biological cells, for example by homologous recombination (HR), is a powerful tool for genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al. (1992) Mol Gen Genet [Molecular and General Genetics] 231: 186-93) and insects (Dray and Gloor, 1997, Genetics [Genetics] 147: 689-99). Homologous recombination can also be achieved in other organisms. For example, in the parasitic protozoan Leishmania, at least 150bp-200bp of homology is required for homologous recombination (Papadopoulou and Dumas, (1997) Nucleic Acids Res [Nucleic Acids Research] 25: 4278-86). In the filamentous fungus Aspergillus nidulans, gene replacement has been achieved with only 50 bp of flanking homology (Chaveroche et al. (2000) Nucleic Acids Res 28:e97). Targeted gene replacement has also been demonstrated in the ciliate Tetrahymena thermophila (Gaertig et al., (1994) Nucleic Acids Res 22:5391-8). In mammals, homologous recombination using pluripotent embryonic stem cell (ES) lines that can be grown in culture, transformed, selected, and introduced into mouse embryos has been most successful in mice (Watson et al. 1992 Recombinant DNA, 2nd ed., Scientific American Books distributed by WH Freeman & Co.).

[0289] Gene targeting

[0290] The guide polynucleotide / Cas system described herein can be used for gene targeting.

[0291] Typically, DNA targeting can be performed by cutting one or both strands at a specific polynucleotide sequence in a cell with any engineered Cas polypeptide disclosed herein in association with a suitable polynucleotide component. Once single-strand breaks or double-strand breaks are induced in the DNA, the cell's DNA repair mechanism is activated to repair the break via non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes that result in modifications at the target site.

[0292] The length of the DNA sequence at the target site can vary, and includes, for example, a target site of at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more 30 nucleotides in length. It is also possible that the target site can be palindromic, that is, the sequence on one strand is the same when read in the opposite direction on the complementary strand. The nick / cleavage site can be within the target sequence, or the nick / cleavage site can be outside the target sequence. In another variation, the cleavage can occur at nucleotide positions that are exactly opposite to each other to produce a blunt-ended cleavage, or in other cases, the nicks can be staggered to produce single-stranded overhangs, also known as "sticky ends", which can be 5' overhangs or 3' overhangs. Active variants of genomic target sites can also be used. Such active variants may comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a given target site, wherein these active variants retain biological activity and are therefore capable of being recognized and cleaved by a Cas endonuclease.

[0293] Assays that measure single-strand breaks or double-strand breaks at a target site caused by an endonuclease are known in the art, and generally measure the overall activity and specificity of the agent on a DNA substrate comprising the recognition site.

[0294] The targeting method herein can be carried out in such a way that two or more DNA target sites are targeted, for example, in the method. This method can optionally be characterized as a multiplexing method. In certain embodiments, two, three, four, five, six, seven, eight, nine, ten or more target sites can be targeted simultaneously. The multiplexing method is typically carried out by the targeting method herein, wherein a plurality of different RNA components are provided, each of which is designed to guide the polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0295] Gene Editing

[0296] The process of editing a genomic sequence by combining DSB and modification templates generally includes: introducing a DSB inducer or a nucleic acid encoding a DSB inducer (which recognizes a target sequence in a chromosomal sequence and is capable of inducing a DSB in a genomic sequence) into a host cell, and at least one polynucleotide modification template comprising at least one nucleotide change when compared to the nucleotide sequence to be edited. The polynucleotide modification template may further comprise a nucleotide sequence flanking the at least one nucleotide change, wherein the flanking sequence is substantially homologous to the chromosomal region flanking the DSB. Genome editing using DSB inducers (such as Cas-gRNA complexes) has been described, for example, in US20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and WO / 2016 / 025131 published on February 18, 2016.

[0297] Some uses of the guide RNA / Cas endonuclease system have been described (see, for example: US20150082478 A1 published on March 19, 2015, WO 2015026886 published on February 26, 2015, and US20150059010 published on February 26, 2015) and include, but are not limited to, modification or replacement of a target nucleotide sequence (such as a regulatory element), insertion of a target polynucleotide, gene knockout, gene knockin, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a target protein, amino acid and / or protein fusion, and gene silencing caused by expression of an inverted repeat sequence in a target gene.

[0298] Proteins can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. For example, amino acid sequence variants of one or more proteins can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations include, for example, Kunkel, (1985) Proc. Natl. Acad. Sci. USA 82:488-92; Kunkel et al. (1987) Meth Enzymol 154:367-82; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York), and references cited therein. Guidance on amino acid substitutions that are unlikely to affect the biological activity of a protein is found, for example, in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC [National Biomedical Research Foundation, Washington, DC, USA]. Conservative substitutions, such as exchanging one amino acid for another with similar properties, would be preferred. Conservative deletions, insertions, and amino acid substitutions are expected to produce no fundamental changes in the characteristics of the protein, and the effect of any substitution, deletion, insertion, or combination thereof can be evaluated by conventional screening assays. Assays for double-strand-break-inducing activity are known, and generally measure the overall activity and specificity of an agent against a DNA substrate comprising a target site.

[0299] Methods for genome editing with Cas endonucleases and complexes of Cas endonucleases and guide polynucleotides are described herein. After characterizing the guide RNA and PAM sequences, endonucleases and associated CRISPR RNA (crRNA) components can be used to modify chromosomal DNA in other organisms including plants. In order to promote optimal expression and nuclear localization (for eukaryotic cells), the complex containing the gene can be optimized as described in WO 2016186953 published on November 24, 2016, and then delivered to the cell as a DNA expression cassette by methods known in the art. The components necessary for containing the active complex can also be delivered as RNA (with or without modifications to protect RNA from degradation) or as capped or uncapped mRNA (Zhang, Y. et al., 2016, Nat. Commun. [Natural Communications] 7: 12617) or Cas protein guide polynucleotide complexes (disclosed in WO 2017070032 on April 27, 2017), or any combination thereof. In addition, one or more parts of the complex and crRNA can be expressed from a DNA construct, while the other components are delivered as RNA (with or without modifications to protect the RNA from degradation) or as capped or uncapped mRNA (Zhang et al. 2016 Nat. Commun. [Natural Communications] 7: 12617) or Cas protein-guided polynucleotide complexes (disclosed in WO 2017070032 on April 27, 2017) or any combination thereof. For in vivo production of crRNA, tRNA-derived elements can also be used to recruit endogenous RNases to cut crRNA transcripts into mature forms that can guide the complex to its DNA target site, for example, as described in WO 2017105991 disclosed on June 22, 2017. The nickase complex can be used alone or in combination to generate single or multiple DNA nicks on one or both DNA strands. In addition, the cleavage activity of Cas endonucleases can be inactivated by changing the key catalytic residues in the cleavage domain (Sinkunas, T. et al. 2013, EMBO J [Journal of the European Molecular Biology Association] 32: 385-394), thereby generating an RNA-guided helicase that can be used to enhance homology-directed repair, induce transcriptional activation, or reshape local DNA structure. In addition, the activities of the Cas cleavage and helicase domains can be knocked out and used in combination with other DNA shearing, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancement, DNA integration, DNA inversion, and DNA repair agents.

[0300] The transcription direction of tracrRNA for the CRISPR-Cas system (if present) and other components of the CRISPR-Cas system (e.g., variable targeting domains, crRNA repeats, loops, anti-repeat sequences) can be derived as described in WO 2016186946 published on November 24, 2016 and WO 2016186953 published on November 24, 2016.

[0301] As described herein, once the appropriate guide RNA requirements are established, the PAM preference of each new system disclosed herein can be checked. If the cleavage complex results in degradation of the randomized PAM library, the complex can be converted into a nicking enzyme by mutagenesis of key residues or by assembling the reaction in the absence of ATP to invalidate the ATPase-dependent helicase activity, as previously described (Sinkunas, T. et al. 2013, EMBO J. [Journal of the European Molecular Biology Association] 32: 385-394). Double-stranded DNA breaks can be generated using two regions of PAM randomization separated by two pre-spacer targets, which can be captured and sequenced to check the PAM sequence that supports the cleavage of each complex.

[0302] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein into a cell, and identifying at least one cell having a modification at the target, wherein the modification at the target site is selected from the group consisting of: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, chemical alteration of at least one nucleotide, and (v) any combination of (i)-(iv).

[0303] The nucleotide to be edited can be located inside or outside the target site recognized and cut by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification on the target site recognized and cut by the Cas endonuclease. In another embodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900 or 1000 nucleotides between at least one nucleotide to be edited and the genomic target site.

[0304] Knockouts can be produced by indels (insertion or deletion of nucleotide bases in the target DNA sequence by NHEJ), or by specific removal of sequences that reduce or completely abolish sequence function at or near the targeted site.

[0305] Guide polynucleotide / Cas endonuclease-induced targeted mutations can occur in nucleotide sequences located inside or outside the genomic target site recognized and cleaved by the Cas endonuclease.

[0306] The method for editing a nucleotide sequence in the genome of a cell may be a method by restoring the function of a non-functional gene product without using an exogenous selectable marker.

[0307] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, the method comprising introducing at least one PGEN described herein and at least one donor DNA into the cell, wherein the donor DNA comprises a polynucleotide of interest, and optionally, the method further comprises identifying at least one cell that has integrated the polynucleotide of interest into or near the target site.

[0308] In one aspect, the methods disclosed herein can employ homologous recombination (HR) to provide for integration of a polynucleotide of interest at a target site.

[0309] A variety of methods and compositions can be used to produce cells or organisms with a polynucleotide of interest inserted into a target site through the activity of a CRISPR-Cas system component as described herein. In a method described herein, a polynucleotide of interest is introduced into an organism cell via a donor DNA construct. As used herein, "donor DNA" is a DNA construct comprising a polynucleotide of interest to be inserted into a target site of a Cas endonuclease. The donor DNA construct further comprises a first homology region and a second homology region flanking the polynucleotide of interest. The first homology region and the second homology region of the donor DNA have homology to the first and second genomic regions present in the target site of a cell or organism genome or flanking the target site, respectively.

[0310] Donor DNA can be tethered to the guide polynucleotide. Tethered donor DNA can allow co-localization of target and donor DNA, can be used for genome editing, gene insertion and targeted genome regulation, and can also be used to target late mitotic cells, in which the function of endogenous HR machinery is expected to be greatly reduced (Mali et al. 2013, Nature Methods [Natural Methods] Vol. 10: 957-963).

[0311] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and include total length and / or having a length between about 1 bp-20 bp, 20 bp-50 bp, 50 bp-100 bp, 75 bp-150 bp, 100 bp-250 bp, 150 bp-300 bp, 200 bp-400 bp, 250 bp-500 bp, 300 bp-600 bp, 350 bp-750 bp, 400 bp-800 bp, 450 bp- The target site can be a target site having a length of 1-20 bp, 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 11 bp, 12 bp, 13 bp, 14 bp, 15 bp, 16 bp, 17 bp, 18 bp, 19 bp, and 20 bp. The amount of homology can also be described by the percentage sequence identity over the complete comparison length of two polynucleotides, which includes about at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% percentage sequence identity. Sufficient homology includes polynucleotide length, overall percentage sequence identity, and optionally any combination of conserved regions or local percentage sequence identity of continuous nucleotides, for example, enough homology can be described as a region of 75bp-150bp with at least 80% sequence identity to the region of the target locus.Sufficient homology can also be described by the predicted ability of two polynucleotides to hybridize specifically under high stringency conditions, see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, New York); 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).

[0312] Episomal DNA molecules can also be ligated into double-strand breaks, for example, T-DNA is integrated into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J. 17:6086-95). Once the sequence around the double-strand break is altered, for example by mature exonuclease activity involving double-strand breaks, the gene conversion pathway can restore the original structure, provided that homologous sequences are available, such as homologous chromosomes in non-dividing somatic cells, or sister chromatids after DNA replication (Molinier et al. (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA repair templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0313] In one embodiment, the present disclosure comprises a method for editing a nucleotide sequence in the genome of a cell, the method comprising introducing at least one PGEN described herein and a polynucleotide modification template, wherein the polynucleotide modification template comprises at least one nucleotide modification of the nucleotide sequence, and the method optionally further comprises selecting at least one cell comprising the edited nucleotide sequence.

