Cas endonuclease and guide RNA variants with improved efficiency

Abstract

Compositions and methods are provided for genomic modification of target sequences in the genome of a cell using novel engineered Cas endonucleases. The engineered Cas polypeptides and related methods, systems, and compositions disclosed herein can be used in guide polynucleotide / endonuclease systems to modify or alter target sequences in the genome of a cell or organism. Also provided are novel effector and endonuclease systems, such as guide polynucleotide / endonuclease systems comprising endonucleases, and elements comprising such systems. Compositions and methods for guide polynucleotide / endonuclease systems comprising at least one endonuclease, optionally covalently or noncovalently linked to or assembled with at least one additional protein subunit, and compositions and methods for directly delivering an endonuclease as a ribonucleotide protein are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 352,100, filed June 14, 2022, U.S. Provisional Patent Application No. 63 / 378,897, filed October 10, 2022, U.S. Provisional Patent Application No. 63 / 481,458, filed January 25, 2023, and U.S. Provisional Patent Application No. 63 / 497,314, filed April 20, 2023, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Reference to an electronically submitted sequence listing An official copy of the Sequence Listing is submitted electronically herewith as an XML file named 9213-WO-PCT_ST26v2, having a size of 422,257 bytes, created on June 13, 2023. The Sequence Listing contained in this XML document is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to the field of molecular biology, and in particular to compositions of a novel RNA-guided Cas endonuclease system, as well as compositions and methods for editing or modifying the genome of a cell. [Background technology]

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

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

[0006] Despite the identification and characterization of some of these systems, there remains a need to engineer novel effectors and systems for editing endogenous and previously introduced heterologous polynucleotides and to demonstrate activity in eukaryotes, particularly animals and plants.

[0007] Described herein are novel engineered Cas polypeptides and endonucleases, as well as methods and compositions for their use. Summary of the Invention

[0008] Disclosed herein are compositions of novel engineered Cas polypeptides and methods of use thereof. These Cas polypeptides can be guided by a guide polynucleotide to target double-stranded DNA in a PAM-dependent manner. In some embodiments, the engineered Cas polypeptide is an active endonuclease capable of introducing a cleavage at a target site in the target double-stranded DNA. In some embodiments, the Cas polypeptide comprises one or more mutations that prevent the Cas polypeptide from making double-stranded cleavage but allow single-stranded cleavage. In some embodiments, the Cas polypeptide comprises one or more mutations that prevent the Cas polypeptide from cleaving either or both strands of a double-stranded polynucleotide, while retaining the ability to bind to a target polynucleotide sequence.

[0009] In one aspect, novel engineered Cas polypeptides are provided, comprising at least one zinc finger-like domain, at least one bridge-helix-like domain, three split RuvC domains (comprising non-contiguous RuvC-I, RuvC-II, and RuvC-III domains), and optionally comprising a heterologous polynucleotide. Also provided are synthetic compositions comprising the novel engineered Cas polypeptides or endonucleases. Many aspects of the disclosed compositions and methods provide at least one component that is optimized for expression in eukaryotic cells, particularly plant, fungal, or animal cells. In particular examples, the Cas polypeptide has fewer than about 500 amino acids.

[0010] In all embodiments of the novel Cas polypeptides disclosed herein, the polypeptides are engineered to contain an amino acid sequence that differs from a native effector protein obtained or derived from Syntrophomonas palmitatica.

[0011] In a first aspect, an engineered Cas polypeptide, or a polynucleotide encoding an engineered Cas polypeptide, is provided, wherein the engineered Cas polypeptide, when aligned with SEQ ID NO: 14 and compared to the amino acid position numbers of SEQ ID NO: 14, comprises 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, and glutamine at position 85. In some examples of this first aspect, the engineered Cas polypeptide, when aligned with SEQ ID NO: 14 and compared to the amino acid position numbers of SEQ ID NO: 14, comprises at least one, at least two, at least three, at least four, at least five, at least six, or seven of the following relative to the amino acid position numbers of 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, and glutamine at position 230. lysine, 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.

[0012] In a second aspect, there is provided an engineered Cas polypeptide, or a polynucleotide encoding the engineered Cas polypeptide, 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 and compared to the amino acid position numbering 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, and glutamine at position 85, wherein the engineered polypeptide is capable of site-specific binding to a target site in 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, SEQ ID NOs: 309-323, and SEQ ID NO: 362.

[0013] Further examples of each of the aforementioned first or second aspects are provided, wherein the modified Cas polypeptide, or polynucleotide encoding the modified Cas polypeptide, is provided such that the modified 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 when aligned with SEQ ID NO:14 and compared to the amino acid position numbering of SEQ ID NO:14: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, proline at position 120, and amino acid at position 149. aspartic acid 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.

[0014] In yet another example of each of the above first or second aspects, when aligned with SEQ ID NO: 14 and compared to the amino acid position numbering of SEQ ID NO: 14, the engineered Cas polypeptide does not contain at least one of the following: an aspartic acid or glutamic acid at position 38, an aspartic acid at position 79, a proline at position 120, an aspartic acid at position 149, a glycine at position 226, a glycine at position 230, a histidine at position 293, a serine at position 298, Phenylanine 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.

[0015] In certain embodiments of the foregoing examples of the first or second aspect, the modified Cas polypeptide comprises less than 500 amino acids in length. In certain embodiments of the foregoing first or second aspect, the modified Cas polypeptide is a Cas endonuclease.

[0016] Also provided are embodiments of each of the above first or second aspects, wherein the engineered Cas polypeptide comprises at least one zinc finger-like domain, at least one bridge helix-like domain, three split RuvC domains (comprising non-contiguous RuvC-I, RuvC-II, and RuvC-III domains), and optionally comprises a heterologous polynucleotide.

[0017] In a third aspect, the CRISPR-Cas polypeptide comprises a CRISPR-Cas polypeptide or a polynucleotide encoding the CRISPR-Cas polypeptide, wherein the CRISPR-Cas polypeptide comprises at least 250, 250 to 300, at least 300, 300 to 350, at least 350, 350 to 400, at least 400, or more than 400 consecutive amino acids and at least 50%, 50% to 55%, or at least 100 consecutive amino acids of a sequence selected from the group consisting of SEQ ID NOs: 15 to 47, SEQ ID NOs: 49 to 56, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NOs: 309 to 323, and SEQ ID NO: 362. Synthetic compositions are provided that include sequences that share at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity.

[0018] Any of the methods or compositions herein may further comprise a heterologous polynucleotide, which 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-modified template optionally comprising at least one nucleotide modification compared to a polynucleotide sequence in a cell; a transgene; a guide RNA; a guide DNA; a guide RNA-DNA hybrid; an endonuclease; a nuclear localization signal; and a cellular transit peptide.

[0019] In another aspect, methods are provided for using any of the compositions disclosed herein. In some embodiments, methods are provided that include a Cas polypeptide or endonuclease of the present disclosure (i.e., any of the embodiments of the first, second, or third aspects) binding to a target sequence of a polynucleotide, e.g., in the genome of a cell or in vitro. In some embodiments, the Cas polypeptide or endonuclease of the present disclosure forms a complex with a guide polynucleotide, e.g., a guide RNA (gRNA), which may be a single-stranded guide RNA (sgRNA). In some embodiments, the complex recognizes and binds to a polynucleotide at or near the target sequence, and optionally generates a nick (single-stranded) or a break (double-stranded) therein. In some embodiments, the nick or break is repaired by non-homologous end joining (NHEJ). In some embodiments, the nick or break is repaired by homology-directed repair (HDR) or homologous recombination (HR) using a polynucleotide-modified template or donor DNA molecule.

[0020] In any aspect, the modified Cas polypeptide or endonuclease can be used to bind, nick, and / or cleave a polynucleotide comprising a target sequence at a temperature below about 45° C., e.g., about 40° C. or less, about 37° C. or less, about 35° C. or less, about 30° C. or less, about 28° C. or less, or about 25° C. or less. Accordingly, methods are also provided that include contacting a polynucleotide with any modified Cas polypeptide or endonuclease disclosed herein (e.g., any Cas polypeptide of the first, second, or third aspects disclosed herein) and generating a cleavage in the polynucleotide at a temperature below about 45° C., e.g., about 40° C. or less, about 35° C. or less, about 30° C. or less, about 28° C. or less, or about 25° C. or less. This cleavage can be used to generate a targeted modification or altered target site (such as a base edit, deletion, or insertion) in the polynucleotide. Thus, in some examples, the modified Cas polypeptide or endonuclease of the first, second, or third aspects disclosed herein provides greater activity (binding, targeting efficiency, and / or endonuclease activity) than wild-type Cas-alpha10 endonuclease (SEQ ID NO: 48) or its base variant (SEQ ID NO: 14) at temperatures below about 45°C, such as about 40°C or less, about 35°C or less, about 30°C or less, about 28°C or less, or about 25°C or less.

[0021] The novel engineered Cas endonucleases described herein can generate double-stranded breaks in any prokaryotic or eukaryotic cell in or adjacent to a target polynucleotide that contains an appropriate PAM and to which the engineered Cas endonuclease is directed by a guide polynucleotide. In some examples, the cell is a plant cell, an animal cell, or a fungal cell. In some examples, the plant cell is selected from the group consisting of corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, tobacco, Arabidopsis, safflower, and tomato.

[0022] In another aspect, the modified Cas polypeptides described herein comprise one or more mutations that result in nuclease-inactivated or dead Cas polypeptides. For example, the modified Cas polypeptides disclosed herein can be altered to include an alanine at position 228, an alanine at relative position 327, or an alanine at position 434 when aligned with SEQ ID NO: 14 or 48 and compared to the amino acid position numbering of SEQ ID NO: 14 or 48. The inactivated modified Cas polypeptides of the present disclosure can be linked to an effector or effector protein, which can be a molecule that recognizes, binds to, and / or cleaves or nicks a polynucleotide target. The inactivated modified Cas polypeptides of the present disclosure can be linked to a base-editing molecule, such as a deaminase, for targeted base editing. The inactivated engineered Cas polypeptides of the present disclosure, optionally linked to an effector or effector protein, can be used for targeted delivery of effector molecules at temperatures of about 45°C or less, about 40°C or less, about 37°C or less, about 35°C or less, about 30°C or less, or about 25°C or less.

[0023] In yet another aspect, provided is a method of modifying 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 or 48 and compared to the amino acid position numbering of SEQ ID NO: 14 or 48, the target Cas polypeptide includes one or more of the following amino acid modifications: 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 modified Cas polypeptide is capable of site-specific binding to a target site in a polynucleotide, thereby producing the modified Cas polypeptide. Optionally, in some examples of this embodiment, the target Cas polypeptide is further modified such that, when aligned with SEQ ID NO: 14 or 48 and compared to the amino acid position numbering of SEQ ID NO: 14 or 48, the target Cas polypeptide includes 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 position 226, glycine at position 230, histidine at position 293. lysine at position 298, 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.