[0314] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to allow editing (modification) of the target genomic nucleotide sequence. (See also US20150082478 published on March 19, 2015 and WO 2015026886 published on February 26, 2015).

[0315] The polynucleotides of interest and / or traits can be stacked together in a complex trait locus as described in WO 2012129373 published on September 27, 2012 and WO 2013112686 published on August 1, 2013. The guide polynucleotide / Cas9 endonuclease system described herein provides an efficient system for generating double strand breaks and allows for the stacking of traits in a complex trait locus.

[0316] The guide polynucleotide / Cas system for mediating gene targeting as described herein can be used in the following methods for guiding heterologous gene insertion and / or generating complex trait loci comprising multiple heterologous genes in a manner similar to that disclosed in WO 2012129373 published on September 27, 2012, wherein the guide polynucleotide / Cas system as disclosed herein is used to replace the introduction of the target gene using a double-strand break inducing agent. By inserting independent transgenes within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or even 5 centimorgans (cM) of each other, these transgenes can be bred as a single genetic locus (e.g., see US20130263324 published on October 3, 2013 or WO2012129373 published on March 14, 2013). After selecting plants comprising transgenes, plants comprising (at least) one transgene can be crossed to form F1 comprising two transgenes. In the progeny from these F1 (F2 or BC1), 1 / 500 of the progeny will have two different transgenes recombined on the same chromosome. The composite locus can then be bred to a single genetic locus with two transgenic traits. This process can be repeated to stack as many traits as possible.

[0317] Further uses of the guide RNA / Cas endonuclease system have been described (see, e.g., US20150082478 published on March 19, 2015, WO 2015026886 published on February 26, 2015, US20150059010 published on February 26, 2015, WO 2016007347 published on January 14, 2016, and PCT application WO2016025131 published on February 18, 2016) and include, but are not limited to, modification or replacement of a nucleotide sequence of interest (such as a regulatory element), insertion of a polynucleotide of interest, gene knockout, gene knockin, modification of splice sites and / or introduction of alternative splice sites, modification of a nucleotide sequence encoding a protein of interest, amino acid and / or protein fusions, and gene silencing caused by expression of an inverted repeat sequence in a gene of interest.

[0318] The characteristics produced by the gene editing compositions and methods described herein can be evaluated. Chromosome intervals associated with the target phenotype or trait can be identified. Various methods well known in the art can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are extended to cover markers that will be linked to genes controlling the target trait. In other words, the chromosomal interval is extended so that any marker located within the interval (including the end markers defining the boundaries of the interval) can be used as markers for specific traits. In one embodiment, the chromosomal interval contains at least one QTL, and in addition, more than one QTL can indeed be included. Multiple QTLs very close in the same interval can confuse the association of a specific marker with a specific QTL, because a marker can be shown to be linked to more than one QTL. On the contrary, for example, if two very close markers show co-segregation with the desired phenotypic trait, it is sometimes unclear whether each of those markers identifies the same QTL or identifies two different QTLs. The term "quantitative trait locus" or "QTL" refers to a DNA region associated with the differential expression of a quantitative phenotypic trait in at least one genetic background (e.g., in at least one breeding population). The region of the QTL encompasses or is closely linked to one or more genes that affect the trait under consideration. "Alleles of a QTL" can include multiple genes or other genetic factors, such as haplotypes, in a continuous genomic region or linkage group. The alleles of a QTL can represent haplotypes within a specified window, where the window is a continuous genomic region that can be defined and tracked with a set of one or more polymorphic markers. Haplotypes can be specified to be defined by the unique fingerprint of the alleles at each marker within the window.

[0319] Introducing CRISPR-Cas system components into cells

[0320] The methods described herein do not depend on a particular method for introducing a sequence into an organism or cell, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the organism. Introducing includes reference to incorporating a nucleic acid into a eukaryotic or prokaryotic cell, wherein the nucleic acid can be incorporated into the genome of the cell, and includes reference to nucleic acids, proteins, or polynucleotide-protein complexes (PGEN, RGEN) being provided transiently (directly) to the cell.

[0321] Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are not limited to, microinjection, electroporation, stable transformation methods, transient transformation methods, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated introduction, transfection, transduction, cell penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical application, sexual hybridization, sexual breeding, and any combination thereof.

[0322] For example, a guide polynucleotide (guide RNA, cr nucleotides + tracr nucleotides, guide DNA and / or guide RNA-DNA molecules) can be directly introduced into cells (transiently) as single-stranded or double-stranded polynucleotide molecules. Guide RNA (or crRNA + tracrRNA) can also be indirectly introduced into cells by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding a guide RNA (or crRNA + tracrRNA), which is operably linked to a specific promoter capable of transcribing the guide RNA (crRNA + tracrRNA molecule) in the cell. The specific promoter can be, but is not limited to, an RNA polymerase III promoter that allows transcription of RNA with precisely defined, unmodified 5'- and 3'-ends (Ma et al. 2014, Mol. Ther. Nucleic Acids [Molecular Therapy - Nucleic Acids] 3: e161; DiCarlo et al. 2013, Nucleic Acids Res. [Nucleic Acids Research] 41: 4336-4343; WO 2015026887 published on February 26, 2015). Any promoter capable of transcribing the guide RNA in a cell can be used, and these promoters include heat shock / heat-inducible promoters operably linked to a nucleotide sequence encoding the guide RNA.

[0323] Plant cells are distinct from animal cells (eg, human cells), fungal cells (eg, yeast cells), and protoplasts, including, for example, in that they contain a plant cell wall that can serve as a barrier to the delivery of components.

[0324] Delivery of Cas endonucleases, and / or guide RNAs, and / or ribonucleoprotein complexes, and / or polynucleotides encoding any one or more of the foregoing into plant cells can be achieved by methods known in the art, such as, but not limited to, rhizobium-mediated transformation (e.g., Agrobacterium, Ochrobactrum), particle-mediated delivery (particle bombardment), polyethylene glycol (PEG)-mediated transfection (e.g., protoplasts), electroporation, cell-penetrating peptides, or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery.

[0325] The Cas endonuclease herein can be introduced into the cell by directly introducing the Cas polypeptide itself (referred to as direct delivery of the Cas endonuclease), mRNA encoding the Cas protein and / or guiding the polynucleotide / Cas endonuclease complex itself, using any method known in the art. The Cas endonuclease can also be introduced into the cell indirectly by introducing a recombinant DNA molecule encoding the Cas endonuclease. Using any method known in the art, the endonuclease can be introduced into the cell instantaneously, or the endonuclease can be incorporated into the genome of the host cell. Cell penetrating peptides (CPPs) as described in WO2016073433 disclosed on May 12, 2016 can be used to promote the uptake of endonucleases and / or guided polynucleotides into cells. Any promoter capable of expressing the Cas endonuclease in the cell can be used, and these promoters include heat shock / heat-inducible promoters operably connected to the nucleotide sequence encoding the Cas endonuclease.

[0326] Direct delivery of polynucleotide modified templates into plant cells can be achieved by particle-mediated delivery, and any other direct delivery method, such as but not limited to polyethylene glycol (PEG)-mediated protoplast transfection, whisker-mediated transformation, electroporation, particle bombardment, cell penetrating peptides or mesoporous silica nanoparticles (MSN)-mediated direct protein delivery can be successfully used to deliver polynucleotide modified templates in eukaryotic cells (e.g., plant cells).

[0327] Donor DNA can be introduced by any means known in the art. Donor DNA can be provided by any transformation method known in the art (including, for example, Agrobacterium-mediated transformation or biolistic particle bombardment). Donor DNA can be transiently present in the cell, or can be introduced via a viral replicon. In the presence of Cas endonucleases and target sites, donor DNA is inserted into the genome of the transformed plant.

[0328] The direct delivery of any one of the guided Cas system components can be accompanied by the direct delivery (co-delivery) of other mRNAs that can promote the enrichment of cells receiving the guidance polynucleotide / Cas endonuclease complex components and / or visualization. For example, direct co-delivery of mRNAs encoding phenotypic markers (such as, but not limited to, transcriptional activators such as CRC (Bruce et al. 2000 The Plant Cell [Plant Cell] 12: 65-79)) can be achieved by restoring the function of non-functional gene products without using exogenous selectable markers to select and enrich cells, as described in WO2017070032 published on April 27, 2017.

[0329] Introducing the guide RNA / Cas endonuclease complex described herein (representing the cleavable complex described herein) into a cell includes introducing the components of the complex into the cell individually or in combination, the introduction being directly (direct delivery as RNA (for guides) and proteins (for Cas endonucleases and protein subunits or functional fragments thereof)) or via a recombinant construct expressing these components (guide RNA, Cas endonucleases, protein subunits or functional fragments thereof). Introducing the guide RNA / Cas endonuclease complex (RGEN) into a cell includes introducing the guide RNA / Cas endonuclease complex into the cell as a ribonucleotide-protein. The ribonucleotide-protein can be assembled prior to introduction into a cell as described herein. Components comprising guide RNA / Cas endonuclease ribonucleotide proteins (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) can be assembled in vitro or by any method known in the art prior to introduction into a cell (the cell is targeted for genome modification as described herein).

[0330] The direct delivery of RGEN ribonucleoprotein allows genome editing at the target site in the genome of the cell, which can be followed by rapid degradation of the complex, and only allows the complex to exist briefly in the cell. This short-lived presence of the RGEN complex may cause the off-target effect to be reduced. In contrast, the delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences can cause RGEN to be constantly expressed from these plasmids, and this constant expression can strengthen the off-target effect (Cradick, TJ et al. (2013) Nucleic Acids Res [Nucleic Acids Research] 41: 9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. [Natural Biotechnology] 31: 822-826).

[0331] Direct delivery can be achieved by combining any one of the components of a guide RNA / Cas endonuclease complex (RGEN) (standing for a cleavage-ready complex as described herein, such as at least one guide RNA, at least one Cas protein, and optionally at least one additional protein) with a delivery matrix comprising microparticles, such as but not limited to gold particles, tungsten particles, and silicon carbide whisker particles (see also WO 2017070032 published on April 27, 2017). The delivery matrix can comprise any of these components, such as a Cas endonuclease, attached to a solid matrix (e.g., a particle for bombardment).

[0332] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein forming the guide RNA / Cas endonuclease complex are introduced into the cell as RNA and protein, respectively.

[0333] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein forming the guide RNA / Cas endonuclease complex and at least one protein subunit of the complex are introduced into the cell as RNA and protein, respectively.

[0334] 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 (cleavage-ready complex) and at least one protein subunit of the complex are preassembled in vitro and introduced into a cell as a ribonucleotide-protein complex.

[0335] Protocols for introducing polynucleotides, polypeptides or polynucleotide-protein complexes (PGEN, RGEN) into eukaryotic cells such as plants or plant cells are known and include microinjection (Crossway et al. (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543), meristem transformation (U.S. Pat. No. 5,736,369), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-6), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), whisker-mediated transformation (Ainley et al. 2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide [Properties and Applications of Silicon Carbide] (2011), 345-358. Editor: Gerhardt, Rosario. Publisher: InTech, 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 and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-6; Weissinger et al. (1988) Ann Rev Genet 22:421-77; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol 87:671-4 (soybean); Finer and McMullen, (1991) In vitro Cell Dev Biol 27P:175-82 (soybean); Singh et al. (1998) Theor Appl Genet [Theoretical and Applied Genetics] 96:319-24 (soybean); Datta et al. (1990) Biotechnology [Biotechnology] 8:736-40 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci.USA [Proceedings of the National Academy of Sciences of the United States of America] 85:4305-9 (maize); Klein et al. (1988) Biotechnology [Biotechnology] 6:559-63 (maize); U.S. Patents Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol [Plant Physiol] 91:440-4 (maize); Fromm et al. (1990) Biotechnology [Biotechnology] 8:833-9 (maize); Hooykaas-Van Slogteren et al. (1984) Nature 311:763-4; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al. (1985) The Experimental Manipulation of Ovule Tissues, Chapman et al., eds. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell Rep 9:415-8) and Kaeppler et al., (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); 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 transformed by Agrobacterium tumefaciens).

[0336] Alternatively, polynucleotides can be introduced into plants or plant cells by contacting cells or organisms with viruses or viral nucleic acids. Typically, such methods involve incorporating polynucleotides into viral DNA or RNA molecules. In some instances, the target polypeptide can be initially synthesized as a part of a viral polyprotein, and then the viral polyprotein is processed in vivo or in vitro by proteolytic processing to produce a desired recombinant protein. It is known to express polynucleotides in plants and to express proteins encoded therein (involving viral DNA or RNA molecules), see, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931.

[0337] For maize, the cell transformation methods disclosed herein include, for example, Agrobacterium-mediated delivery, Ensifer-based delivery, nanoparticle-mediated delivery, and particle-mediated biolistic delivery, as well as methods utilizing protoplasts (Sardesai and Subramanyan (2018) Agrobacterium Biology: From Basic Science to Biotechnology. Cham: Springer International Publishing, 463-488, Rathore et al. (2019) Transgenic Plants: Methods and Protocols. New York, NY: Springer New York, 37-48, Wang et al. (2019) Molecular Plant. [Molecular Plant] 12, 1037-1040, Rhodes et al. (1988) Science. [Science] 240, 204-207 and Golovkin et al. (1993) Plant Science [Plant Science]. 90, 41-52). In this example, Cas-α endonuclease and guide RNA plasmid expression cassettes were co-delivered into 9-10 day old maize immature embryos using particle-mediated biolistic transformation (Svitashev et al. (2015) Plant Physiology. 169, 931-945 and Karvelis et al. (2015) Genome Biology. 16, 253) or co-delivered into maize immature embryos via a single transfer DNA (T-DNA) using Agrobacterium as previously described (Lowe et al. (2018) In vitro cellular & developmental biology-Plant. 54, 240-252).

[0338] For Saccharomyces cerevisiae, several methods (lithium acetate, polyethylene glycol (PEG), heat shock, electroporation, biolistics, etc.) can be used for transformation (Kawai et al. (2010) Bioengineered Bugs. 1:395-403).

[0339] A variety of transient transformation methods can be used to provide or introduce polynucleotides or recombinant DNA constructs into prokaryotic and eukaryotic cells or organisms. Such transient transformation methods include, but are not limited to, directly introducing polynucleotide constructs into cells.

[0340] Nucleic acids and proteins can be provided to cells by any method, including methods using molecules to promote the uptake of any or all components (e.g., cell penetrating peptides and nanocarriers) of the directed Cas system (proteins and / or nucleic acids). See also US20110035836 published on February 10, 2011 and EP 2821486 A1 published on January 7, 2015.

[0341] Other methods for introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts may be used, including plastid transformation methods, and methods for introducing polynucleotides into tissues from seedlings or mature seeds.

[0342] "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 and is not integrated into the genome of the organism, or that a polypeptide is introduced into an organism. Transient transformation indicates that the introduced composition is only temporarily expressed or present in the organism.

[0343] A variety of methods can be used to identify those cells with altered genomes at or near the target site without using a screenable marker phenotype. Such methods can be considered to be direct analysis of the target sequence to detect any changes in the target sequence, including but not limited to PCR methods, sequencing methods, nuclease digestion, Southern blotting, and any combination thereof.

[0344] Cells and plants

[0345] The polynucleotides and polypeptides disclosed herein can be introduced into cells. Cells include, but are not limited to, human, non-human, animal, mammalian, bacterial, fungal, insect, yeast, unconventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein. Any plant (including monocots and dicots and plant elements) can be used with the compositions and methods described herein.

[0346] Examples of monocots that can be used include, but are not limited to, corn (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet, Pennisetum glaucum), sorghum (Panicum miliaceum), foxtail millet (Setaria italica), foxtail millet (Eleusine coracana), wheat (Triticum species, e.g., Triticum vulgare), aestivum, einkorn wheat (Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), bananas (Musa spp.), palms, ornamentals, turf grasses, and other grasses.

[0347] Examples of dicots that can be used include, but are not limited to, soybean (Glycine max), Brassica species (such as, but not limited to, rapeseed or canola) (Brassica napus, B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum, Gossypium barbadense), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), potato (Solanum tuberosum).

[0348] Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), bananas (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), kiwi fruit (Olea europaea), walnut ... europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), vegetables, ornamentals and conifers.

[0349] Vegetables that can be used include tomatoes (Tomato), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumber such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums.

[0350] Conifers that can be used include pines, such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs, such as silver fir (Abies vancouverii); amabilis) and balsam fir (Abies balsamea); and cedars, such as western red cedar (Thuja plicata) and Alaskan yellow cedar (Chamaecyparis nootkatensis).

[0351] In certain embodiments of the present disclosure, fertile plants are plants that produce live male and female gametes and are self-fertile. Such self-fertile plants can produce progeny plants without the contribution of gametes from any other plant and the genetic material contained therein. Other embodiments of the present disclosure may involve the use of non-self-fertile plants because the plant does not produce viable or otherwise fertile male or female gametes or both.

[0352] The present disclosure can be used for breeding of plants comprising one or more introduced traits or edited genomes.

[0353] A non-limiting example of how two traits can be stacked into a genome at a genetic distance of, for example, 5 cM from each other is described below: A first plant comprising a first transgenic target site integrated into a first DSB target site within a genomic window and not having a first genomic locus of interest is crossed with a second transgenic plant comprising a genomic locus of interest at a different genomic insertion site within the genomic window, and the second plant does not comprise the first transgenic target site. Approximately 5% of the progeny of the plant from this cross will have within the genomic window the first transgenic target site integrated into the first DSB target site and the first genomic locus of interest integrated at a different genomic insertion site. Progeny plants with two sites in a defined genomic window can be further crossed with a third transgenic plant comprising a second transgenic target site integrated into a second DSB target site, and / or a second genomic locus of interest within a defined genomic window and lacking the first transgenic target site and the first genomic locus of interest. Progeny with the first transgenic target site, the first genomic locus of interest, and the second genomic locus of interest integrated at different genomic insertion sites within the genomic window are then selected. Such methods can be used to generate transgenic plants comprising complex trait loci having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or more transgenic target sites integrated into the DSB target site and / or genomic loci of interest integrated at different sites within a genomic window. In this way, a variety of complex trait loci can be generated.

[0354] Cells and Animals

[0355] The polynucleotides and polypeptides disclosed herein can be introduced into animal cells. Animal cells can include, but are not limited to, organisms of the following phyla, including Chordata, Arthropoda, Mollusca, Annelida, Cnidaria, or Echinodermata; organisms of the following classes, including mammals, insects, birds, amphibians, reptiles, or fish. In some aspects, the animal is a human, mouse, C.elegans, rat, fruit fly (Drosophila spp.), zebrafish, chicken, dog, cat, guinea pig, hamster, chicken, Japanese rice fish, sea lamprey, pufferfish, tree frog (e.g., Xenopus spp.), monkey, or chimpanzee. Specific cell types contemplated include haploid cells, diploid cells, germ cells, neurons, muscle cells, endocrine or exocrine cells, epithelial cells, muscle cells, tumor cells, embryonic cells, hematopoietic cells, osteocytes, germ plasm cells, somatic cells, stem cells, pluripotent stem cells, induced pluripotent stem cells, progenitor cells, meiotic cells, and mitotic cells. In some aspects, multiple cells from an organism can be used.

[0356] The disclosed novel engineered Cas polypeptides can be used to edit the genome of animal cells in various ways. On the one hand, it may be desirable to delete one or more nucleotides. On the other hand, it may be desirable to insert one or more nucleotides. On the one hand, it may be desirable to replace one or more nucleotides. On the other hand, it may be desirable to modify one or more nucleotides by covalent or non-covalent interaction with another atom or molecule.

[0357] Genome modification via the disclosed engineered Cas polypeptides can be used to achieve genotypic and / or phenotypic changes in target organisms. Such changes are preferably related to the improvement of the target phenotype or physiologically important characteristics, the correction of endogenous defects, or the expression of a certain type of expression marker. In some aspects, the target phenotype or physiologically important characteristics are related to the overall health, adaptability or fertility of the animal, the ecological adaptability of the animal, or the relationship or interaction between the animal and other organisms in the environment.In some aspects, the phenotype or physiologically important characteristic of interest is selected from the group consisting of improved overall health, disease reversal, disease modification, disease stabilization, disease prevention, treatment of parasitic infections, treatment of viral infections, treatment of retroviral infections, treatment of bacterial infections, treatment of neurological disorders (such as but not limited to: multiple sclerosis), endogenous genetic defects (such as but not limited to: metabolic disorders, rickets, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Barth syndrome, breast cancer, Charcot-Marie-Tooth, colon cancer, Cri du chat, Crohn's disease, cystic fibrosis, Dercum Disease, Down Syndrome, Duane Syndrome, Duchenne Muscular Dystrophy, Factor V Leiden Thrombophilia Thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington disease, Klinefelter syndrome, Marfan syndrome, myotonic dystrophy, neurofibromatosis, Noonan syndrome, osteogenesis imperfecta, Parkinson disease, phenylketonuria, Poland Anomaly, porphyria, progeria, prostate cancer, retinitis pigmentosa, severe combined immunodeficiency (SCID), sickle cell disease, skin cancer, spinal muscular atrophy, Tay-Sachs, thalassemia, trimethylaminuria, Turner syndrome, velocardiofacial syndrome, WAGR syndrome, and Wilson disease. Disease), treatment of innate immune disorders (such as, but not limited to, immunoglobulin subclass deficiencies), treatment of acquired immune disorders (such as, but not limited to, AIDS and other HIV-related disorders), treatment of cancer, and treatment of diseases, including rare or "orphan" conditions, for which no effective treatment options are available by other means.

[0358] Cells genetically modified using the compositions or methods disclosed herein can be transplanted into a subject for purposes such as gene therapy, for example, to treat a disease or as an antiviral, antipathogen or anticancer therapeutic, for production of genetically modified organisms in agriculture, or for biological research.

[0359] In vitro polynucleotide detection, binding and modification

[0360] In some aspects, compositions disclosed herein can be further used as (in some aspects together with one or more separated polynucleotide sequences) for compositions used in in vitro methods. The one or more separated polynucleotide sequences can include one or more target sequences for modification. In some aspects, the one or more separated polynucleotide sequences can be genomic DNA, PCR products or synthetic oligonucleotides.

[0361] Composition

[0362] Modification of the target sequence can be in the form of nucleotide insertion, nucleotide deletion, nucleotide substitution, addition of atomic molecules to existing nucleotides, nucleotide modification, or binding of heterologous polynucleotides or polypeptides to the target sequence. Insertion of one or more nucleotides can be accomplished by including a donor polynucleotide in the reaction mixture: the donor polynucleotide is inserted into the double-stranded break produced by the engineered Cas endonuclease disclosed herein. Insertion can be via non-homologous end joining or via homologous recombination.

[0363] In one aspect, the sequence of the target polynucleotide is known prior to modification and is compared to one or more sequences of one or more polynucleotides produced by treatment with an engineered Cas endonuclease. In one aspect, the sequence of the target polynucleotide is unknown prior to modification and treatment with an engineered Cas endonuclease is used as part of a method to determine the sequence of the target polynucleotide.

[0364] Polynucleotide modifications with engineered Cas polypeptides can be accomplished using a full-length polypeptide identified from the Cas locus, or a fragment, modification, or variant of a polypeptide identified from the Cas locus. In some aspects, the Cas polypeptide variant is a polypeptide having at least 80% identity to any one of SEQ ID NO:15-47. In some aspects, the Cas polypeptide variant is a functional variant of any one of SEQ ID NO:15-47. In some aspects, the Cas polypeptide variant is a functional fragment of any one of SEQ ID NO:15-47. In some aspects, the Cas polypeptide is a Cas endonuclease polypeptide that recognizes the PAM sequence of N(T>W>C)TTC. In some aspects, the Cas polypeptide variant is provided by a polynucleotide encoding the Cas polypeptide.

[0365] In some aspects, the engineered Cas polypeptide can be selected from the group consisting of: an engineered or modified wild-type Cas endonuclease ortholog, a functional Cas endonuclease ortholog variant, a functional engineered Cas polypeptide fragment, a fusion protein comprising an active or inactivated engineered Cas polypeptide variant, an engineered Cas polypeptide further comprising one or more nuclear localization sequences (NLS) at the C-terminus or N-terminus or both the N-terminus and the C-terminus, a biotinylated engineered Cas polypeptide, an engineered Cas endonuclease nickase, an engineered Cas polypeptide ortholog endonuclease, an engineered Cas polypeptide further comprising a histidine tag, and a mixture of any two or more of the foregoing.