[0024] For example, in certain examples of the foregoing embodiments, the method includes aligning a target Cas polypeptide (e.g., a Cas polypeptide less than 500 amino acids in length) to SEQ ID NO: 14 or 48, identifying amino acid residues in the target Cas polypeptide that correspond 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 amino acid residues in the target Cas polypeptide 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 comprises identifying a target Cas polypeptide amino acid residue corresponding to one or more 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 SEQ ID NO: 14 or 48, and modifying the target Cas polypeptide amino acid residue to one or more of the following: aspartic acid or glutamic acid at position 38, aspartic acid at position 79, and a nucleotide sequence corresponding to one or more of the following amino acid residues: proline at position 149, 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. In these methods, the target Cas polypeptide can be (i) 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 (i) and (ii).In certain examples, the modified Cas polypeptide provides greater activity (binding, targeting efficiency, and / or endonuclease activity) than the unmodified target Cas polypeptide at temperatures below about 45°C, e.g., temperatures of about 40°C or less, about 35°C or less, about 30°C or less, about 28°C or less, or about 25°C or less.

[0025] This method of altering a target Cas polypeptide can include, for example, modifying a nucleic acid sequence encoding the target Cas polypeptide to include one or more altered codons that encode one or more of the following amino acid alterations when aligned with SEQ ID NOs: 14 and 48 and compared to the amino acid position numbering of SEQ ID NOs: 14 and 48: 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 the altered Cas polypeptide. Optionally, the method includes aligning with SEQ ID NOs: 14 and 48 and detecting the following amino acid changes when compared to the amino acid position numbers of SEQ ID NOs: 14 and 48: 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, and asparagine at position 325. , glutamic acid at position 327, valine at position 338, cysteine at position 376, cysteine at 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.

[0026] Also disclosed is 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 the egRNA comprises: (i) a nucleotide sequence at 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, 162-163, 164-165, 166-167, 168-169, 170-171, 172-173, 174-175, 176-177, 178-179, 179-200, 179-201, 180-181, 182-183, 183-184, 184-185, 185-186, 186-187, 187-188, 188-189, 189-200, 190-201, 191-202, 192-203, 193-204, 194-205, 195-206, 196-207, 197-208, 198-209, 199-210, 199-211, 200-212, 2010-213, 2010-214, 2010-215, 2020-216, 2020-217, 2030-218, 2040-21 (ii) one of the sequences of SEQ ID NOs: 62-156, 192-207, 260-284, 324-327, or 346-355; or (iii) an aptamer inserted at position 1, 3, 4, 5, 6, 7, 9, 10, or 11 of the Cas polypeptide recognition domain of an sgRNA. Thus, the disclosed egRNAs can contain deletions, substitutions, or insertions 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, or 167-169, or any combination thereof. Such modified guide (egRNA) fragments and their functionality (e.g., effect on target DNA cleavage efficiency) are shown in Tables 6, 7, 8, 9, 10, 11, and 12 herein.

[0027] In some examples of any of the aforementioned systems comprising a Cas polypeptide and a modified guide RNA (egRNA), the Cas polypeptide is a modified Cas polypeptide disclosed herein, for example, (i) a Cas polypeptide that, when aligned with SEQ ID NO: 14, contains one or more of the following amino acid changes relative to the amino acid position numbering of 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, and glutamine at position 85; (ii) a sequence that has 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, contains one or more of the amino acid changes (iii) the engineered Cas polypeptide comprises one or more of the following amino acids, relative to amino acid position number: 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-specific binding to a target site in a polynucleotide); (iv) the engineered Cas polypeptide is capable of site-specific binding to a target site in a polynucleotide and (v) 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, SEQ ID NOs: 309-323, or SEQ ID NO: 362. Specific examples of systems comprising modified Cas polypeptides and modified guide RNAs (egRNAs) are disclosed herein in Tables 6, 7, 8, 9, 10, 11, and 12, respectively.

[0028] The present disclosure can be more fully understood from the following detailed description and the accompanying figures and sequence listing, which form a part of this application. [Brief explanation of the drawings]

[0029] [Figure 1]Cas endonuclease variant yeast expression vectors are shown (where: 1 = ROX3 CAN1 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-alpha10 sgRNA ADE2 targeting domain (SEQ ID NO: 7); 8 = sequence encoding Cas-alpha10 sgRNA sequence CAN1 targeting domain (SEQ ID NO: 8); 9 = sequence encoding Cas-alpha10 sgRNA targeting ADE2 (SEQ ID NO: 9); 10 = sequence encoding Cas-alpha10 sgRNA targeting CAN1 (SEQ ID NO: 10)). [Figure 2] Overview of targeted cleavage detection in S. cerevisiae. Targeted cleavage and cellular repair result in the formation of a nonfunctional ade2 gene, which leads to adenine auxotrophy and a switch from a white to a red / pink cellular phenotype. This color switch allows for the identification of phenotypic differences and can be used to select for cells expressing Cas endonuclease variants and / or associated guide RNAs with improved targeted DSB activity. Colonies with functional ade2 genes are white. Colonies with nonfunctional ade2 genes are red. Red and white variegated colonies indicate multiple sectors containing nonfunctional ade2 genes. Colonies with a single sector containing a nonfunctional ade2 gene are white with red sections. [Figure 3] Overview of targeted cleavage detection in S. cerevisiae. Targeted cleavage of the CAN1 gene and non-functional repair of cells confers resistance to L-canavanine, thereby enabling positive selection of Cas endonuclease variants with improved double-strand cleavage activity. [Figure 4]

[0023] Figure 14 is the polypeptide sequence of a Cas endonuclease (SEQ ID NO: 14) containing several modifications that allow function at lower temperatures, and which was further modified in the Examples described herein. Key catalytic residues are shown in bold, underlined font. The zinc finger domain is shown in dashed underline. [Figure 5]

[0023] Figure 6 is the polypeptide sequence (SEQ ID NO: 60) of a modified Cas polypeptide with improved activity, as shown in the Examples herein. Key catalytic residues are shown in bold, underlined font. The zinc finger domain is shown in dashed underline. Double underlined letters indicate residues that have been further altered relative to the base Cas endonuclease (SEQ ID NO: 14) used herein. [Figure 6] Two schematic diagrams of human-optimized expression cassettes are shown: the top shows the cassette for the Cas-alpha nuclease, and the bottom shows the expression cassette for the Cas-alpha sgRNA. [Figure 7] Shown are modified sgRNAs containing MS2 recognition sequences incorporated at the 5' end, at the apex of the stem-loop, and within the 5'-GAAA-3' linker connecting the tracrRNA and crRNA of the sgRNA; also shown are examples of positions 1-12 into which aptamer or Cas6 sequences have been inserted as described in Table 9 herein. [Figure 8] Two schematic diagrams of Zea mays optimized expression cassettes are shown: the top shows the cassette for Cas-alpha nuclease, and the bottom shows the expression cassette for Cas-alpha sgRNA. [Figure 9] 1 is a graph showing the difference in cleavage between base variant Cas endonuclease SEQ ID NO: 14 and wild-type Cas endonuclease SEQ ID NO: 157 at different temperatures. [Figure 10] 1 is a graph showing the difference in cleavage between variant Cas endonucleases SEQ ID NO: 14 and SEQ ID NO: 48 at different temperatures. [Figure 11] 1 is a graph showing the difference in cleavage between variant Cas endonucleases SEQ ID NO: 14 and SEQ ID NO: 362 at different temperatures, demonstrating that SEQ ID NO: 362 has significantly improved cleavage activity at 25°C. DETAILED DESCRIPTION OF THE INVENTION

[0030] Table 1 provides a description of the sequences referenced herein. The isolated polynucleotides, nucleic acids, and nucleotide sequences disclosed herein further encompass all complementary forms (e.g., reverse complements) of each sequence disclosed for such constructs. The sequence descriptions and the sequence listing accompanying this specification comply with the rules governing the disclosure of nucleotide and amino acid sequences in patent applications as set forth in 37 C.F.R. §§ 1.821 and 1.835.

[0031] [Table 1]

[0032] [Table 2]

[0033] [Table 3]

[0034] [Table 4]

[0035] [Table 5]

[0036] [Table 6]

[0037] [Table 7]

[0038] [Table 8]

[0039] [Table 9]

[0040] The optimum temperature of native Cas endonucleases exceeds the typical biological temperature of some organisms, such as plants and yeast. This results in wild-type Cas endonucleases (e.g., SEQ ID NO: 48) requiring a heat shock of approximately 45°C for optimal activity. Depending on the application, it may be advantageous to modify this characteristic. 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, as well as proteins (domains) containing endonuclease functionality. Also provided are compositions and methods for direct delivery of endonucleases, cleavage-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 the genome of a cell, gene editing, and insertion of a polynucleotide of interest into the genome of a cell. The identified variants will improve genome editing outcomes in various cell types, including humans, and aid in the widespread adoption of this small RNA-guided Cas nuclease.

[0041] Terms used in the claims and specification are defined as set forth below unless otherwise specified. It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0042] As used herein, "nucleic acid" refers to a polynucleotide, including single- or double-stranded polymers of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can also include fragments and modified nucleotides. Thus, the terms "polynucleotide," "nucleic acid sequence," "nucleotide sequence," and "nucleic acid fragment" are used interchangeably to refer to polymers of RNA, and / or DNA, and / or RNA-DNA that are single- or double-stranded, and optionally contain synthetic, non-natural, or modified nucleotide bases. Nucleotides (usually found in their 5'-monophosphate form) are referred to by the single letter designation: "A" for adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" for cytosine or deoxycytosine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "I" for inosine, and "N" for any nucleotide.

[0043] The term "genome," as applied to prokaryotic and eukaryotic cells or organismal cells, encompasses not only chromosomal DNA found in the nucleus, but also organelle DNA found within cellular components of the cell (e.g., mitochondria or plastids).

[0044] "Open reading frame" is abbreviated as ORF.

[0045] The term "selectively hybridizes" includes reference to a nucleic acid sequence that hybridizes to a specific nucleic acid target sequence under stringent hybridization conditions to a detectably greater extent (e.g., at least twice background) than to non-target nucleic acid sequences, to the substantial exclusion of non-target nucleic acids. Selectively hybridizing sequences typically have at least about 80% sequence identity or 90% sequence identity, and up to 100% sequence identity (i.e., fully complementary) with each other.

[0046] The terms "stringent conditions" or "stringent hybridization conditions" include reference to conditions under which a probe will selectively hybridize to its target sequence in an in vitro hybridization assay. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of hybridization and / or washing conditions, it is possible to identify target sequences that are 100% complementary to a probe (homologous probing). Alternatively, stringency conditions can be adjusted to allow some sequence mismatch so that lower similarities are detected (heterologous probing). Generally, probes are less than about 1000 nucleotides in length, optionally less than 500 nucleotides in length. Typically, stringent conditions would be conditions with a pH of 7.0-8.3, a temperature of at least about 30°C for short probes (e.g., 10-50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides), and a salt concentration of about 1.5 M Na ion, typically about 0.01-1.0 M Na ion (or other salt). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Examples of low stringency conditions include hybridization at 37°C in a buffer consisting of 30-35% formamide, 1 M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by a wash with 1x-2x SSC (20x SSC = 3.0 M NaCl / 0.3 M trisodium citrate) at 50-55°C. Examples of moderate stringency conditions include hybridization in 40-45% formamide, 1 M NaCl, and 1% SDS at 37° C., followed by washing with 0.5× to 1× SSC at 55-60° C. Examples of high stringency conditions include hybridization in 50% formamide, 1 M NaCl, and 1% SDS at 37° C., followed by washing with 0.1× SSC at 60-65° C.

[0047] "Homology" refers to similar DNA sequences. For example, a "region homologous to a genomic region" found on donor DNA refers to a region of DNA that has a similar sequence to a given "genomic region" in the genome of a cell or organism. The homologous region can be of any length sufficient to promote homologous recombination at the target site of cleavage. For example, the homologous region can be at least 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 30, 5 to 35, 5 to 40, 5 to 45, 5 to 50, 5 to 55, 5 to 60, 5 to 65, 5 to 70, 5 to 75, 5 to 80, 5 to 85, 5 to 90, 5 to 95, 5 to 100, 5 to 200, 5 to 300, 5 to 400, 5 to 500, 5 to 600, 5 to 750, 5 to 850, 5 to 900, 5 to 950, 5 to 1000, 5 to 2000, 5 to 3000, 5 to 4000, 5 to 5000, 5 to 6000, 5 to 7500, 5 to 8000, 5 to 9500, 5 to 1000, 5 to 2000, 5 to 3000, 5 to 4000, 5 to 5000, 5 to 6000, 5 to 6000, 5 to 7500, 5 to 8000, 5 to 9500, 5 to 1000, 5 to 2000, 5 to 3000, 5 to 4000, The length may be 700, 5 to 800, 5 to 900, 5 to 1000, 5 to 1100, 5 to 1200, 5 to 1300, 5 to 1400, 5 to 1500, 5 to 1600, 5 to 1700, 5 to 1800, 5 to 1900, 5 to 2000, 5 to 2100, 5 to 2200, 5 to 2300, 5 to 2400, 5 to 2500, 5 to 2600, 5 to 2700, 5 to 2800, 5 to 2900, 5 to 3000, 5 to 3100 bases or more. "Sufficient homology" refers to sufficient structural similarity between two polynucleotide sequences to act as substrates for a homologous recombination reaction. This structural similarity includes the total length of each polynucleotide fragment and the sequence similarity of the polynucleotides. Sequence similarity can be described as percent sequence identity over the entire length of the sequence and / or percent sequence identity over conserved regions and portions of the length of the sequence, including localized similarities such as consecutive nucleotides with 100% sequence identity.

[0048] As used herein, a "genomic region" is a portion of a chromosome in the genome of a cell, which lies on either side of a target site or includes a portion of the target site. This genomic region may be at least 5-10, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, 5-50, 5-55, 5-60, 5-65, 5-70, 5-75, 5-80, 5-85, 5-90, 5-95, 5-100, 5-200, 5-300, 5-400, 5-500, 5-600, 5-700, 5-800, 5-900, 5-1000, 5-1100, 5-1200, 5-1300, The genomic region may comprise 5 to 1400, 5 to 1500, 5 to 1600, 5 to 1700, 5 to 1800, 5 to 1900, 5 to 2000, 5 to 2100, 5 to 2200, 5 to 2300, 5 to 2400, 5 to 2500, 5 to 2600, 5 to 2700, 5 to 2800, 5 to 2900, 5 to 3000, 5 to 3100, or more bases, such that the genomic region has sufficient homology to undergo homologous recombination with a corresponding homologous region.

[0049] As used herein, "homologous recombination" (HR) involves the exchange of DNA fragments between two DNA molecules at homologous sites. The frequency of homologous recombination is influenced by many factors. The amount of homologous recombination and the relative proportions of homologous and non-homologous recombination vary among different organisms. Generally, the length of the homologous region affects the frequency of homologous recombination events; the longer the region of homology, the higher the frequency. The length of the homologous region required to observe homologous recombination also varies among species. Often, at least 5 kb of homology has been utilized, although homologous recombination has been observed using as little as 25-50 bp. For example, Singer et al., (1982) Cell 31:25-33; Shen and Huang, (1986) Genetics 112:441-57; Watt et al., (1985) Proc. Natl. Acad. Sci. USA 82:4768-72, Sugawara and Haber, (1992) Mol Cell Biol. 12:563-75; Rubnitz and Subramani, (1984) Mol Cell Biol 4:2253-8; Ayares et al., (1986) Proc. Natl. Acad. Sci. USA 83:5199-203; Liskay et al. (1987) Genetics 115:161-7.

[0050] "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.

[0051] The term "percentage of sequence identity" refers to a numerical value determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence within this comparison window may contain additions or deletions (i.e., gaps) relative to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue occurs to obtain the number of matched positions, dividing this number of matched positions by the total number of positions within the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Useful examples of percent sequence identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage between 50% and 100%. These identities may be determined using any of the programs described herein.

[0052] Sequence alignments and percent identity or similarity calculations may be determined using various comparison methods designed to detect homologous sequences, including, but not limited to, the MegAlign™ program in the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). In the context of this application, when sequence analysis software is used for analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" refers to any set of values or parameters that initially load with the software when first initialized.

[0053] The "Clustal V method of alignment" is referred to as Clustal V (described in Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al. (1992) Comput Appl Biosci 8:189-191) and corresponds to the alignment method found in the MegAlign™ program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. The default parameters for pairwise alignment and percent identity calculation of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5, and DIAGONALS SAVED=5. For nucleic acids, these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4, and DIAGONALS SAVED=4. After alignment of sequences using the Clustal V program, a "percent identity" can be obtained by consulting the "sequence distance" table in the same program. The "Clustal W method of alignment" is referred to as Clustal W (Higgins and Sharp, (1989) CABIOS 5:151-153; Higgins et al. (1992) Comput Appl Biosci 8:189-191) and corresponds to the alignment method found in the MegAlign™ v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, WI). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Sequences (%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB).After aligning sequences using the Clustal W program, "percent identity" can be obtained by consulting the "sequence distance" table in the same program. Unless otherwise specified, the sequence identity / similarity values shown herein refer to values obtained using GAP version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: nucleotide sequence identity and similarity percentages are obtained using a gap creation penalty weight of 50, a gap extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; amino acid sequence identity and similarity percentages are obtained using a gap creation penalty weight of 8, a gap extension penalty weight of 2, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA 89:10915). GAP uses the algorithm of Needleman and Wunsch (1970) J Mol Biol 48:443-53 to find a global alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and uses gap creation and extension penalties in units of matched bases to create an alignment with the maximum number of matched bases and the fewest gaps. "BLAST" is a search algorithm provided by the National Center for Biotechnology Information (NCBI) used to find regions of similarity between biological sequences. This program compares nucleotide or protein sequences with sequence databases and calculates the statistical significance of matches to identify sequences sufficiently similar to a query sequence that the similarity is not expected to occur randomly. BLAST reports the identified sequences and their local alignment to the query sequence. It will be appreciated by those skilled in the art that many levels of sequence identity are useful in identifying polypeptides from other species or that are naturally or synthetically modified, such polypeptides having the same or similar function or activity.Useful examples of percent identity include, but are not limited to, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any percentage between 50% and 100%. Indeed, any amino acid identity between 50% and 100%, e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity may be useful in describing the present disclosure.

[0054] Polynucleotide and polypeptide sequences, their variants, and the structural relationships of these sequences can be described by the terms "homology," "homologous," "substantially identical," "substantially similar," and "substantially corresponding," which are used interchangeably herein. These refer to polypeptide or nucleic acid sequences in which changes in one or more amino acids or nucleotide bases do not affect the function of the molecule (e.g., its ability to mediate gene expression or its ability to produce a particular phenotype). These terms also refer to modifications of nucleic acid sequences in which the functional properties of the resulting nucleic acid are not substantially altered compared to the initial, unmodified nucleic acid. These modifications include deletions, substitutions, and / or insertions of one or more nucleotides in the nucleic acid fragment. Substantially similar nucleic acid sequences can be defined by their ability to hybridize (under moderate stringency conditions, e.g., 0.5×SSC, 0.1% SDS, 60° C.) to the sequences exemplified herein, or to any portion of the nucleotide sequences disclosed herein, and are functionally equivalent to any of the nucleic acid sequences disclosed herein. Stringency conditions can be adjusted to screen for fragments with moderate similarity, such as homologous sequences from distantly related organisms, against fragments with high similarity, such as genes replicating functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions.

[0055] A "centimorgan" (cM) or "map unit" is the distance between two polynucleotide sequences, linkage genes, markers, target sites, loci, or any pair thereof, where 1% of meiotic outcomes are recombinations. Thus, a centimorgan is equivalent to the distance that equates to an average recombination frequency of 1% between two linkage genes, markers, target sites, loci, or any pair thereof.

[0056] An "isolated" or "purified" nucleic acid molecule, polynucleotide, polypeptide, or protein, or biologically active portion thereof, is substantially or essentially free from components that normally co-occur with or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide, polypeptide, or protein is substantially free of other cellular material and culture medium if produced by recombinant techniques, or substantially free of chemical precursors and other chemicals if chemically synthesized. Optimally, an "isolated" polynucleotide (optimally, a protein-encoding sequence) is free of sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide). For example, in various embodiments, an isolated polynucleotide can comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. An isolated polynucleotide can be purified from the cell in which it naturally occurs. Conventional nucleic acid purification methods known to those skilled in the art can be used to obtain isolated polynucleotides. The term also encompasses recombinant polynucleotides and chemically synthesized polynucleotides.

[0057] The term "fragment" refers to a contiguous set of nucleotides or amino acids. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 contiguous nucleotides. In one embodiment, a fragment is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 contiguous amino acids. A fragment may or may not exhibit function of a sequence that shares some percent identity over the entire length of the fragment.

[0058] The terms "functionally equivalent fragment" and "functionally equivalent fragment" are used interchangeably herein. These terms refer to a portion or subsequence of an isolated nucleic acid fragment or polypeptide that exhibits the same activity or function as the longer sequence from which it is derived. In one example, a fragment retains the ability to alter gene expression or produce a particular phenotype, regardless of whether the fragment encodes an active protein. For example, a fragment may be used to engineer a gene that will produce a desired phenotype in an altered plant. A gene may be engineered to be used in an inhibitory manner by linking the nucleic acid fragment, whether it encodes an active enzyme or not, in a sense or antisense orientation to a plant promoter sequence.