[0366] In some aspects, the engineered Cas polypeptide is a fusion protein that further comprises a nuclease domain, a transcriptional activator domain, a transcriptional repressor domain, an epigenetic modification domain, a cleavage domain, a nuclear localization signal, a cell penetration domain, a translocation domain, a marker, or a transgene that is heterologous to the target polynucleotide sequence or to the cell from which the target polynucleotide sequence was obtained or derived.

[0367] In some aspects, multiple engineered Cas polypeptides may be desired. In some aspects, the multiple may include engineered Cas polypeptides derived from different source organisms or derived from different loci within the same organism. In some aspects, the multiple may include engineered Cas polypeptides with different binding specificities to target polynucleotides. In some aspects, the multiple may include engineered Cas endonucleases with different cutting efficiencies. In some aspects, the multiple may include engineered Cas polypeptides with different PAM specificities. In some aspects, the multiple may include engineered Cas polypeptides with different molecular compositions, i.e., polynucleotides encoding engineered Cas polypeptides and polypeptides as engineered Cas polypeptides.

[0368] The engineered Cas polypeptides disclosed herein can be used with a guide polynucleotide, which can be provided as a single guide RNA (sgRNA), a chimeric molecule comprising a tracrRNA, a chimeric molecule comprising a crRNA, a chimeric RNA-DNA molecule, a DNA molecule, or a polynucleotide comprising one or more chemically modified nucleotides.

[0369] The storage conditions of the engineered Cas polypeptide and / or guidance polynucleotide include parameters of temperature, material state and time. In some aspects, the Cas polypeptide and / or guidance polynucleotide are stored at about -80 degrees Celsius, about -20 degrees Celsius, about 4 degrees Celsius, about 20-25 degrees Celsius, or about 37 degrees Celsius. In some aspects, the Cas polypeptide and / or guidance polynucleotide are stored in the form of a liquid, a frozen liquid, or a lyophilized powder. In some aspects, the Cas polypeptide and / or guidance polynucleotide are stable for at least one day, at least one week, at least one month, at least one year, or even more than one year.

[0370] Any or all possible polynucleotide components of the reaction (e.g., guide polynucleotides, donor polynucleotides, polynucleotides optionally encoding Cas polypeptides) can be provided as part of a vector, construct, linearized or circularized plasmid, or as part of a chimeric molecule. Each component can be provided to the reaction mixture alone or together. In some aspects, one or more polynucleotide components are operably linked to a heterologous non-coding regulatory element that regulates its expression.

[0371] The method for modifying a target polynucleotide comprises combining a minimum of elements into a reaction mixture comprising: an engineered Cas polypeptide (or a variant, fragment or other related molecule as described above), a guide polynucleotide (which comprises a sequence that is substantially complementary to or selectively hybridizes with a target polynucleotide sequence of a target polynucleotide), and a target polynucleotide for modification. In some aspects, the engineered Cas polypeptide is provided as a polypeptide. In some aspects, the engineered Cas polypeptide is provided as a Cas polypeptide polynucleotide. In some aspects, the guide polynucleotide is provided as: an RNA molecule, a DNA molecule, an RNA: DNA hybrid, or a polynucleotide molecule comprising chemically modified nucleotides.

[0372] Storage buffer or reaction mixture of any one of components can be optimized for stability, efficacy or other parameters. Other components of storage buffer or reaction mixture may include buffer composition, Tris, EDTA, dithiothreitol (DTT), phosphate buffered saline (PBS), sodium chloride, magnesium chloride, HEPES, glycerol, BSA, salt, emulsifier, detergent, chelating agent, redox agent, antibody, nuclease-free water, protease and / or viscosity agent. In some aspects, storage buffer or reaction mixture further include a buffer solution with at least one of the following components: HEPES, MgCl2, NaCl, EDTA, protease, proteinase K, glycerol, nuclease-free water.

[0373] Incubation conditions will vary according to desired result.Temperature is preferably at least 10 degrees Celsius, between 10 degrees Celsius and 15 degrees Celsius, at least 15 degrees Celsius, between 15 degrees Celsius and 17 degrees Celsius, at least 17 degrees Celsius, between 17 degrees Celsius and 20 degrees Celsius, at least 20 degrees Celsius, between 20 degrees Celsius and 22 degrees Celsius, at least 22 degrees Celsius, between 22 degrees Celsius and 25 degrees Celsius, at least 25 degrees Celsius, between 25 degrees Celsius and 27 degrees Celsius, at least 27 degrees Celsius, between 27 degrees Celsius and 30 degrees Celsius, at least 30 degrees Celsius, between 30 degrees Celsius and 32 degrees Celsius, at least 32 degrees Celsius, between 32 degrees Celsius and 35 degrees Celsius, at least 35 degrees Celsius, at least 36 degrees Celsius, at least 37 degrees Celsius, at least 38 degrees Celsius, at least 39 degrees Celsius, at least 40 degrees Celsius or even greater than 40 degrees Celsius. The incubation time is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or even greater than 10 minutes.

[0374] Before, during or after incubation, the sequence or sequences of the one or more polynucleotides in the reaction mixture can be determined by any method known in the art. In one aspect, the sequence or sequences of the one or more polynucleotides purified from the reaction mixture are compared to the sequence of the target polynucleotide before combination with the engineered Cas polypeptide disclosed herein to determine modification of the target polynucleotide.

[0375] The kit may include any one or more of the compositions disclosed herein that can be used for in vitro or in vivo polynucleotide detection, binding and / or modification. The kit includes an engineered Cas polypeptide disclosed herein (e.g., an engineered Cas endonuclease) or a polynucleotide encoding the engineered Cas polypeptide or endonuclease, optionally further comprising a buffer component capable of achieving effective storage, and one or more additional compositions, which can introduce the engineered Cas polypeptide into a cell or system comprising a heterologous polynucleotide, wherein the engineered Cas polypeptide can achieve modification, addition, deletion or substitution of at least one nucleotide of the heterologous polynucleotide. In another aspect, the engineered Cas polypeptide disclosed herein can be used to enrich one or more polynucleotide target sequences from a mixed pool. In another aspect, the engineered Cas polypeptide disclosed herein can be fixed on a matrix for use in in vitro target polynucleotide detection, binding and / or modification.

[0376] For the purpose of storage, purification and / or characterization, the engineered Cas endonuclease can be attached, associated or attached to a solid substrate. Examples of solid substrates include, but are not limited to, filters, chromatographic resins, assay plates, test tubes, cryogenic vials, etc. The Cas endonuclease can be fully purified and stored in a suitable buffer solution, or lyophilized.

[0377] Detection Methods

[0378] Methods for detecting engineered Cas polypeptide:guide polynucleotide complexes bound to a target polynucleotide may include any method known in the art, including but not limited to microscopy, chromatographic separation, electrophoresis, immunoprecipitation, filtration, nanopore separation, microarrays, and those described below.

[0379] DNA Electrophoretic Mobility Shift Assay (EMSA): studies proteins bound to known DNA oligonucleotide probes and assesses the specificity of the interaction. The technique is based on the principle that protein-DNA complexes migrate more slowly than free DNA molecules when subjected to polyacrylamide or agarose gel electrophoresis. Since the migration of the DNA is retarded upon protein binding, the assay is also called a gel retardation assay. Adding a protein-specific antibody to the bound components results in a larger complex (antibody-protein-DNA) that migrates even slower during electrophoresis, which is called a supershift and can be used to confirm the protein's identity.

[0380] DNA pull-down assay uses a DNA probe labeled with a high affinity tag (e.g., biotin), which allows recovery or fixed probes. DNA probes can be compounded with the protein of the cell lysate from a similar reaction used in EMSA and then used to purify using agarose or magnetic beads. Then from DNA eluted protein, and detected by Western blotting or identified by mass spectrometry. Alternatively, affinity tag labeled protein can be used, or the antibody separation DNA-protein complex (similar to super-variable assay) for the target protein can be used. In this case, the unknown DNA sequence bound to the protein is detected by Southern blotting or PCR analysis.

[0381] The reporter assay provides real-time in vivo readout of the translation activity of the target promoter.Reporter gene is a fusion of a target promoter DNA sequence and a reporter gene DNA sequence (the reporter gene DNA sequence is customized by the researcher and encoded with a protein that can detect characteristics, such as firefly / Rainilla luciferase or alkaline phosphatase).These genes only produce enzymes when the target promoter is activated.Enzyme then catalyzes substrates to produce light or color changes that can be detected by spectral instruments.The signal from the reporter gene is used as an indirect determinant for the translation of the endogenous protein driven by the same promoter.

[0382] Microplate capture and detection assays use immobilized DNA probes to capture specific protein-DNA interactions and confirm protein identity and relative amounts with target-specific antibodies. Typically, DNA probes are immobilized on the surface of a 96- or 384-well microplate coated with streptavidin. Cell extracts are prepared and added to allow binding proteins to bind to the oligonucleotides. The extracts are then removed and each well is washed several times to remove non-specifically bound proteins. Finally, the proteins are detected using specific antibodies labeled for detection. This method is very sensitive and can detect less than 0.2 pg of target protein / well. This method can also be used for oligonucleotides labeled with other tags (e.g., primary amines that can be fixed on microplates coated with amine-reactive surface chemicals).

[0383] DNA footprinting is one of the most widely used methods to obtain detailed information about individual nucleotides in protein–DNA complexes or even inside living cells. In such experiments, chemicals or enzymes are used to modify or digest DNA molecules. When sequence-specific proteins bind to DNA, they can protect the binding sites from modification or digestion. This can subsequently be visualized by denaturing gel electrophoresis, where the unprotected DNA is cleaved more or less randomly. It therefore appears as a "ladder" of bands, and the protein-protected sites have no corresponding bands and look like footprints in the banding pattern. From this footprint, the specific nucleoside at the protein-DNA binding site can be identified.

[0384] Microscopy techniques include optical, fluorescence, electron and atomic force microscopy (AFM).

[0385] Chromatin immunoprecipitation (ChIP) assays covalently bind proteins to their DNA targets, which can then be unlinked and characterized separately.

[0386] Systematic Evolution of Ligands by Exponential Enrichment (SELEX) exposes the target protein to a random library of oligonucleotides. Those that bind are isolated and amplified by PCR.

[0387] Although the present invention has been clearly shown and described with reference to preferred embodiments and various alternative embodiments, it will be appreciated by those skilled in the art that various changes may be made to it in form and detail without departing from the spirit and scope of the present invention. For example, although the following specific examples may use specific target sites or target organisms to illustrate the methods and embodiments described herein, the principles in these examples may be applied to any target site or target organism. Therefore, it should be understood that the scope of the present invention is covered by the embodiments of the present invention described herein and in the specification, rather than by the specific examples exemplified below. For all purposes, all cited patents, applications, and publications mentioned in this application are incorporated herein by reference in their entirety, to the extent that each is individually and specifically incorporated by reference, except for any definition, subject disclaimer, or denial, and unless the incorporated material is inconsistent with the extent explicitly disclosed herein, in such a case, the language in the present disclosure shall prevail.

[0388] Examples

[0389] The following are examples of specific embodiments that disclose some aspects of the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. With regard to the numbers used (e.g., amounts, temperatures, etc.), efforts have been made to ensure their accuracy, but some experimental errors and deviations should still be allowed.

[0390] Example 1: Cas-α nuclease and guide RNA expression cassette

[0391] In this example, methods for generating Cas-α endonuclease and guide RNA expression cassettes in Saccharomyces cerevisiae, Zea mays, and Homo sapiens cells are described.

[0392] For Saccharomyces cerevisiae, the gene encoding the Cas-α endonuclease was yeast codon optimized. To promote nuclear localization of the optimized Cas-α endonuclease protein, a nucleotide sequence encoding the simian virus 40 (SV40) monotypic nuclear localization signal (NLS) (PKKKRKV (SEQ ID NO: 13)) was added to the 5' end and / or the 3' end. The nucleotide sequences of the optimized Cas-α endonuclease gene and NLS variants were then synthesized and operably cloned into a low copy yeast plasmid DNA containing a CEN6 replication origin (ORI) between the ROX3 promoter and the CYC1 terminator (GenScript). Examples of yeast optimized Cas-α nuclease expression cassettes and references to the sequences described herein can be found at Figure 1 Found in.