[0059] A "gene" includes a nucleic acid fragment that expresses a functional molecule, such as, but not limited to, a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. A "native gene" refers to a gene found in its natural location with its own regulatory sequences.

[0060] The term "endogenous" refers to a sequence or other molecule that is naturally present in a cell or organism. In one aspect, an endogenous polynucleotide is one that is normally found in the genome of the cell, i.e., is not heterologous.

[0061] An "allele" is one of several alternative forms of a gene occupying a given locus on a chromosome. If all alleles present at a given chromosomal locus are the same, the plant is homozygous at that locus. If alleles present at a given chromosomal locus are different, the plant is heterozygous at that locus.

[0062] A "coding sequence" refers to a polynucleotide sequence that encodes a specific amino acid sequence. A "regulatory sequence" refers to a nucleotide sequence located upstream of a coding sequence (5' non-coding sequences), within a coding sequence, or downstream of a coding sequence (3' non-coding sequences), that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, promoters, translation leader sequences, 5' untranslated sequences, 3' untranslated sequences, introns, polyadenylation target sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0063] A "mutated gene" is a gene that has been altered by human intervention. Such a "mutated gene" has a sequence that differs from that of the corresponding non-mutated gene by the addition, deletion, or substitution of at least one nucleotide. In certain embodiments of the present disclosure, the mutant gene comprises an alteration resulting from the guide polynucleotide / Cas endonuclease system disclosed herein. A mutant plant is a plant that comprises a mutant gene.

[0064] As used herein, a "targeted mutation" refers to a mutation in a gene (referred to as a target gene), including a native gene, made by altering a target sequence within the target gene using any method known to those of skill in the art, including methods involving the inducible Cas endonuclease system disclosed herein.

[0065] The terms "knockout," "gene knockout," and "genetic knockout" are used interchangeably herein. Knockout refers to a cellular DNA sequence that has been partially or completely disabled by targeting with a Cas protein; for example, the DNA sequence prior to the knockout may have encoded an amino acid sequence or had a regulatory function (e.g., a promoter).

[0066] The terms "knock-in," "gene knock-in," "gene insertion," and "genetic knock-in" are used interchangeably herein. Knock-in refers to replacing or inserting a DNA sequence with a specific DNA sequence in a cell by targeting using a Cas protein (e.g., by homologous recombination (HR), where a suitable donor DNA polynucleotide is also used). Examples of knock-ins include the specific insertion of a heterologous amino acid coding sequence in the coding region of a gene or the specific insertion of a transcriptional regulatory element at a locus.

[0067] "Domain" means a contiguous stretch of nucleotides (which may be RNA, DNA, and / or combined RNA-DNA sequences) or amino acids.

[0068] The term "conserved domain" or "motif" refers to a series of polynucleotides or amino acids that are conserved at specific positions along the aligned sequences of evolutionarily related proteins. While amino acids at other positions may vary between homologous proteins, highly conserved amino acids at specific positions refer to amino acids that are essential for the structure, stability, or activity of a protein. Because they are identified by the high degree of conservation in the aligned sequences of that protein homolog family, 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.

[0069] A "codon-modified gene," or "codon-preferred gene," or "codon-optimized gene" is a gene whose codon usage is designed to mimic the preferred codon usage of a host cell.

[0070] An "optimized" polynucleotide is a sequence that has been optimized for improved expression in a particular heterologous host cell.

[0071] A "plant-optimized nucleotide sequence" refers to a nucleotide sequence that is optimized for expression in plants, particularly a nucleotide sequence that is optimized for increased expression in plants. Plant-optimized nucleotide sequences include codon-optimized genes. Plant-optimized nucleotide sequences can be synthesized by modifying a nucleotide sequence encoding a protein, such as a Cas endonuclease disclosed herein, with one or more plant-preferred codons to improve expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage.

[0072] A "promoter" is a region of DNA involved in the recognition and binding of RNA polymerase and other proteins that initiate transcription. A promoter sequence consists of proximal and more distal upstream elements, the latter often referred to as enhancers. An "enhancer" is a DNA sequence that can stimulate promoter activity and may be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. A promoter may be derived entirely from a native gene or may be composed of different elements derived from different promoters found in nature and / or may contain synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissue or cell types, at different developmental stages, or in response to different environmental conditions. It is further recognized that because the exact boundaries of regulatory sequences in most cases have not been completely defined, DNA fragments of some variation may have identical promoter activity.

[0073] Promoters that cause the expression of most genes in most cell types are commonly referred to as "constitutive promoters." The term "inducible promoter" refers to a promoter that selectively expresses a coding sequence or functional RNA in response to the presence of an endogenous or exogenous stimulus, for example, by a chemical compound (chemical inducer), or in response to environmental, hormonal, chemical, and / or developmental signals. Inducible or regulatable promoters include, for example, promoters that are induced or regulated by light, heat, stress, flooding or drought, salt stress, osmotic stress, plant hormones, wounding, or chemicals such as ethanol, abscisic acid (ABA), jasmonic acid, salicylic acid, or safeners.

[0074] A "translation leader sequence" refers to a polynucleotide sequence located between the promoter sequence and coding sequence of a gene. A translation leader sequence is located upstream of the translation initiation sequence of an mRNA. A translation leader sequence may affect the processing of a primary transcript into mRNA, mRNA stability, or translation efficiency. Examples of translation leader sequences have been reported (e.g., Turner and Foster, (1995) Mol Biotechnol 3:225-236).

[0075] "3' non-coding sequence," "transcription terminator," or "termination sequence" refers to DNA sequences located downstream of a coding sequence, including polyadenylation recognition sequences and other sequences encoding regulatory signals that may affect mRNA processing or gene expression. Polyadenylation signals are usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of a pre-mRNA. The use of different 3' non-coding sequences is exemplified in Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0076] "RNA transcript" refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When an RNA transcript is a perfect complementary copy of a DNA sequence, it is called a primary transcript or pre-mRNA. When an RNA transcript is an RNA sequence resulting from post-transcriptional processing of a primary transcript pre-RNA, it is called a mature RNA or mRNA. "Messenger RNA" or "mRNA" refers to RNA that is free of introns and can be translated into protein by a cell. "cDNA" refers to DNA that is complementary to an mRNA template and synthesized from the mRNA template using reverse transcriptase. cDNA can be single-stranded or converted to double-stranded form using the Klenow fragment of DNA polymerase I. "Sense" RNA refers to an RNA transcript that includes mRNA and can be translated into protein in cells or in vitro. "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and blocks expression of a target gene (see, e.g., U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA can be with any part of a specific gene transcript, i.e., with the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet affects cellular processes. The terms "complement" and "reverse complement" are used interchangeably herein with respect to mRNA transcripts and are intended to define the antisense RNA of the message.

[0077] The term "genome" refers to the entire body of genetic material (genes and non-coding sequences) present in each cell or virus or organelle of an organism; and / or the set of chromosomes inherited as a (haploid) unit from one parent.

[0078] The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is regulated by the other. For example, a promoter is operably linked to a coding sequence if it is capable of controlling the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). A coding sequence can be operably linked to a regulatory sequence in a sense or antisense orientation. In another example, complementary RNA regions can be operably linked, directly or indirectly, to the 5' end of a target mRNA or the 3' end of a target mRNA, or within a target mRNA, or a first complementary region is at the 5' end of the target mRNA and its complement is at the 3' end of the target mRNA.

[0079] Generally, "host" refers to an organism or cell into which a heterologous component (polynucleotide, polypeptide, other molecule, cell) has been introduced. As used herein, "host cell" refers to a eukaryotic cell, a prokaryotic cell (e.g., a bacterial or archaeal cell), or a cell derived from a multicellular organism (e.g., a cell line) cultured as a unicellular entity, in vivo or in vitro, into which a heterologous polynucleotide or polypeptide has been introduced. In some embodiments, the cell is selected from the group consisting of: an archaeal cell, a bacterial cell, a eukaryotic cell, a 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 porcine cell, a bovine cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell. In some examples, the cell is in vitro. In some examples, the cell is in vivo.

[0080] The term "recombinant" refers to the artificial combination of two otherwise separate segments of sequence, for example, by chemical synthesis or the manipulation of isolated segments of nucleic acid by genetic engineering techniques.

[0081] The terms "plasmid," "vector," and "cassette" refer to additional chromosomal elements, often linear or circular, that carry genes that are not part of the cell's central metabolism, usually in the form of double-stranded DNA. Such elements can be autonomously replicating sequences, genome-integrating sequences, phages, or nucleotide sequences of any origin, single- or double-stranded DNA or RNA, in linear or circular form, in which multiple nucleotide sequences are linked or recombined into a unique construct capable of introducing a polynucleotide of interest into a cell. A "transformation cassette" refers to a specific vector containing a gene and having elements in addition to the gene that facilitate transformation of a specific host cell. An "expression cassette" refers to a specific vector containing a gene and having elements in addition to the gene that direct the expression of the gene in a host.

[0082] 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 (e.g., regulatory and coding sequences) that are not necessarily found together in nature. For example, a recombinant DNA construct may contain regulatory and coding sequences that are derived from different sources, or regulatory and coding sequences that are derived from the same source but that are arranged in a manner different from that found in nature. Such a construct may be used by itself or in conjunction with a vector. When a vector is used, the choice of vector will depend on the method used to introduce the vector into a host cell, as is well known to those skilled in the art. For example, a plasmid vector may be used. Those skilled in the art are familiar with the genetic elements that must be present on a vector for successful transformation, selection, and propagation of a host cell. Those skilled in the art will also recognize that different independent transformation events can result in different expression levels and patterns (Jones et al. (1985) EMBO J 4:2411-2418; De Almeida et al. (1989) Mol Gen Genetics 218:78-86), and therefore, many events are typically screened to obtain strains exhibiting the desired expression levels and patterns. Such screening can be performed by standard molecular biological, biochemical, and other analytical methods, including Southern analysis of DNA, Northern analysis of mRNA expression, PCR, real-time quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), immunoblot analysis of protein expression, enzyme or activity analysis, and / or phenotypic analysis.

[0083] The term "heterologous" refers to a difference between the original environment, location, or composition of a particular polynucleotide or polypeptide sequence and its current environment, location, or composition. Non-limiting examples include differences in taxonomic origin (e.g., a polynucleotide sequence obtained from Zea mays is heterologous if inserted into the genome of an Oryza sativa plant or a different subspecies or variety of Zea mays; or a polynucleotide obtained from a bacterium is introduced into a plant cell) or sequence differences (e.g., a polynucleotide sequence obtained from Zea mays, isolated, modified, and reintroduced into a maize plant). As used herein, "heterologous" in reference to a sequence can refer to a sequence originating from a different species, subspecies, or foreign species, or, if from the same species, a sequence whose composition and / or genomic locus has been substantially altered from its native form by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide may be from a species different from the species from which the polynucleotide was derived, or, if from the same / similar species, one or both may be substantially altered from their original form and / or genomic locus, or the promoter may not be the native promoter of the operably linked polynucleotide. Alternatively, one or more of the regulatory regions and / or polynucleotides set forth herein may be entirely synthetic. In another example, a target polynucleotide for cleavage by a Cas endonuclease may be from a different organism than the Cas endonuclease. In another example, the Cas endonuclease and guide RNA may be introduced into a target polynucleotide along with an additional polynucleotide that acts as a template or donor for insertion into the target polynucleotide, where the additional polynucleotide is heterologous to the target polynucleotide and / or the Cas endonuclease.