[0393] Cas α endonucleases are guided by small RNAs (referred to herein as guide RNAs) to cleave double-stranded DNA in the presence of a 5' pre-spacer adjacent motif (PAM) (Bigelyte et al. (2021), Nature Communications. [Natural Communications] 12:6191, Karvelis et al. (2020), Nucleic Acids Research. [Nucleic Acids Research] 48:5016-5023, U.S. Patent Application Publication No. US20200190494 A1, and International Patent Application Publication WO 2022 / 082179). These guide RNAs include sequences that assist Cas-α recognition (referred to as Cas-α recognition domains) and sequences for guiding Cas-α cleavage by base pairing with one strand of the DNA target site (Cas-α variable targeting domains). In order to transcribe the small RNA necessary for the Cas-α endonuclease cleavage activity in Saccharomyces cerevisiae cells, a DNA sequence encoding the hepatitis delta virus ribozyme is first appended to the 3' end of a DNA sequence encoding a Cas-α single guide RNA (sgRNA) (which has a variable targeting domain capable of targeting the yeast ADE2 or CAN1 gene). Next, the SNR52 promoter and SUP4 terminator are operably linked to the ends of the ribozyme and Cas-α encoding sgRNA, and G bp is incorporated into the 3' end of SNR52 to promote transcription of the Cas-α sgRNA. The DNA fragment is then synthesized and cloned into the Saccharomyces cerevisiae CEN6 vector containing the cas-α gene (GenScript). A schematic diagram of the yeast-optimized Cas-α guide RNA expression cassette and references to the sequences described herein are shown in Figure 1 middle.

[0394] The maize Cas-α nuclease expression cassette is generally established as described above, except that the sequence encoding the nuclease is codon-optimized using the maize codon table, the GC content is adjusted using standard techniques, and repetitive sequences and gene instability features, such as miniature inverted repeat transposable elements (MITEs), are removed. The resulting gene (GenScript) is then synthesized and cloned in a Gateway-compatible plasmid DNA containing a polymerase II promoter and terminator by restriction enzyme digestion and ligation. For expression in maize, this includes the maize ubiquitin (UBI) promoter and terminator. In order to further enhance expression, a 5' untranslated region (UTR) (such as, but not limited to, maize UBI 5'UTR) and additional introns (such as UBI maize intron 1 and potato ST-LS1 intron 2) are used, although other introns may also work. A schematic diagram of a maize optimized Cas-α endonuclease expression construct is shown in Figure 8 As shown in the figure above.

[0395] The Cas-α sgRNA expression cassette was generally established as described above, except that the maize U6 promoter and terminator were utilized and a variable targeting domain capable of directing the Cas-α endonuclease to the maize MS26 (SEQ ID NO: 303), CR36 (SEQ ID NO: 304), or D8 (SEQ ID NO: 305) target site was incorporated. A schematic diagram of the maize optimized Cas-α sgRNA expression cassette is shown in Figure 8 As shown in the figure below.

[0396] For testing in human cells, Cas-α endonuclease expression cassettes of SEQ ID NOs: 14, 60, 157, and 362 were constructed, generally as described above, except that the sequence encoding the nuclease was human codon optimized, sequences encoding the ribosome skipping T2A peptide (Ahier et al. (2014) Genetics. [Genetics] 196: 605-613) and green fluorescent protein (GFP) were fused in-frame to the 3' end of the Cas-α endonuclease open reading frame, human beta herpes virus promoter and enhancer were used to drive expression, and sequences from human gamma herpes virus were used to terminate transcription. A schematic diagram of the human optimized Cas-α nuclease expression cassette is shown in Figure 6 As shown in the figure above.

[0397] Next, the Cas-α sgRNA expression cassette was established as described above, except that the human U6 promoter and terminator were utilized, and a variable targeting domain capable of directing the Cas-α endonuclease to the human RUNX (SEQ ID NO: 306), WTAP (SEQ ID NO: 307), or DNMT (SEQ ID NO: 308) target site was incorporated. A schematic diagram of the human optimized Cas-α sgRNA expression cassette is shown in Figure 6 As shown in the figure below.

[0398] Example 2: Cas-α nuclease and guide RNA expression cassette transformation

[0399] In this example, methods for transforming Cas-α endonuclease and guide RNA expression cassettes into Saccharomyces cerevisiae, Zea mays, and Homo sapiens cells are described.

[0400] For Saccharomyces cerevisiae, a method similar to the lithium cation-based method was used, using the Freeze-EZ Yeast Transformation II Kit (Zymo Research). Saccharomyces cerevisiae competent cells were generated by culturing Saccharomyces cerevisiae (BY4742 (Baker et al. (1998) Yeast. 14: 115-132) (ATCC)) in yeast extract-peptone-glucose (YPD) liquid medium (Gibco) to mid-log phase (corresponding to OD 600nm of 0.8-1.0) according to the manufacturer's instructions. Next, the cells were precipitated by centrifugation (500xg for 4 minutes), the culture medium was decanted, the precipitate was gently washed with 10 ml of EZ 1 solution, and the cells were spun down again before removing the wash solution. The cells were then resuspended in 1 ml of EZ 2 solution, aliquoted, and stored at -70°C or used in the next step. Next, 0.5 μg-1 μg (less than 5 μl) of CEN6 yeast plasmid DNA containing Cas-α nuclease and single guide RNA expression cassette is added to 50 μl competent cells for transformation. Optionally, double-stranded DNA repair templates (0.5 μl, 50 μM) with homology to the expected Cas-α double-strand break site flank are also included. After gently mixing the DNA, 500 μl EZ 3 solution is added. The cells are then incubated at 30 ° C for 60-90 minutes. The cells are flicked or vortexed 3-4 times during the duration of incubation. After transformation, cells were cultured in YPD liquid medium for about 3 hours, pelleted, washed once with 1 ml sterile water, resuspended in 1 ml sterile water, and then about 200 μl was plated onto selective media (e.g., but not limited to, 6.7 g / L Yeast Nitrogen Medium without Amino Acids (Becton Dickinson), 20 g / L Glucose (Phytotechnology Labs), 1.92 g / L Yeast Histidine Dropout Medium (MP Biomedicals), and 20 g / L Bacto Agar (Becton Dickinson). To determine the most suitable culture conditions for activity, cells were incubated at 30°C until colonies formed, or incubated overnight at a range of temperatures (typically 25°C, 30°C, 37°C, and 45°C) and then returned to 30°C until colony growth was visible.

[0401] For maize transformation, particle-mediated biolistic transformation, DNA expression cassettes were coprecipitated onto 0.6 μM (average size) gold particles using TransIT-2020. The gold particles coated with DNA were precipitated by centrifugation, washed with anhydrous ethanol and redispersed by ultrasound. After ultrasound treatment, 10 μl of gold particles coated with DNA were loaded onto a macrocarrier and air-dried. Biolistic transformation was performed using a PDS-1000 / He Gun (Bio-Rad) with a 425 psi rupture disk. To promote cell division, BabyBoom ​​(BBM) and Wuschel2 (WUS2) genes expressed from non-constitutive promoters, maize phospholipid transferase protein and maize auxin inducer, respectively, are also co-delivered with Cas-α endonuclease and guide RNA expression cassettes (Lowe et al. (2018) In vitro cellular & developmental biology-Plant. [In vitro cell and developmental biology-plant] 54: 240-252). Visual marker DNA expression cassettes encoding fluorescent proteins (e.g., yellow fluorescent protein) can also be co-delivered to facilitate the selection of uniformly transformed tissues. In addition, chemical selectable markers (e.g., but not limited to neomycin phosphotransferase II) can also be delivered with the above plasmids to select transformed cells. In order to check Cas-α nuclease-guide RNA activity, the transformed tissue can be incubated at 28°C (the standard for particle gun transformation) or a temperature range below or above 28°C.

[0402] For Agrobacterium delivery, the Cas-α nuclease and associated sgRNA expression cassettes are Gateway cloned into T-DNA, which optionally already contains genes encoding morphogenic transcription factors Bbm and Wus2, which are operably linked to Zm-PLTP and Axig1 promoters, respectively. The resulting T-DNA is then placed into LBA4404, a thymine auxotrophic strain of Agrobacterium tumefaciens containing Vir9, a separate plasmid encoding Bo542 virulence genes (US20170121722 A1 and WO 2017 / 078836). The resulting Agrobacterium strain is then used to transform immature maize embryos (approximately 2 mm in size) of nine to ten days old, which are immersed in a 700A liquid medium containing the strain (optical density (OD) of 0.4 to 2.0 at 550nm) for 5 minutes, then removed from the culture medium, and placed in a solid co-culture medium, overnight at 21°C in the dark. After T-DNA delivery, the embryo is transferred to a resting culture medium 13266R and grown in the dark at 28°C for 5-7 days. Next, the coleoptile is removed, and the embryo is transferred to a selection culture medium for 11-16 days and maintained in the dark at 28°C. They are then moved to a maturation culture medium and incubated in the dark at 28°C for 14-25 days, then incubated under illumination for 2-5 days. Next, they are transferred to a rooting culture medium and incubated under 26-28°C illumination for 14-28 days, with the culture medium being renewed as needed. To examine Cas-α nuclease-guide RNA activity, transformed tissues were incubated at 28°C (standard for Agrobacterium transformation) or at a range of temperatures below or above 28°C.

[0403] Human cell transformation was performed by culturing HEK293T cells (ATCC catalog number CRL-3216) in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U / ml) / streptomycin (100 ug / ml) at 37°C in 5% CO2. The cells were then seeded into 96-well plates at a density of 1.8×104 cells / well. After about one day of growth, cells were transfected with plasmids encoding nucleases and U6-guide RNA expression cassettes using FuGENE HD (Promega) according to the manufacturer's instructions. Briefly, on the day of transfection, 0.450 μg of plasmid DNA (0.353 μg of Cas-α nuclease and 0.099 μg of sgRNA expression cassette) was diluted in a final volume of 20 μl of room temperature Opti-MEM (Gibco), mixed, and 1.35 μl of FuGENE HD was added directly to the solution (undiluted transfection reagent was not allowed to contact the sides or bottoms of the tubes or plates) to achieve a 3:1 ratio of transfection reagent to plasmid DNA. The resulting mixture was then incubated at room temperature for 10 minutes, 20 μl was added to each HEK293T inoculation well, and gently mixed by pipetting or using a shaker. The transfected cells were then grown at 37°C, 5% CO2 for 4 days, and GFP expression was monitored by fluorescence microscopy. Experiments were performed in three to twelve independent replicates, and transfections omitting Cas-α10 endonuclease and / or sgRNA plasmid DNA expression cassettes were used as negative controls.

[0404] Example 3: Detection of Cas-α and guide RNA variants with improved cleavage of cellular DNA targets

[0405] In this example, methods are described for detecting Cas-α endonuclease and guide RNA variants with improved double-stranded DNA target cleavage at low dose or expression.

[0406] In one approach, Cas-α nuclease variants and associated single guide RNAs (sgRNAs) were encoded on low-copy (2-5 copies per cell) Saccharomyces cerevisiae CEN6 autonomously replicating yeast plasmid DNA (Karim et al. (2013) FEMS Yeast Research. [FEMS Yeast Research] 13: 107-116) and expressed using the ROX3 promoter (which has previously been shown to support weak gene expression) (Generoso et al. (2016) Journal of Microbiological Methods. [Microbiological Methods Journal] 127: 203-205). Variants with improved cell-targeted DSB activity were then selected by targeting the ADE2 and / or CAN1 genes in Saccharomyces cerevisiae (BY4742, genotype-MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0). See Figure 2 and Figure 3 .

[0407] When targeting the ADE2 gene, by monitoring the non-functional ade2 gene ( Figure 2 ) formation caused by the transition from white to red (pink) cell phenotype to select Cas-α nuclease and guide RNA target cutting. When regarded as a colony and depending on how fast the ADE2 gene is destroyed, the red coloration varies from completely red in the originally white colony to observing one or more smaller red sectors. These phenotypic patterns are also used to quantify the activity differences between variants. Colonies scored as completely red, colonies containing several sectors or only one red sector are counted and divided by the total number of colonies present. In some cases, colonies are not counted, but images of yeast colonies are taken with a Nikon Digital Sight Ds-Fi1 camera (Nikon, Japan) and NIS Elements BR software (version 4.00.07) (Nikon, Japan), and analyzed by first determining the total area of ​​yeast (in pixels) and then calculating the total percentage of red using a custom script.