[0084] The term "expression," as used herein, refers to the production of a functional end-product (e.g., mRNA, guide RNA, or protein) in precursor or mature form.

[0085] A "mature" protein refers to a post-translationally processed polypeptide (i.e., a polypeptide from which any pre- or propeptides present in the primary translation product have been removed).

[0086] "Precursor" protein refers to the primary product of translation of mRNA (i.e., with pre- and propeptides still present). The pre- and propeptides may be, but are not limited to, subcellular localization signals. "CRISPR" (clustered regularly interspaced short palindromic repeats) loci refer to specific loci that encode components of DNA cleavage systems used, for example, by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327:167-170; WO 2007 / 025097, published March 1, 2007).

[0087] CRISPR loci can consist of CRISPR arrays containing short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.

[0088] As used herein, an "effector" or "effector protein" is a protein whose activities include recognizing, binding to, and / or cleaving or nicking a polynucleotide target. An effector or effector protein can also be an endonuclease. The "effector complex" of a CRISPR system includes Cas proteins that are involved in recognizing and binding to the crRNA and the target. Some of the component Cas proteins may further include domains involved in cleaving the target polynucleotide.

[0089] The term "Cas protein" refers to a protein encoded by a Cas (CRISPR-associated) gene. Cas proteins include proteins encoded by genes at the cas locus and include adaptation molecules and interference molecules. Interference molecules of bacterial adaptive immune complexes include endonucleases. Cas endonucleases described herein contain one or more nuclease domains. Cas endonucleases include, but are not limited to, the novel Cas endonuclease proteins disclosed herein, Cas9 protein, Cpf1 (Cas12) protein, C2c1 protein, C2c2 protein, C2c3 protein, Cas3, Cas3-HD, Cas5, Cas7, Cas8, Cas10, or combinations or complexes thereof. A Cas protein, when complexed with a suitable polynucleotide component, can be a "Cas endonuclease" or "Cas effector protein" that can recognize, bind to, and optionally nick or cleave all or part of a specific polynucleotide target sequence. Cas endonucleases of the present disclosure include those that have one or more RuvC nuclease domains.A Cas protein is further defined as a functional fragment or functional variant of a naturally occurring Cas protein, or a fragment or variant of at least 50, 50-100, at least 100, 100-150, at least 150, 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 500, or more than 500 consecutive amino acids of a naturally occurring Cas protein, and at least 50%, 50%-55%, at least 55%, 55%-60% , at least 60%, 60%-65%, at least 65%, 65%-70%, at least 70%, 70%-75%, at least 75%, 75%-80%, at least 80%, 80%-85%, at least 85%, 85%-90%, at least 90%, 90%-95%, at least 95%, 95%-96%, at least 96%, 96%-97%, at least 97%, 97%-98%, at least 98%, 98%-99%, at least 99%, 99%-100%, or 100% sequence identity, and retains at least some activity of the native sequence.

[0090] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a Cas endonuclease are used interchangeably herein to refer to a portion or subsequence of a Cas endonuclease of the present disclosure that retains the ability to recognize, bind to, and optionally unwind, nick, or cleave (introduce a single- or double-stranded break within) a target site. This portion or subsequence of a Cas endonuclease may include a complete or partial (functional) peptide of any one of its domains, for example, but not limited to, an entire functional portion of the Cas3 HD domain, an entire functional portion of the Cas3 helicase domain, or an entire functional portion of a protein (such as, but not limited to, Cas5, Cas5d, Cas7, and Cas8b1).

[0091] 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 a variant of the Cas effector protein disclosed herein that retains the ability to recognize, bind to, and optionally unwind, nick, or cleave all or part of a target sequence.

[0092] Cas endonucleases can also include multifunctional Cas endonucleases. The terms "multifunctional Cas endonuclease" and "multifunctional Cas endonuclease polypeptide" are used interchangeably herein and include reference to a single polypeptide having a Cas endonuclease function (comprising at least one protein domain capable of acting as a Cas endonuclease) and at least one other function, such as, but not limited to, a complexing function (comprising at least a second protein domain capable of complexing with another protein). In one aspect, a multifunctional Cas endonuclease comprises at least one additional protein domain (internal, upstream (5'), downstream (3'), or both internal 5' and 3', or any combination thereof) relative to the domains typical of a Cas endonuclease.

[0093] The terms "cascade" and "cascade complex" are used interchangeably herein and include reference to a multisubunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP). A cascade is a PNP that relies on a polynucleotide for complex assembly and stability and for identification of target nucleic acid sequences. A cascade functions as a surveillance complex that finds and optionally binds to target nucleic acids complementary to the variable targeting domain of a guide polynucleotide.

[0094] The terms "cleavage-ready cascade," "cr cascade," "cleavage-ready cascade complex," "cr cascade complex," "cleavage-ready cascade system," "CRC," and "cr cascade system" are used interchangeably herein and include reference to a multi-subunit protein complex that can assemble with a polynucleotide to form a polynucleotide-protein complex (PNP), wherein one of the cascade proteins is a Cas endonuclease that can recognize, bind to, and optionally unwind, nick, or cleave all or part of a target sequence.

[0095] The terms "5'-cap" and "7-methylguanylate (m7G) cap" are used interchangeably herein. 7-methylguanylate residues are located at the 5' end of messenger RNA (mRNA) in eukaryotic cells. RNA polymerase II (Pol II) transcribes mRNA in eukaryotic cells. Messenger RNA capping generally occurs as follows: the extreme 5' phosphate group of an mRNA transcript is removed by an RNA terminal phosphatase, leaving two terminal phosphates. Guanosine monophosphate (GMP) is added to the terminal phosphate of the transcript by a guanylyltransferase, leaving a 5'-5' triphosphate-linked guanine at the end of the transcript. Finally, the 7-nitrogen of this terminal guanine is methylated by a methyltransferase.

[0096] The term "not having a 5'-cap" is used herein to refer to RNA that has, for example, a 5'-hydroxyl group instead of a 5'-cap. Such RNA may be referred to, for example, as "decapped RNA." Because 5'-capped RNA undergoes nuclear export, decapped RNA can accumulate more fully in the nucleus after transcription. One or more RNA components herein are decapped.

[0097] As used herein, the term "guide polynucleotide" relates to a polynucleotide sequence that can form a complex with a Cas endonuclease (e.g., a Cas endonuclease described herein) and enables the Cas endonuclease to recognize, optionally bind to, and optionally cleave a DNA target site. A guide polynucleotide sequence may be an RNA sequence, a DNA sequence, or a combination thereof (an RNA-DNA combination sequence).

[0098] The terms "functional fragment," "functionally equivalent fragment," and "functionally equivalent fragment" of a guide RNA, crRNA, or tracrRNA are used interchangeably herein and refer to a portion or subsequence of a guide RNA, crRNA, or tracrRNA of the present disclosure that retains the ability to function as a guide RNA, crRNA, or tracrRNA, respectively.

[0099] 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 that retain the ability to function as a guide RNA, crRNA, or tracrRNA, respectively.

[0100] The terms "single guide RNA" and "sgRNA" are used interchangeably herein and refer to a synthetic fusion of two RNA molecules: a crRNA (CRISPR RNA) containing a variable targeting domain (linked to a tracr mate sequence that hybridizes to the tracrRNA) fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA may comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of a Type II CRISPR / Cas system that can form a complex with a Type II Cas endonuclease, and the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, allowing the Cas endonuclease to recognize, optionally bind to, and optionally nick or cleave (introduce a single- or double-strand break) the DNA target site.

[0101] The terms "variable targeting domain" or "VT domain" are used interchangeably herein and comprise a nucleotide sequence that can hybridize to (be complementary to) one strand (nucleotide sequence) of a double-stranded DNA target site. The percent complementarity between the first nucleotide sequence domain (VT domain) and the target sequence can be at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 63%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The variable targeting domain can be at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the variable targeting domain comprises a contiguous stretch of 12 to 30 nucleotides. The variable targeting domain can be comprised of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence, or any combination thereof.

[0102] The terms "Cas endonuclease recognition domain" or "CER domain" (of a guide polynucleotide) are used interchangeably herein and comprise a nucleotide sequence that interacts with a Cas endonuclease polypeptide. A CER domain comprises a (trans-acting) tracr nucleotide mate sequence followed by a tracr nucleotide sequence. A CER domain can be composed of a DNA sequence, an RNA sequence, a modified DNA sequence, a modified RNA sequence (see, e.g., U.S. Patent Application Publication No. 2015 / 0059010A1, published February 26, 2015), or any combination thereof.

[0103] As used herein, the terms "guide polynucleotide / Cas endonuclease complex," "guide polynucleotide / Cas endonuclease system," "guide polynucleotide / Cas complex," "guide polynucleotide / Cas system," and "inducible Cas system," "polynucleotide-guided endonuclease," "PGEN," are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease that can form a complex, where 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 cleave (introduce a single- or double-strand break) the DNA target site. The guide polynucleotide / Cas endonuclease complex herein can comprise a Cas protein and a suitable polynucleotide component from any known CRISPR system (Horvath and Barrangou, 2010, Science 327:167-170; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al. 2015, Cell 163, 1-13; Shmakov et al. 2015, Molecular Cell 60, 1-13).

[0104] The terms "guide RNA / Cas endonuclease complex," "guide RNA / Cas endonuclease system," "guide RNA / Cas complex," "guide RNA / Cas system," "gRNA / Cas complex," "gRNA / Cas system," "RNA-guided endonuclease," and "RGEN" are used interchangeably herein and refer to at least one RNA component and at least one Cas endonuclease that can form a complex, wherein the guide RNA / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, allowing the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single- or double-strand break) the DNA target site.

[0105] The terms "target site," "target sequence," "target site sequence," "target DNA," "target locus," "genomic target site," "genomic target sequence," "genomic target locus," and "protospacer" are used interchangeably herein and refer to a polynucleotide sequence, such as, but not limited to, a nucleotide sequence in a chromosome, episome, locus, or any other DNA molecule in the genome of a cell (e.g., chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA), that a guide polynucleotide / Cas endonuclease complex can recognize, bind to, and optionally nick or cleave. A target site can be an endogenous site in the genome of a cell, or the target site can be heterologous to the cell and therefore not naturally occurring in the genome of the cell, or the target site can be found in a genomic location heterologous to where it occurs in nature. As used herein, the terms "endogenous target sequence" and "native target sequence" are used interchangeably herein and refer to a target sequence that is endogenous or native to the genome of a cell and is present in the endogenous or natural location of this 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 may be located at a different location in the genome of the cell (i.e., a non-endogenous or non-native location).

[0106] As used herein, a "protospacer adjacent motif" (PAM) refers to a short nucleotide sequence adjacent to the target sequence (protospacer) recognized (targeted) by the guide polynucleotide / Cas endonuclease system described herein. The Cas endonuclease may not be able to successfully recognize a target DNA sequence unless the target DNA sequence is followed by a PAM sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence 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.

[0107] The terms "altered target site," "altered target sequence," "modified target site," and "altered target sequence" are used interchangeably herein and refer to a target sequence disclosed herein that contains at least one alteration when compared to an unaltered target sequence. Such "alteration" may include, for example: (i) a substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, (iv) a chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0108] "Modified nucleotide" or "edited nucleotide" refers to a nucleotide sequence of interest that contains at least one alteration when compared to its unmodified nucleotide sequence. Such "alterations" include, for example, (i) a substitution of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, (iv) a chemical alteration of at least one nucleotide, or (v) any combination of (i)-(iv).

[0109] 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.

[0110] 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.

[0111] The term "polynucleotide modification template" includes a polynucleotide that contains at least one nucleotide modification compared to the nucleotide sequence to be edited. The nucleotide modification can be a substitution, addition, or deletion of at least one nucleotide. Optionally, the polynucleotide modification template can further include a homologous nucleotide sequence adjacent to the at least one nucleotide modification, which provides sufficient homology to the desired nucleotide sequence to be edited.

[0112] The term "plant-optimized Cas endonuclease" herein refers to a Cas protein (e.g., a multifunctional Cas protein) encoded by a nucleotide sequence that is optimized for expression in a plant cell or plant.

[0113] The terms "plant-optimized nucleotide sequence encoding a Cas endonuclease," "plant-optimized construct encoding a Cas endonuclease," and "plant-optimized polynucleotide encoding a Cas endonuclease" are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, 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.

[0114] The term "plant" generally includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and their progeny. Plants are monocotyledonous or dicotyledonous. Plant cells include, but are not limited to, cells derived from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. "Plant element" is intended to refer to a whole plant or plant component, which may include differentiated and / or undifferentiated tissues (e.g., but not limited to, plant tissues, parts, and cell types). In one embodiment, the plant element is one of the following: whole plants, seedlings, meristems, ground tissues, vascular tissue, epidermal tissue, seeds, leaves, roots, shoots, stems, flowers, fruits, stolons, bulbs, tubers, corms, stems, shoots, shoots, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, callus tissue). It should be noted that because protoplasts lack a cell wall, they are not technically "intact" plant cells (as found in nature with all their components). The term "plant organ" refers to a plant tissue or group of tissues that constitutes a morphologically and functionally independent part of a plant. As used herein, "plant element" is synonymous with plant "part" and refers to any part of a plant, may include separate tissues and / or organs, and may be used interchangeably with the term "tissue" throughout. Similarly, "plant reproductive element" is intended to generally refer to any part of a plant (e.g., without limitation, a seed, seedling, root, shoot, cutting, shoot, scion, stolon, bulb, tuber, corm, kettle, or sprout) that can initiate other plants by sexual or asexual reproduction of the plant. Plant elements may be present in a plant, or in a plant organ, tissue culture, or cell culture.

[0115] "Progeny" includes any subsequent generation of the plant.

[0116] As used herein, the term "plant part" refers to plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant mass, and intact plant cells in a plant or plant part (e.g., embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, grains, ears, cobs, pods, stalks, roots, root tips, anthers, etc., and these parts themselves). Grain is intended to mean mature seeds produced by growers for purposes other than the cultivation or propagation of the species. Progeny, variants, and mutants of the regenerated plants are also within the scope of the present invention, provided that such portions contain the introduced polynucleotide.

[0117] The terms "monocotyledonous" or "monocot" refer to a subclass of angiosperms, also known as the "monocotyledoneae," whose seeds typically contain only one primary leaf or cotyledon. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0118] The terms "dicotyledonous" or "dicot" refer to a subclass of angiosperms, also known as "dicotyledoneae," whose seeds typically contain only two primary leaves or cotyledons. The term includes reference to whole plants, plant elements, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and their progeny.

[0119] As used herein, a "male sterile plant" is a plant that does not produce viable or otherwise fertile male gametes. As used herein, a "female sterile plant" is a plant that does not produce viable or otherwise fertile female gametes. It is recognized that male sterile plants and female sterile plants can be female fertile and male fertile, respectively. Furthermore, it is recognized that male fertile (other than female fertile) plants can produce viable offspring when crossed with female fertile plants, and female fertile (other than male fertile) plants can produce viable offspring when crossed with male fertile plants.

[0120] As used herein, the term "non-conventional yeast" refers to any yeast that is not a Saccharomyces yeast species (e.g., S. cerevisiae) or a Schizosaccharomyces yeast species (see "Non-Conventional Yeasts in Genetics, Biochemistry and Biotechnology: Practical Protocols," K. Wolf, KD Breunig, G. Barth, Eds., Springer-Verlag, Berlin, Germany, 2003).

[0121] In the context of this disclosure, the terms "hybridized" or "crossing" or "crossing" refer to the fusion of gametes by pollination to produce offspring (i.e., cells, seeds, or plants). The term encompasses both sexual crossing (the pollination of one plant with another) and selfing (self-pollination, i.e., the pollen and ovules (or microspores and megaspores) are from the same plant or genetically identical plants).

[0122] The term "introgression" refers to the transfer of a desired allele at a genetic locus from one genetic background to another. For example, introgression of a desired allele at a particular locus can be transferred to at least one progeny plant by sexual mating between two parent plants, where at least one of the parent plants has the desired allele in its genome. Alternatively, for example, transfer of the allele can occur by recombination between two donor genomes, e.g., in fused protoplasts, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, for example, a transgene, an altered (mutated or edited) native allele, or a selected allele of a marker or QTL.

[0123] The term "isogenic line" is a comparative term, referring to a genetically identical but differently treated reference organism. In one example, two genetically identical maize plant embryos may be separated into two distinct groups, one of which has undergone a treatment (such as the introduction of a CRISPR-Cas effector endonuclease) and the other of which is a control that has not undergone such treatment. Therefore, any phenotypic differences between the two groups may be due solely to the treatment and not to any inherent nature of the plant's endogenous genetic makeup.

[0124] "Introducing" is intended to mean providing a polynucleotide, or polypeptide, or polynucleotide-protein complex to a target, such as a cell or organism, in such a way that the component enters the interior of a cell of the organism or into the cell itself.

[0125] A "polynucleotide of interest" includes any nucleotide sequence that encodes a protein or polypeptide that improves the desirability, i.e., agronomically beneficial, trait of a crop. Polynucleotides of interest include, but are not limited to, polynucleotides that encode agronomically important traits, herbicide resistance, insecticide resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, crop characteristics, commercial products, phenotypic markers, or any other trait of agricultural or commercial importance. Polynucleotides of interest may also be utilized in sense or antisense orientation. Additionally, multiple polynucleotides of interest may be utilized together, or "stacked," to provide additional benefits.

[0126] A "complex trait locus" includes a genomic locus that has multiple transgenes genetically linked to each other.

[0127] The compositions and methods herein may provide plants with improved "agronomic traits," or "agronomically important traits," or "agronomically beneficial traits," which may include, but are not limited to, disease resistance, drought tolerance, heat tolerance, cold tolerance, salt tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, increased yield, enhanced health, improved vigor, improved growth, improved photosynthetic capacity, nutritional enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of metabolites, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, altered seed nutrient composition, compared to an isogenic plant that does not include modification with the methods or compositions herein.

[0128] "Agronomic trait potential" is intended to mean the ability of a plant element, at a certain point in its life cycle, to exhibit a phenotype (preferably an improved agronomic trait) or to transmit said phenotype to another related plant element of the same plant.

[0129] The terms "reduced," "less," "slower," and "increased," "faster," "enhanced," and "greater," as used herein, refer to a decrease or increase in a property of a modified plant element or resulting plant compared to an unmodified plant element or resulting plant. For example, the decrease in the property may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5% to 10%, at least 10%, 10% to 20%, at least 15%, at least 20%, 20% to 30%, at least 25%, at least 30%, 30% to 40%, at least 35%, at least 40%, 40% to 50%, at least 45%, at least 50%, 50% to 60%, at least about 60%, 60% to 70%, 70% to 80%, at least 75%, at least about 80%, 80% to 90%, at least about 90%, 90% to 100%, at least 100%, 100% to 200%, at least 200%, at least about 300%, at least about 400%, or more less than an untreated control. Thus, the increase in the property may be at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 5% to 10%, at least 10%, 10% to 20%, at least 15%, at least 20%, 20% to 30%, at least 25%, at least 30%, 30% to 40%, at least 35%, at least 40%, 40% to 50%, at least 45%, at least 50%, 50% to 60%, at least about 60%, 60% to 70%, 70% to 80%, at least 75%, at least about 80%, 80% to 90%, at least about 90%, 90% to 100%, at least 100%, 100% to 200%, at least 200%, at least about 300%, at least about 400%, or more, over an untreated control.

[0130] As used herein, the term "before," in reference to sequence position, refers to the presence of one sequence upstream or 5' of another sequence.

[0131] The abbreviations mean as follows: "sec" means seconds, "min" means minutes, "h" means hours, "d" means days, "μL" means microliters, "mL" means milliliters, "L" means liters, "μM" means micromolar, "mM" means millimolar, "M" means moles, "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.

[0132] Classification of CRISPR-Cas systems CRISPR-Cas systems have been classified according to the sequence and structural analysis of their components. Several CRISPR / Cas systems have been described, including class 1 systems with multisubunit effector complexes (including types I, III, and IV) and class 2 systems with single-protein effectors (including types II, V, and VI) (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al. 2015, Cell 163, 1-13; Shmakov et al. 2015, Molecular Cell 60, 1-13; Haft et al. 2005, Computational Biology, PLoS Comput Biol 1(6):e60; and Koonin et al. 2017, Curr Opinion Microbiology 37:67-78).

[0133] The CRISPR-Cas system comprises, at a minimum, a CRISPR RNA (crRNA) molecule and at least one CRISPR-associated (Cas) protein, which form a crRNA-ribonucleoprotein (crRNP) effector complex. The CRISPR-Cas locus contains an array of identical repeats interspersed with DNA-targeting spacers encoding the crRNA components and an operon-like unit of cas genes encoding the Cas protein components. The resulting ribonucleoprotein complex recognizes polynucleotides in a sequence-specific manner (Jore et al. 2011, Nature Structural & Molecular Biology 18, 529-536). The crRNA functions as a guide RNA, directing the sequence-specific binding of the effector (protein or complex) to double-stranded DNA sequences by base-pairing with the complementary DNA strand and forming a so-called R-loop, while displacing the non-complementary strand (Jore et al. 2011).