[0408] For the CAN1 experiment, Cas-α nuclease variants with improved on-target DSB activity were selected by plating on media containing L-canavanine, a toxic analog of arginine. In this assay, on-target cleavage and non-functional repair of the CAN1 gene ( Figure 3) provides resistance to L-canavanine by disrupting the transport of L-canavanine into cells (Whelan et al. (1979) Genetics. [Genetics] 91: 35-51). Therefore, if the CAN1 gene is cut and non-functionally repaired, L-canavanine will not be transported across the cell membrane and the cell will survive; on the contrary, if the CAN1 gene is not cut, L-canavanine will enter, causing cell death ( Figure 3 ). To adjust the selection pressure, growth in YPD medium was varied between 2-7 hours before plating on L-canavanine (60 mg / mL). In some experiments, additional selection was also incorporated by adjusting the growth temperature between 30°C and 37°C depending on the Cas-α nuclease thermal preference and the desired selection pressure.

[0409] In some cases, both the ADE2 and CAN1 genes in Saccharomyces cerevisiae were targeted for Cas-α endonuclease target cleavage. Here, both the disrupted ade2 red cell phenotype and resistance to L-canavanine were used to select for Cas-α nuclease variants with improved targeted DSB activity.

[0410] Once an improved Cas-α variant is identified, it is transferred to 5 ml of yeast liquid medium without histidine (such as, but not limited to, 6.7 g / L yeast nitrogen substrate without amino acids (Becton Dickinson Medical), 20 g / L glucose (Plant Technology Laboratories), and 1.92 g / L yeast histidine dropout medium (MP Biomedical)) and incubated overnight with shaking at 30°C. The CEN6 plasmid encoding one or more variants is then isolated using the Yeast Plasmid Miniprep 96 Kit (Zemo Research). After purification, the plasmid is transformed into TransforMax EPI300 (Lucigen) Escherichia coli (E. coli) competent cells according to the manufacturer's instructions and plated onto selective media (such as, but not limited to, 6.7 g / L yeast nitrogen substrate without amino acids (Becton Dickinson Medical), 20 g / L glucose (Plant Technology Laboratories), 1.92 g / L yeast histidine dropout medium (MP Biomedical), and 20 g / L Bacto agar (Becton Dickinson Medical)). In order to explain the possibility of multiple CEN6 vectors being transformed and maintained in a single yeast cell, 4-6 colonies were selected from each E. coli transformation and used to inoculate 2 ml 2X YT medium (Sigma-Aldrich) or an equivalent containing kanamycin. The culture was then grown overnight at 37 ° C with shaking, and then half of the culture was subjected to rolling circle amplification and Sanger sequencing (Eurofins Scientific) using primers specific to the region directly adjacent to or internal to the cas-α gene. After sequencing, plasmid DNA was isolated from E. coli culture (the remaining half) (shown to contain different variants) using Qiagen Spin Miniprep kit (Qiagen). Finally, about 1 μg of each plasmid was re-transformed back into Saccharomyces cerevisiae, and red cell phenotype and / or L-canavanine resistance were evaluated to confirm improvement. To make comparisons between improved variants and directed evolved Cas-α endonucleases, all variants were typically subjected to additional yeast transformations and reassessed for their ability to recognize and cleave the ADE2 and / or CAN1 target sites.

[0411] In the second assay, the Cas-α nuclease and sgRNA target cutting efficiency were evaluated by measuring the frequency of small insertion or deletion (indel) mutations caused by cell repair of DNA target cutting of chromosomes in maize or human cells. To this end, experiments similar to those described in Svitashev et al., 2015, Karvelis et al., 2015 and Bigelyte et al., 2021 were performed.

[0412] For maize, two types of assays were used. In the first, transformed immature maize embryos were harvested 2 - 10 days after transformation, genomic DNA was extracted and the presence of mutations indicative of RNA-guided Cas-α editing was examined at the Cas-α target by Ampli-seq. Briefly, for each experiment, 20 - 30 of the most uniformly transformed immature embryos were harvested based on their fluorescence. Next, total genomic DNA was extracted and the region surrounding the expected target site was PCR amplified and deep sequenced by two rounds of PCR using a high-fidelity PCR premix (New England Biolabs, Inc., M0531L) plus sequences necessary for amplicon-specific barcoding and Illumina sequencing (using "tailed" primers). The resulting reads were then examined for the presence of mutations at the expected cleavage sites by comparison with a control experiment in which the small RNA expression cassette was omitted from the transcript. Sequence reads containing putative indels were further validated as true mutations by confirming their absence from the control dataset.

[0413] For the second maize assay, regenerated plants were sampled and the DNA targets were examined for editing evidence using Ampli-Seq. This was done as described above, but to assess heritability, the frequencies of edited sequence reads and wild-type sequence reads were also calculated for each plant. Since maize is diploid, for targeted mutations, plants with approximately 50% and 100% mutant reads could be assumed to be heterozygous and homozygous, respectively (Zhang et al., (2014) Plant Biotechnology Journal. 12:797 - 807).

[0414] For human cells, cells were washed twice with PBS two to four days after transfection and lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 0.2% Tween 20, 0.2 mg / ml Proteinase K). Next, the presence of targeted mutations at the RUNX and WTAP targets was evaluated using T7 endonuclease I (Guschin, D.Y. et al. (2010) In: Mackay J., Segal D. (Eds.) Engineered Zinc Finger Proteins. Methods in Molecular Biology (Methods and Protocols), Vol. 649. Humana Press, Totowa, NJ) and Illumina deep sequencing as described above.

[0415] Example 4: Library Design and Generation

[0416] In this example, methods for designing and generating Cas-α endonuclease variants for improved cleavage of double-stranded DNA targets are described.

[0417] In one approach, saturation mutagenesis was performed to generate a Cas-α10 variant, SEQ ID NO: 14 ( Figure 4 ) are introduced at every position of the entire amino acid sequence of all other amino acids (19 in total). In a second approach, the beneficial changes identified herein are incorporated (alone or in combination) into a variant that already contains one or more beneficial changes, and their combined effect is determined. In a third approach, the amino acid substitutions in a variant containing two or more changes are reverted to the wild-type residue, the improvement in cleavage activity is determined, and if two or more beneficial reversions are identified, they are combined and the improvement is re-determined.

[0418] For the first library method, GenPlus gene synthesis technology (GenScript) was used to change Figure 1 The codons in the cas-α nuclease gene (SEQ ID NO: 2) in the yeast expression plasmid shown are used to encode different amino acids. For library methods two and three, the cas-α nuclease gene encoding amino acid substitutions and / or reversions was generated by synthesis (GenScript) and / or introduced using site-directed mutagenesis as described earlier (Kunkel (1985) Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States] 82, 488-492).

[0419] Example 5: Cas-α endonuclease variants with improved cellular activity

[0420] In this example, Cas-α endonuclease variants with improved double-stranded DNA cleavage activity are described. All variants generated in Example 4 were tested. These include variants with all possible amino acid substitutions over the entire length of the protein (positions 1-497, relative to SEQ ID NO: 14), variants containing combinations of beneficial changes, and variants consisting of a combination of beneficial changes and one or more amino acids that are returned to wild-type residues. The assay was performed to capture variants with improved desired activity.

[0421] In maize, the amino acid substitutions of Cas-α10 variants SEQ ID NO:14, A40G, E81G, A87K, T190K, T217H, K298S, H306F, T335R and I405N were respectively returned to wild-type residues (see equivalent positions in SEQ ID NO:157) and their effects on targeted mutagenesis were evaluated. In these experiments, immature embryos were stored at 28 ° C until one day after particle-mediated transformation, and then they were incubated at 33 ° C for 72 hours and harvested for analysis. In addition, plasmids encoding the guide RNA recognition domain of Cas-α10sgRNA SEQ ID NO:61 targeting MS26 or CR36 sites were co-delivered with plasmids encoding the corresponding Cas-α10 nuclease variants. As shown in Table 2, several amino acids (G81E, S298K, F306H, and N405I) resulted in an increase in the frequency of targeted mutagenesis when changed back to the wild-type residue.

[0422] Table 2

[0423]

[0424] The foregoing results identify a set of amino acid substitutions that, if reverted to wild-type residues, can be used to engineer Cas-α polypeptide variants that provide significantly improved DNA cleavage activity and targeting efficiency. Cas-α polypeptide variants with improved DNA cleavage activity and targeting efficiency include, but are not limited to, SEQ ID NOs: 48, 313-323, and 362.

[0425] In yeast, when the Cas-α10 nuclease and guide RNA (recognition domain from the original Cas-α10 sgRNA (SEQ ID NO: 61)) were encoded on low-copy CEN6 yeast plasmid DNA and the Cas nuclease was expressed from the weak ROX3 promoter, all possible amino acid substitutions over the entire length of the protein (positions 1-497, relative to SEQ ID NO: 14) were next screened for those amino acid substitutions that improved double-stranded DNA target cleavage activity. Initial selection was performed by targeting the ADE2 and CAN1 Saccharomyces cerevisiae genes, with a growth temperature regimen of 1 hour at 30°C, then 2 hours at 37°C, or 1 hour at 30°C, then 4 hours at 37°C. After growth, cells were plated on medium containing L-canavanine, placed at 37°C overnight, and then grown at 30°C until visible colony growth. Variants that survived L-canavanine and exhibited the ade2 red cell phenotype were selected and sequenced. In order to visualize the changes in the ade2 red cell phenotype relative to the starting Cas-α nuclease variants ( Figure 4, SEQ ID NO: 14), the experiment was then set up using only ADE2 screening. To this end, cells were plated on adenine-deficient medium immediately after transformation and placed overnight at 37°C and then grown at 30°C until colony growth appeared (about 2 days). Photos of S. cerevisiae colonies were taken and the total percentage of ade2 red phenotypes was calculated by image analysis using a custom script. The total percentage of red S. cerevisiae produced by each variant was divided by the total percentage of red S. cerevisiae produced from the original protein ( Figure 4 , SEQ ID NO: 14) was used to calculate the fold improvement in targeted DNA cleavage (Table 3).

[0426] Table 3

[0427]

[0428] Combinations of the changes listed in Table 3 were next tested to identify those that provided additional improvements using ADE2 screening and overnight incubation at 37°C as described above. Certain combinations also incorporated the G81E revertant (SEQID NO:48) identified in the maize experiment. Based on visual assessment of the ade2 red phenotype, several combinations showed further improvement in targeted DNA cleavage activity (Table 4). To further distinguish each variant, overnight incubation at 37°C was omitted, and cells were incubated immediately at 25°C or 30°C after transformation until colonies were visible. As shown in Table 4, all combinations improved activity at 30°C; and M45S+F54D+G81E+K85Q+G470D (SEQ ID NO:50) provided an improvement relative to the original protein ( Figure 4 , SEQ ID NO: 14) had the highest activity gain (1.52-fold at 37°C, 29.05-fold at 30°C, and 29.58-fold at 25°C).

[0429] Table 4 lists amino acid substitutions that, when combined, further improved double-stranded DNA target cleavage activity. S. cerevisiae experiments were performed as described above, and the fold improvement in targeted DNA cleavage relative to the native protein was calculated as described above.

[0430] Table 4

[0431]

[0432]

[0433] K85Q was also predicted to be involved in PAM recognition using AlphaFold. Therefore, for M45S+F54D+G81E+K85Q+G470D (SEQ ID NO: 50) and T34E+M45S+F54D+G81E+K85Q+G470D (SEQ ID NO: 54), it was converted back to the K residue and retested using two ADE2 target sites. The test was performed as described above, and the fold improvement in targeted DNA cleavage relative to the original protein was calculated as described above. The results shown in Table 5 show that changing the K residue has a negligible effect on the activity at the initial ADE2 target (ADE2-CR2, SEQ ID NO: 57) with 5'-ATTC-3' PAM. However, when used for the second ADE2 target site with a 5'-GTTC-3' PAM (ADE2-CR1, SEQ ID NO:58), the restored K residue greatly improved activity, as shown in Table 5, where T34E+M45S+F54D+G81E+G470D (SEQ ID NO:60) ( Figure 5 ) demonstrated the greatest fold improvement for both targets.