[0134] The RNA transcript (pre-crRNA) of the CRISPR locus is specifically cleaved at the repeat sequence by CRISPR-associated (Cas) endoribonucleases in Type I and Type 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.

[0135] Various cas genes, encoding proteins with various domains, exist in various CRISPR systems. The cas operon contains genes encoding one or more effector endonucleases and other Cas proteins. Protein subunits include those described in Makarova et al. 2011, Nat Rev Microbiol. 2011 9(6):467-477; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; and Koonin et al. 2017, Current Opinion Microbiology 37:67-78. Domain types include those involved in expression (pre-crRNA processing, e.g., Cas6 or RNase III), buffering (including effector modules for crRNA and target binding and domains for target cleavage), adaptation (spacer insertion, e.g., Cas1 or Cas2), and assistance (regulatory, helper, or unknown function). Some domains may serve multiple purposes; for example, Cas9 contains a domain for endonuclease function and a domain for target cleavage.

[0136] The Cas endonuclease is guided by a single CRISPR RNA (crRNA) through direct RNA-DNA base pairing and recognizes DNA target sites 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).

[0137] Class I CRISPR-Cas systems Class I CRISPR-Cas systems include types I, III, and IV. A distinctive feature of Class I systems is the presence of an effector endonuclease complex instead of a single protein. The Cascade complex contains an RNA recognition motif (RRM) and a nucleic acid-binding domain, which is the core fold of the diverse RAMP (repeat-associated mysterious protein) protein superfamily (Makarova et al. 2013, Biochem Soc Trans 41, 1392-1400; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). RAMP protein subunits include Cas5 and Cas7 (which comprise the scaffold of the crRNA-effector complex), where the Cas5 subunit binds to the 5' handle of the crRNA and interacts with the large subunit, and Cas6, which is often loosely associated with the effector complex and generally functions as a repeat-specific RNase in pre-crRNA processing (Charpentier et al. FEMS Microbiol Rev 2015, 39:428-441; Niewoehner et al. 2016 RNA, 22:318-329).

[0138] Type I CRISPR-Cas systems contain a complex of effector proteins called Cascade (CRISPR-associated complex for antiviral defense), including at least Cas5 and Cas7. The effector complex functions with a single CRISPR RNA (crRNA) and Cas3 to defend against invading viral DNA (Brouns, SJJ et al. Science 321:960-964; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type I CRISPR-Cas loci contain the signature gene cas3 (or variants cas3' or cas3"), which encodes a metal-dependent nuclease with a single-stranded DNA (ssDNA)-stimulating superfamily 2 helicase that has demonstrated the ability to unwind double-stranded DNA (dsDNA) and RNA-DNA duplexes (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Following target recognition, the Cas3 endonuclease is recruited to the Cascade-crRNA-target DNA complex to cleave and degrade the DNA target (Westra, E.R. et al. (2012) Molecular Cell 46:595-605; Sinkunas, T. et al. (2011) EMBO J. 30:1335-1342; and Sinkunas, T. et al. (2013) EMBO J. 32:385-394). In some type I systems, Cas6 may be the active endonuclease involved in crRNA processing, and Cas5 and Cas7 function as non-catalytic RNA-binding proteins, whereas in type I-C systems, crRNA processing may be catalyzed by Cas5 (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type I systems are divided into seven subtypes (Makarova et al. 2011, Nat Rev Microbiol. 2011 9(6):467-477; Koonin et al. 2017, Curr Opinion Microbiology 37:67-78).A modified type I CRISPR-associated complex (Cascade) for adaptive antiviral defense has been described (WO 2013 / 098244 published July 4, 2013) that includes at least the protein subunits Cas7, Cas5, and Cas6, where one of these subunits is synthetically fused to the Cas3 endonuclease or the modified restriction endonuclease FokI.

[0139] Type III CRISPR-Cas systems, containing multiple cas7 genes, target either ssRNA or ssDNA and function as either an RNase or a target RNA-activated DNA nuclease (Tamulaitis et al. 2017 Trends in Microbiology 25(10)49-61). The Csm (type III-A) and Cmr (type III-B) complexes function as RNA-activated single-stranded (ss)DNases that couple target RNA binding / cleavage with ssDNA degradation. Upon infection with foreign DNA, CRISPR RNA (crRNA)-guided binding of the Csm or Cmr complex to nascent transcripts recruits the Cas10 DNase to actively transcribed phage DNA, resulting in degradation of both the transcript and the phage DNA, but not the host DNA. The Cas10 HD domain is responsible for the ssDNase activity, and the Csm3 / Cmr4 subunits are responsible for the endoribonuclease activity of the Csm / Cmr complex. The 3' flanking sequence of the target RNA is important for the ssDNase activity of Csm / Cmr: base pairing with the 5' handle of the crRNA protects the host DNA from degradation.

[0140] Type IV systems contain typical type I cas5 and cas7 domains in addition to a cas8-like domain, but can lack the CRISPR array that is characteristic of most other CRISPR-Cas systems.

[0141] Class II CRISPR-Cas systems Class II CRISPR-Cas systems include types II, V, and VI. A distinctive feature of class II systems is the presence of a single Cas effector protein instead of an effector complex.

[0142] Type II and V Cas proteins contain a RuvC endonuclease domain that adopts an RNase H fold. Type II CRISPR / Cas systems use crRNA and tracrRNA (trans-activating CRISPR RNA) to guide the Cas endonuclease to its DNA target. The crRNA contains a spacer region that is complementary to one strand of the double-stranded DNA target and a region that base-pairs with the tracrRNA (trans-activating CRISPR RNA) to form an RNA duplex that allows the Cas endonuclease to cleave the DNA target, leaving blunt ends. The spacer is obtained through a poorly understood process involving the Cas1 and Cas2 proteins. Type II CRISPR-Cas loci generally contain the cas1 and cas2 genes in addition to the cas9 gene (Chylinski et al. 2013, RNA Biology 10:726-737; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). Type II CRISPR-Cas loci may encode tracrRNAs that are partially complementary to repeats within each CRISPR array and may contain other proteins such as Csn1 and Csn2. The presence of cas9 adjacent to the cas1 and cas2 genes is characteristic of type II loci (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).

[0143] Type V CRISPR / Cas systems contain a single Cas endonuclease, Cpf1 (Cas12) (Koonin et al. 2017 Curr Opinion Microbiology 37:67-78), which, unlike Cas9, is an active RNA-guided endonuclease that does not necessarily require an additional trans-activating CRISPR (tracr) RNA for target cleavage.

[0144] Type VI CRISPR-Cas systems contain two HEPN (higher eukaryotic and prokaryotic nucleotide-binding) domains, but also contain the cas13 gene, which encodes a nuclease lacking either the HNH or RuvC domains, and thus are independent of tracrRNA activity. The majority of the HEPN domain contains a conserved motif that constitutes a metal-independent endo-RNase active site (Anantharam et al. 2013 Biol Direct 8:15). This feature suggests that type VI systems act on RNA targets instead of the DNA targets common to other CRISPR-Cas systems.

[0145] Novel Cas endonuclease CRISPR-Cas system Disclosed herein is a novel CRISPR-Cas system, its components, and methods of using said components, which includes a novel Cas effector protein, the Cas endonuclease.

[0146] The novel CRISPR-Cas system components described herein may include one or more subunits from different Cas systems, subunits derived from or modified from multiple different bacterial or archaeal prokaryotes, and / or synthetic or engineered components.

[0147] Described herein is a newly identified CRISPR-Cas system that includes novel sequences for the cas genes. Additionally, novel cas genes and proteins are described.

[0148] One feature of the novel Cas endonuclease system is the locus structure, which contains the endonuclease gene upstream of the CRISPR array but does not contain the cas1, cas2, or cas4 genes.

[0149] CRISPR-Cas system components Cas proteins Numerous proteins can be encoded in the CRISPR cas operon, including proteins involved in adaptation (spacer insertion), interference (effector module target binding, target nicking or cleavage—e.g., endonuclease activity), expression (pre-crRNA processing), regulation, and more.

[0150] Two proteins, Cas1 and Cas2, are conserved in many CRISPR systems (see, for example, Koonin et al. 2017, Curr Opinion Microbiology 37:67-78). Cas1 is a metal-dependent DNA-specific endonuclease that generates double-stranded DNA fragments. In some systems, Cas1 forms a stable complex with Cas2, which is essential for spacer acquisition and insertion in CRISPR systems (Nunez et al. 2014, Nature Str Mol Biol 21:528-534).

[0151] Many other proteins, including Cas4 (which may have similarity to RecB nuclease), have been identified in various systems and are thought to play a role in capturing novel viral DNA sequences for incorporation into CRISPR arrays (Zhang et al. 2012, PLOS One 7(10):e47232).

[0152] Some proteins may encompass multiple functions, for example, Cas9, the signature protein of class 2 type II systems, has been demonstrated to be involved in pre-crRNA processing, target binding, and target cleavage.

[0153] In some natural systems, such as the Cas endonuclease CRISPR system from Syntrophomonas palmitatica, no genes encoding Cas1, Cas2, or Cas4 proteins were detected near the endonuclease gene.

[0154] Cas endonucleases and effectors Endonucleases are enzymes that cleave phosphodiester bonds within polynucleotide chains, including restriction endonucleases, which cleave DNA at specific sites without damaging bases. Examples of endonucleases include restriction endonucleases, meganucleases, TAL effector nucleases (TALENs), zinc finger nucleases, and Cas (CRISPR-associated) effector endonucleases.

[0155] Cas endonucleases, either as single effector proteins or in effector complexes with other components, unwind DNA duplexes at target sequences and, optionally, cleave at least one DNA strand when mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) complexed with the Cas effector protein. Such recognition and cleavage of a target sequence by a Cas endonuclease typically occurs when a precise protospacer adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, the Cas endonucleases herein may lack DNA cleavage or nicking activity but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component (see also U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015).

[0156] Cas endonucleases can occur as individual effectors (class 2 CRISPR systems) or as part of larger effector complexes (class I CRISPR systems).

[0157] Described Cas endonucleases include, but are not limited to, Cas3 (characteristic of class 1 type I systems), Cas9 (characteristic of class 2 type II systems), and Cas12 (Cpf1) (characteristic of class 2 type V systems).