[0434] Table 5

[0435]

[0436]

[0437] In addition, AlphaFold was used to predict that F54D is likely to change the oligomeric state of Cas-α10 and return to phenylalanine. The targeted mutation efficiency of the new variant T34E+M45S+G81E+G470D (SEQ ID NO: 362) was then compared with T34E+M45S+F54D+G81E+G470D (SEQ ID NO: 60) in human cells (at 37 ° C). As shown in Table 6, T34E+M45S+G81E+G470D (SEQ ID NO: 362) showed enhanced targeted mutagenesis at DNMT and WTAP sites, and performed comparable to T34E+M45S+G81E+G470D (SEQ ID NO: 362) at the RUNX target.

[0438] Table 6

[0439]

[0440] Next, the double-stranded DNA target cleavage activity of SEQ ID NO: 157, SEQ ID NO: 14, SEQ ID NO: 48, and SEQ ID NO: 362 was evaluated biochemically similar to that described in Gasiunas et al. (2020) Nat Commun [Nature · Communication] 11: 5512. Briefly, Cas-α10 protein was expressed and purified as described in Bigelyte et al., 2021, and sgRNA was generated using the HiScribe T7 Fast High Yield RNA Synthesis Kit (NEB). The sgRNA was then diluted in 1X reaction buffer (Tris, pH 7.5, 200nM NaCl, 1mM DTT, 1mM EDTA, 10mM MgCl2, and 0.01mM ZnCl2) and heat denatured at 90°C for 3 minutes, followed by slow cooling to 12°C. Next, the sgRNA was combined with purified Cas-α10 nuclease pre-diluted in 1X reaction buffer, incubated at room temperature for 5-10 minutes, then moved to the desired reaction temperature for 2-3 minutes, and then mixed with 5'-FAM labeled double-stranded DNA substrate. The reaction was performed in 10 μl of 1X reaction buffer, with the final concentrations of sgRNA, Cas-α nuclease, and DNA substrate being 200 nM, 200 nM, and 10 nM, respectively. After 30 minutes, the reaction was quenched using stop buffer (1X reaction buffer, 67.5 mM EDTA (final concentration), 0.1 U / μl proteinase K (final concentration), and the extent of target cleavage was determined using capillary electrophoresis after dilution 4X in nuclease-free water.

[0441] The effect of temperature on nuclease activity was preliminarily evaluated. Fig. 9 As shown, the wild-type Cas-α10 nuclease (SEQ ID NO: 157) robustly cleaves its double-stranded DNA substrate (fraction of cleaved targets > 0.6) over a temperature range of 42°C to 58°C. This can be contrasted with SEQ ID NO: 14, SEQ ID NO: 48, and SEQ ID NO: 362, which have reduced requirements for high temperatures ( Figure 9-11 In addition, as a result of the engineering described herein, the temperature range over which SEQ ID NO:48 effectively cleaves its target is expanded to 30°C-48°C ( ) compared to SEQ ID NO:14, which has a narrower temperature preference (37°C-43°C). Fig.10 Finally, the thermal spectrum of SEQ ID NO: 362 was further shifted toward lower temperatures, demonstrating efficient substrate cleavage at temperatures as low as 25°C ( Fig.11 ).

[0442] The foregoing results identify a collection of amino acid changes that can be used to engineer Cas-α polypeptide variants that provide significantly improved DNA cleavage activity, targeting efficiency, and / or PAM preference. Figure 4 , SEQ ID NO: 14), the functional improvement of the disclosed variants is significant. The results also show that the disclosed variants and amino acid changes can broaden the possible application range and range of organisms, where such engineered Cas-α polypeptide variants can be used as Cas effectors (e.g., endonucleases), including for targeted genome editing.

[0443] Example 6: Cas-α sgRNA variants with improved cellular activity

[0444] In this example, Cas-α sgRNA variants with improved double-stranded DNA cleavage activity are described.

[0445] In one method, three nucleotides of the Cas-α10 sgRNA recognition domain (SEQ ID NO: 61) were systematically deleted in one nucleotide overlapping interval, and the ability to cut the ADE2 gene was evaluated as described in Example 5 above, except that two yeast plasmids were used. The first contained a CEN6 ORI and a Cas-α10 endonuclease (SEQ ID NO: 14) expression cassette, while the second contained a 2 micron ORI and an sgRNA expression cassette. The DNA sequence encoding the sgRNA variant was generated in Saccharomyces cerevisiae by inverse PCR and seamless cloning using a 2 micron plasmid (which encodes an sgRNA (SEQ ID NO: 62) capable of guiding Cas-α10 endonuclease to cut at the ADE2-CR2 target site). The experiment was performed at 37°C, and the percentage of the red cell phenotype produced by ADE2-CR2 target cutting was normalized to the original Cas-α sgRNA (SEQ ID NO: 62). Values ​​of about 1.00 represent comparable activity to the original sgRNA, while values ​​above or below about 1.00 correspond to increased or decreased cleavage of the ADE2-CR2 target, respectively. The results of deletions of different three nucleotide regions of the Cas-α10 sgRNA recognition domain are shown in Table 7.

[0446] Table 7

[0447]

[0448]

[0449]

[0450] The results in the above Table 7 (percentage of red yeast) show that the trinucleotide deletions at positions 1-3, 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 147-149, 149-151, 151-153, 153-155, 157-159, 159-161, 163-165 and 167-169 are tolerated. In particular, the deletions at 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 149-151, 151-153, 153-155, 157-159, 163-165 and 167-169 lead to an increased efficiency of target cleavage at the ADE2-CR2 site.

[0451] Next, deletions in the sgRNA recognition domain that improved or had negligible effects on DNA target cleavage activity were combined and tested for improvement in human cells. They were introduced into the recognition domains of four sgRNA designs (Cas-α10 original sgRNA (SEQ ID NO: 61), Cas-α10 sgRNA MS10 (SEQ ID NO: 324), Cas-α10 sgRNA MS13 (SEQ ID NO: 325), and Cas-α10 sgRNA design 5 (SEQ ID NO: 310 in WO-2022082179-A2) (SEQ ID NO: 326)), as listed in Tables 8 and 9. Since Cas-α10sgRNA MS10, Cas-α10sgRNA MS13 and Cas-α10sgRNA design 5 already contain deletions at positions 1-3, 3-5, 5-7, 7-9, 147-149, 149-151 and 151-153, these nucleotides do not need to be removed (Tables 8 and 9). In addition, deletions at positions 157-159 and 167-169 were not tested using Cas-α10sgRNA MS13 because there were no regions covering these deletions (Tables 8 and 9). In addition, in some cases, the MS13 solution was further modified by replacing the sequence connecting the tracrRNA and crRNA portions or sgRNA with 5'-GAAA-3' tetraloop (SEQ ID NO: 327) (Tables 8 and 9).

[0452] At the RUNX target, the new sgRNA solution derived from design 5 sgRNA in combination with Cas-α10 endonuclease variants (SEQ ID NO: 14 and SEQ ID NO: 362) had the greatest effect on enhancing targeted indel frequency, with the highest mutagenesis frequency obtained using design 5_157-159 (Table 8).

[0453] Table 8

[0454]

[0455]

[0456]

[0457] At the WTAP site, design 5sgRNA produced the highest targeted mutation frequency when paired with Cas-α10 endonucleases SEQ ID NO: 14 and 3362 (Table 8). When used in combination with Cas-α10 nuclease SEQ ID NO: 157, design 5_157-159 produced the highest targeted mutagenesis rate (Table 9).

[0458] Table 9

[0459]

[0460]

[0461]

[0462] In a second study, RNA polypeptides were added to Cas-α sgRNA. For example, sequences that form hairpin-like secondary structures were incorporated, including the hairpin-like RNA recognition sequence of the MS2 phage coat protein (SEQ ID NO: 208). The secondary structure of Cas-α sgRNA was predicted using the Vienna fold (Gruber et al. (2008) Nucleic Acids Res. [Nucleic Acids Research] 36: W70-74). The MS2 recognition sequence was then incorporated into the 5' end, the top of the stem loop, and the 5'-GAAA-3' linker connecting the tracrRNA and crRNA of the sgRNA, such as Figure 7 As shown. To facilitate proper folding of the aptamer, additional sequences were incorporated on a per sequence addition basis (e.g., multiple G or C nucleotides on either side of the MS2 recognition sequence, arranged in a manner that base pairs with each other). The ability of the solution to direct Cas-α endonuclease target recognition and cleavage at one target in a DNMT gene was tested in human cells using the evolved Cas-α10 nuclease SEQ ID NO:14. Three independent transfections were performed for each sgRNA variant; negative controls included experiments in which the Cas-α10 endonuclease and sgRNA plasmid DNA expression cassettes were omitted.

[0463] As shown in Table 10, sgRNAs incorporating MS2 aptamers were tolerated at positions 1, 3, 5, 7, 10, 11, and 12, with improvements in targeted mutagenesis observed at position 1 relative to the original sgRNA. To confirm that the results were not sequence dependent, a hairpin-forming RNA recognition sequence (SEQ ID NO: 209) from the Cas6 protein of Pseudomonas aeruginosa (Sternberg et al. (2012) RNA. 18: 661-672) was also incorporated into the same position of the Cas-α sgRNA and tested. Similar trends in targeted mutagenesis were observed at positions 1, 3, 4, 5, 6, 7, 9, 10, and 11, showing equivalent or better targeted mutagenesis frequencies than the original sgRNA solutions (Table 10).

[0464] Table 9

[0465]

[0466] In the third study, the targeted mutagenesis efficiency of Cas-α10 endonuclease variants SEQ ID NOs: 14, 48, 60 and 157 at two target sites MS26 and D8 was compared when tested with two sgRNA recognition domains (SEQ ID NOs: 61 and 327). The experiment was carried out in maize using Agrobacterium (Agro) transformation as described in Example 2. Two temperature schemes were performed. The first placed Agro-transformed embryos (once a day) at 45°C for 4 hours on days 3, 4 and 5 after infection (3x4 hours at 45°C), and the second was incubated continuously at 37°C (1-3 days at 37°C) on days 1-3 after infection. For both schemes, the embryos were placed at 28°C before and after temperature treatment. In initial tests, embryos were inoculated with Agro (OD of 0.4) and then inoculated with 10% sgRNA (0.5% sgRNA). 550 ) were harvested seven days after incubation and the percentage of targeted mutations was determined as described in Example 3.

[0467] Cas-α10 endonuclease SEQ ID NO:48 combined with sgRNA recognition domain SEQ ID NO:327 showed the highest frequency of targeted mutagenesis at two target sites and two temperature treatments (Table 11). This resulted in an improvement of 11.50-fold and 12.61-fold compared to the wild-type nuclease (SEQ ID NO:157) at MS26 and D8 targets in combination with sgRNA recognition domain SEQ ID NO:61 using 45°C temperature treatment (3x4 hours 45°C). Under the 37°C regimen (1-3 days 37°C), 1053.00 and 13.15-fold enhancements were observed at MS26 and D8 sites, respectively.

[0468] Table 11

[0469]

[0470]

[0471] To confirm the findings in Table 11, TO plants were regenerated for several experiments and the percentage of plants with predicted germline mutations was calculated as described in Example 3. The results confirmed that the Cas-α10 endonuclease variant SEQ ID NO: 48, when paired with the sgRNA recognition domain SEQ ID NO: 327, produced the highest frequency of targeted mutagenesis among the combinations initially tested (Table 12).

[0472] Table 12

[0473]

Claims

1. An engineered Cas polypeptide comprising a sequence having 90% amino acid sequence identity to SEQ ID NO: 14 and one or more of the following amino acids at positions relative to alignment with SEQ ID NO: 14: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamic acid at position 81, or glutamine at position 85, wherein the engineered Cas polypeptide is capable of site-specifically binding to a target site of a polynucleotide.

2. An engineered Cas polypeptide comprising: (a) C-terminal trisection RuvC domain and three zinc finger motifs; and (b) one or more of the following amino acids at positions relative to alignment with SEQ ID NO: 14: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamic acid at position 81, or glutamine at position 85; wherein the engineered Cas polypeptide does not comprise at least one of the following amino acids at positions relative to alignment with SEQ ID NO: 14: phenylalanine at position 38, alanine at position 40, histidine at position 79, alanine at relative position 87, threonine at relative position 335, cysteine ​​at position 409, glutamic acid at position 421, lysine at position 467, or glutamate at position 468, and wherein the engineered Cas polypeptide is capable of site-specific binding to a target site of a polynucleotide.

3. An engineered Cas polypeptide as described in claim 1 or 2, wherein the polypeptide has at least 95% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 14-56 or a group consisting of SEQ ID NOs: 59 and 60, or a group consisting of SEQ ID NOs: 309-323 and SEQ ID NO:

362.

4. The engineered Cas polypeptide of any one of claims 1-3, further comprising at least one of the following amino acids at positions aligned with SEQ ID NO: 14: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at position 226, glycine at position 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at position 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at position 376, cysteine ​​at position 379, cysteine ​​at position 395, cysteine ​​at position 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

5. An engineered Cas polypeptide as described in any one of claims 1-4, wherein the engineered Cas polypeptide is an endonuclease with greater activity than SEQ ID NO: 14 at one or more of the following temperatures: about 40 degrees Celsius, about 37 degrees Celsius, about 35 degrees Celsius, about 30 degrees Celsius, about 25 degrees Celsius, or about 20 degrees Celsius.

6. An engineered Cas polypeptide as described in any one of claims 1-5, wherein the engineered Cas polypeptide has greater polynucleotide target site cleavage activity than SEQ ID NO: 14 at a temperature of about 30 degrees Celsius, about 25 degrees Celsius, or about 20 degrees Celsius.

7. An engineered Cas polypeptide as described in any one of claims 1-5, wherein the engineered Cas polypeptide cuts the target site on the polynucleotide with a higher efficiency than SEQ ID NO: 14 at a temperature of about 30 degrees Celsius, about 25 degrees Celsius, or about 20 degrees Celsius.

8. An engineered Cas polypeptide as described in claim 7, wherein the engineered Cas polypeptide cuts the polynucleotide target site with an efficiency at least about 1.5 times higher than SEQ ID NO: 14 at a temperature of about 30 degrees Celsius, about 25 degrees Celsius, or about 20 degrees Celsius.

9. The engineered Cas polypeptide of any one of claims 1-8, wherein the engineered Cas polypeptide has a length of less than about 500 amino acids.

10. The engineered Cas polypeptide of any one of claims 1-9, wherein the engineered Cas polypeptide is in a complex comprising a target site on a double-stranded DNA polynucleotide.

11. An engineered Cas polypeptide as described in any one of claims 1-10, further comprising a guidance polynucleotide containing a variable targeting domain, wherein the variable targeting domain comprises a region complementary to the target site of the polynucleotide.

12. An engineered Cas polypeptide as described in claim 11, wherein the guide polynucleotide variable targeting domain comprises less than 20 nucleotides.

13. An engineered Cas polypeptide as described in claim 11 or 12, wherein the engineered Cas polypeptide recognizes a PAM sequence on a target polynucleotide, and wherein the guide polynucleotide and the Cas polypeptide form a complex that binds to a target site on a double-stranded DNA polynucleotide.

14. An engineered Cas polypeptide as described in any one of claims 1-13, wherein the engineered Cas polypeptide is an endonuclease that cuts double-stranded DNA polynucleotides.

15. An engineered Cas polypeptide as described in any one of claims 1-4 or 9-13, wherein the endonuclease activity of the engineered Cas polypeptide is catalytically inactivated.

16. An engineered Cas polypeptide as described in any one of claims 1-15, wherein the engineered Cas polypeptide recognizes a PAM sequence comprising N(T>W>C)TTC.

17. The engineered Cas polypeptide of any one of claims 1-16, wherein the engineered Cas polypeptide is part of a fusion protein.

18. An engineered Cas polypeptide as described in any one of claims 1-18, wherein the engineered Cas polypeptide is part of a fusion protein, wherein the fusion protein further comprises a heterologous nuclease domain.

19. The engineered Cas polypeptide of any one of claims 1-18, further comprising a deaminase.

20. A synthetic composition comprising the engineered Cas polypeptide of any one of claims 1-18, and further comprising a heterologous polynucleotide.

21. The synthetic composition of claim 20, wherein the heterologous polynucleotide is an expression element, a transgene, a donor DNA molecule, or a polynucleotide modification template.

22. The synthetic composition of claim 20, wherein the heterologous polynucleotide is a temperature-inducible promoter.

23. A synthetic composition comprising: (a) the engineered Cas polypeptide of any one of claims 1-19; (b) a target double-stranded DNA polynucleotide; and (c) a guide polynucleotide comprising a variable targeting domain comprising a region complementary to a target double-stranded DNA polynucleotide; wherein the Cas polypeptide recognizes a PAM sequence on the target double-stranded DNA polynucleotide, wherein the guide polynucleotide and the Cas polypeptide form a complex that binds to the target double-stranded DNA polynucleotide.

24. A polynucleotide encoding an engineered Cas polypeptide as described in any one of claims 1-19 or a synthetic composition as described in any one of claims 20-22.

25. The polynucleotide of claim 24, wherein the polynucleotide encodes the engineered Cas polypeptide and at least one expression element.

26. The polynucleotide of claim 24, wherein the polynucleotide encodes the engineered Cas polypeptide and gene.

27. The engineered Cas polypeptide of any one of claims 1-19, wherein the engineered Cas polypeptide is attached to a solid substrate or the Cas polypeptide is complexed with a guide polynucleotide and the Cas polypeptide / guide polynucleotide complex is attached to a solid substrate.

28. A eukaryotic cell comprising an engineered Cas polypeptide as described in any one of claims 1-19, a synthetic composition as described in any one of claims 20-23, or a polynucleotide as described in any one of claims 24-27.

29. The eukaryotic cell of claim 28, wherein the eukaryotic cell is a plant cell, an animal cell or a fungal cell.

30. The eukaryotic cell of claim 28, wherein the eukaryotic cell is a monocotyledonous plant cell or a dicotyledonous plant cell.

31. The eukaryotic cell of claim 28, wherein the plant cell is a cell from maize, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turf grass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, or tomato.

32. The eukaryotic cell of any one of claims 28-31, wherein the eukaryotic cell is at a temperature of about 40 degrees Celsius or less, about 37 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 25 degrees Celsius or less, or about 20 degrees Celsius or less.

33. A method for introducing targeted editing in a target polynucleotide, the method comprising: (a) providing an engineered Cas polypeptide and a guide polynucleotide as described in any one of claims 11 to 13, wherein the Cas polypeptide / guide polynucleotide forms a complex that recognizes a PAM sequence on the target polynucleotide; as well as (b) contacting the Cas polypeptide / guide polynucleotide complex with the target; and (c) introducing targeted editing in the target polynucleotide.

34. The method of claim 33, wherein the target polynucleotide is a target genomic sequence of a cell, and the method comprises: (i) delivering the Cas polypeptide / guide polynucleotide complex to the cell; (ii) incubating the cells at a temperature of about 37 degrees Celsius or less, about 35 degrees Celsius or less, about 30 degrees Celsius or less, about 25 degrees Celsius or less, or about 20 degrees Celsius or less; (iii) modifying at least one nucleotide in the target genomic sequence of the cell to generate a modified genomic sequence compared to the target genomic sequence of the cell prior to delivering the Cas polypeptide / guide polynucleotide complex; and (iv) generating a whole organism from the cell, wherein the organism comprises the modified genomic sequence.

35. The method of claim 34, wherein the cell is a eukaryotic cell.

36. The method of claim 35, wherein the eukaryotic cell is derived or obtained from an animal, fungus or plant.

37. The method of claim 36, wherein the eukaryotic cell is from a monocot or a dicot.

38. The method of claim 37, wherein the plant is selected from the group consisting of maize, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oats, sugarcane, turf grass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, and tomato.

39. The method of any one of claims 33-38, wherein the variable targeting domain of the guide polynucleotide comprises less than 20 nucleotides.

40. The method of claims 33-39, further comprising providing a heterologous polynucleotide.

41. The method of claim 40, wherein the heterologous polynucleotide is a donor DNA molecule.

42. The method of claim 40, wherein the heterologous polynucleotide is a polynucleotide modification template comprising a sequence that is at least 50% identical to a sequence in the cell.

43. The method of claim 40, wherein the heterologous polynucleotide is an inducible promoter.

44. A kit comprising an engineered Cas polypeptide as described in any one of claims 1-19, a polynucleotide as described in any one of claims 24-26, or an engineered Cas endonuclease as described in claim 27 and a solid substrate.

45. A method for altering a target Cas polypeptide; the method comprising modifying the target Cas polypeptide to include one or more amino acid changes such that when aligned with SEQ ID NO: 14 and numbered relative to the amino acid positions of SEQ ID NO: 14, the modified Cas polypeptide comprises one or more of the following amino acids: glutamate at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamate at position 81, or glutamine at position 85, wherein the modified Cas polypeptide is capable of site-specifically binding to a target site of a polynucleotide.

46. ​​The method of claim 45, wherein the method further comprises modifying the Cas polypeptide so that when aligned with SEQ ID NO: 14 and relative to SEQ ID When the amino acid positions of NO: 14 are numbered, the modified Cas polypeptide further comprises: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, aspartic acid at position 149, glycine at position 226, glycine at position 230, histidine at position 293, serine at position 298, phenylalanine at position 306, glutamic acid at position 329, serine at position 313, asparagine at position 325, glutamic acid at position 327, valine at position 338, cysteine ​​at position 376, cysteine ​​at position 379, cysteine ​​at position 395, cysteine ​​at position 398, cysteine ​​at position 406, lysine or arginine at position 409, asparagine or arginine at position 421, proline at position 430, arginine at position 467, or proline at position 468.

47. The method of claim 45 or 46, wherein the method comprises modifying the nucleic acid sequence encoding the target Cas polypeptide to include one or more altered codons, each altered codon encoding one or more of the following amino acids at positions relative to alignment with SEQ ID NO: 14: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, glutamic acid at position 81, or glutamine at position 85.

48. A system comprising a Cas polypeptide and an engineered guide RNA (egRNA), wherein the egRNA is based on a template guide RNA sequence (sgRNA) comprising a Cas polypeptide recognition domain, and wherein the egRNA comprises (i) a deletion, substitution or insertion at one or more of nucleotide positions 1-3, 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 147-149, 149-151, 151-153, 153-155, 157-159, 159-161, 163-165, or 167-169 of the sgRNA Cas polypeptide recognition domain or (ii) one of SEQ ID NOs: 62-156, SEQ ID NOs: 192-207, SEQ ID NOs: 260-284, SEQ ID NOs: 324-327, SEQ ID NOs: 346-355, or SEQ ID NO:

362.

49. The system of claim 48, wherein the egRNA comprises deletions, substitutions or insertions at positions 3-5, 5-7, 7-9, 59-61, 61-63, 63-65, 149-151, 151-153, 153-155, 157-159, 163-165 and 167-169 of the sgRNA Cas polypeptide recognition domain.

50. The system of claim 48, wherein the egRNA comprises deletions, substitutions, or insertions at positions 1-9 and 146-153 of the sgRNA Cas polypeptide recognition domain.

51. The system of claim 48, wherein the egRNA comprises a deletion, substitution, or insertion at positions 1-9, 6-63, 61-63, 146-153, 157-159, 158-170, 167-169, or a combination thereof, of the sgRNA Cas polypeptide recognition domain.

52. The system of claim 48, wherein the egRNA comprises SEQ ID NOs: 64, 65, 66, 92, 93, 94, 137, 138, 139, 141, 144, and 146.

53. A system comprising a Cas polypeptide and an engineered guide RNA (egRNA), wherein the egRNA is based on a template guide RNA sequence (sgRNA) and wherein the egRNA comprises an aptamer inserted at position 1, 3, 4, 5, 6, 7, 9, 10 or 11 of the sgRNA Cas polypeptide recognition domain.

54. The system of any one of claims 48-53, wherein the Cas polypeptide is an engineered Cas polypeptide as described in any one of claims 1-19.

55. A method of altering a target site in a cell, the method comprising introducing the system of any one of claims 48-54 into a cell, the cell comprising a target site of an engineered guide RNA of the system.

Images