[0158] Cas3 (and its variants Cas3' and Cas3") functions as a single-stranded DNA nuclease (HD domain) and an ATP-dependent helicase. Variants of Cas3 endonuclease can be obtained by disabling the functional activity of one or both domains of the Cas3 endonuclease polypeptide, as previously described (Sinkunas, T. et al. 2013, EMBO J. 32:385-394), a cleavage-ready cascade containing a modified Cas3 endonuclease can be converted into a nickase (since the HD domain is still functional) by abolishing the ATPase-dependent helicase activity by deletion, knockout of the Cas3-helicase domain, or mutagenesis of key residues, or by assembling the reaction in the absence of ATP. Abolishing HD endonuclease activity can be achieved by any method known in the art, including but not limited to, mutagenesis of key residues in the HD domain, thereby converting a cleavage-ready cascade containing a modified Cas3 endonuclease into a helicase. Abolishing both Cas helicase and Cas3 HD endonuclease activity can be achieved by any method known in the art, including but not limited to, mutagenesis of key residues in both the helicase and HD domains, thereby converting a cleavage-ready cascade containing a modified Cas3 endonuclease into a binding protein that binds to a target sequence.

[0159] Cas9 (previously called Cas5, Csn1, or Csx12) is a Cas endonuclease that specifically recognizes and cleaves all or part of a DNA target sequence in complex with cr and tracr nucleotides or a single guide polynucleotide. Cas9 recognizes the 3'GC-rich PAM sequence of target dsDNA. The Cas9 protein contains a RuvC nuclease with an HNH (HNH) nuclease adjacent to the RuvC-II domain. The RuvC nuclease and HNH nuclease can each cleave one DNA strand at the target sequence (the concerted action of both domains results in a DNA double-strand break, while the activity of one domain results in a nick). Generally, the RuvC domain comprises subdomains I, II, and III, with domain I located near the N-terminus of Cas9 and subdomains II and III located in the center of the protein and flanking the HNH domain (Hsu et al. 2013, Cell 157:1262-1278). Cas9 endonucleases are typically derived from type II CRISPR systems, which include DNA cleavage systems that utilize Cas9 endonuclease complexed with at least one polynucleotide component. For example, Cas9 can form a complex with CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In another example, Cas9 can exist in a complex with a single guide RNA (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15).

[0160] Cas12 (previously called Cpf1 and variants c2c1, c2c3, CasX, and CasY) contains a RuvC nuclease domain and generates staggered 5' overhangs on dsDNA targets. Some variants do not require tracrRNA for their functionality, unlike Cas9. Cas12 and its variants recognize 5' AT-rich PAM sequences on target dsDNA. The insertion domain of the Cas12a protein, called Nuc, has been demonstrated to be involved in target strand cleavage (Yamano et al. 2016, Cell 165:949-962). Further mutation studies in other Cas12 proteins demonstrated that the Nuc domain, along with the RuvC domain involved in cleavage, contributes to guidance and target binding (Swarts et al. Mol Cell 2017, 66:221-233 e224).

[0161] Cas endonucleases and effector proteins can be used for targeted genome editing (by single and multiple double-strand breaks and nicks) and targeted genome regulation (by attaching epigenetic effector domains to Cas proteins or sgRNAs). Cas endonucleases can also be engineered to function as RNA-guided recombinases, and by binding RNA, can function as scaffolds for the assembly of multiprotein and nucleic acid complexes (Mali et al. 2013 Nature Methods Vol. 10:957-963).

[0162] Cas endonucleases and their variants A Cas endonuclease or a functional variant thereof is defined as a functional RNA-guided, PAM-dependent dsDNA cleavage protein of less than about 500 amino acids, divided into three subdomains: a C-terminal RuvC catalytic domain containing 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.

[0163] The novel Cas endonuclease variant proteins disclosed herein include an effector protein (endonuclease). Wild-type (WT) Cas endonuclease proteins require a PAM sequence of N(T>W>C)TTC at or near the target site of a target double-stranded polydeoxyribonucleotide.

[0164] Certain functional variants of the Cas endonucleases disclosed herein have double-strand cleavage or single-strand nicking activity, which may be less than, approximately the same as, or even greater than the activity of the WT Cas endonuclease. Varying levels of activity may have different uses depending on the practitioner's desires. A Cas endonuclease or functional variant, when aligned with SEQ ID NO: 14 or SEQ ID NO: 48, has the following amino acid motifs relative to amino acid position numbering in SEQ ID NO: 14 or SEQ ID NO: 48: (i) 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; and (ii) the following amino acid motifs: GxxxG starting at amino acid position 226; ExL starting at amino acid position 327; and Cx starting at amino acid position 376. n C; Cx starting at amino acid position 395 n(C,H), where X is any nucleotide and n is any number. In some embodiments, functional variants do not have a phenylalanine at position 38, an alanine (A) at position 40, a histidine at position 79, a glutamic acid at relative position 81, an alanine at relative position 87, a threonine at relative position 335, a cysteine at position 409, a glutamic acid at position 421, a lysine at position 467, or a glutamic acid at position 468 (where 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, the functional variant, when aligned with SEQ ID NO: 14 or SEQ ID NO: 48, has, relative to the amino acid position numbers of SEQ ID NO: 14 or SEQ ID NO: 48, an aspartic acid or glutamic acid at position 38, an aspartic acid at position 79, a proline at position 120, an aspartic acid at position 149, a glycine at position 226, a glycine at position 230, a histidine at position 293, a serine at position 298, a phenylalanine at position 306, a phenylalanine at position 329, a phenylalanine at position 330, a phenylalanine at position 340, a phenylalanine at position 341, a phenylalanine at position 342, a phenylalanine at position 343, a phenylalanine at position 344, a phenylalanine at position 345, a phenylalanine at position 346, a phenylalanine at position 347, a phenylalanine at position 348, a phenylalanine at position 349, a phenylalanine at position 350, a phenylalanine at position 351, a phenylalanine at position 352, a phenylalanine at position 353, a phenylalanine at position 354, a phenylalanine at position 355, a phenylalanine at position 356, a phenylalanine at position 357, a phenylalanine at position 358, a phenylalanine at position 359, a phenylalanine at position 359, a phenylalanine at position 360, a phenylalanine at position 361, a phenylalanine at position 362, a phenylalanine at position 36 at position 313, serine at position 314, 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.

[0165] A "functional fragment" of a Cas endonuclease variant endonuclease includes at least 50, 50-100, at least 100, 100-150, at least 150, 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 450, or more than 450 consecutive amino acids of any one of SEQ ID NOS: 15-47, and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, at least 65%, 65%-70%, at least 70%, 70%-80%, or more than 80% consecutive amino acids of any one of SEQ ID NOS: 15-47. "A polynucleotide" refers to a polynucleotide of less than 497 amino acids that shares at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity with the amino acid sequence; and the ability to recognize, bind to, or nick one strand of a double-stranded polynucleotide, or the ability to cleave both strands of a double-stranded polynucleotide, or any combination of the foregoing.

[0166] The RuvC domain has been shown in the literature to encompass endonuclease functionality. Cas endonucleases can be isolated or identified from loci that contain Cas endonuclease genes encoding effector proteins and arrays containing multiple repeats.

[0167] Zinc finger motifs are domains that stabilize these folds by coordinating one or more zinc ions, usually by cysteine and histidine side chains. Zinc fingers are named for the pattern of cysteine and histidine residues that coordinate the zinc ion (e.g., C4 means that the zinc ion is coordinated by four cysteine residues, while CH means that the zinc ion is coordinated by three cysteine residues and one histidine residue).

[0168] Cas endonuclease proteins contain one or more zinc finger (ZFN) coordination motifs that can form a zinc-binding domain. Zinc finger-like motifs can assist in the separation of target and non-target strands and the loading of guide RNAs onto DNA targets. Cas endonuclease proteins containing one or more zinc finger motifs can provide additional stability to ribonucleoprotein complexes on target polynucleotides. Cas endonuclease proteins contain a C4 zinc-binding domain or a C3H zinc-binding domain.

[0169] 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.

[0170] Cas endonucleases are RNA-guided endonucleases that can bind to and cleave double-stranded DNA targets that contain (1) a sequence that shares homology with the nucleotide sequence of a guide RNA and (2) a PAM sequence.

[0171] Cas endonucleases function as double-strand break inducers and can also be nickases or single-strand break inducers. In some embodiments, catalytically inactive Cas endonucleases can be used to target or recruit to a target DNA sequence but cannot induce cleavage. In some embodiments, catalytically inactive Cas endonuclease proteins can be used in conjunction with functional endonucleases to cleave a target sequence. In some embodiments, catalytically inactive Cas endonuclease proteins can be combined with base-editing molecules such as deaminase. In some embodiments, the deaminase can be cytidine deaminase. In some embodiments, the deaminase can be adenine deaminase. In some embodiments, the deaminase can be ADAR-2.

[0172] The Cas endonuclease may comprise at least 50, 50-100, at least 100, 100-150, at least 150, 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 450, or more than 450 consecutive amino acids of any one of SEQ ID NOs: 15-47, and at least 50%, 50%-55%, at least 55%, 55%-60%, at least 60%, 60%-65%, at least 65%, Further defined as an RNA-guided double-stranded DNA cleavage protein that shares 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity and retains at least partial endonuclease activity.

[0173] The disclosed modified Cas endonucleases or inactivated modified Cas polypeptides include at least 50, 50-100, at least 100, 100-150, at least 150, 150-200, at least 200, 200-250, at least 250, 250-300, at least 300, 300-350, at least 350, 350-400, at least 400, 400-450, at least 500, 500-550, at least 600, 600-650, at least 650, 650-700, at least 700, 700-750, at least 750, 750-800, at least 800, 800-850, at least 850, 850-900, at least 900, 900 The polynucleotide may be encoded by a polynucleotide sharing at least 50%, 50% to 55%, at least 55%, 55% to 60%, at least 60%, 60% to 65%, at least 65%, 65% to 70%, at least 70%, 70% to 75%, at least 75%, 75% to 80%, at least 80%, 80% to 85%, at least 85%, 85% to 90%, at least 90%, 90% to 95%, at least 95%, 95% to 96%, at least 96%, 96% to 97%, at least 97%, 97% to 98%, at least 98%, 98% to 99%, at least 99%, 99% to 100%, or 100% sequence identity with up to 950, at least 950, 950 to 1000, at least 1000, or more than 1000 contiguous nucleotides.

[0174] Cas endonucleases, effector proteins, or functional fragments thereof for use in the disclosed methods can be isolated from natural sources or from recombinant sources in which genetically engineered host cells have been modified to express a nucleic acid sequence encoding the protein. Alternatively, Cas proteins can be produced using cell-free protein expression systems or synthetically produced. Effector Cas nucleases can be isolated and introduced into heterologous cells or modified from their native form to exhibit a different type or magnitude of activity than their native source. Such modifications include, but are not limited to, fragments, variants, substitutions, deletions, and insertions. Cas endonuclease WT compositions are described in International Publication No. WO 2020 / 123887, published July 16, 2020.

[0175] Fragments and variants of Cas endonucleases and Cas endonuclease effector proteins can be obtained by methods such as site-directed mutagenesis and synthetic construction. Methods for measuring endonuclease activity are well known in the art, including, but not limited to, those described in WO 2013 / 166113, published November 7, 2013, WO 2016 / 186953, published November 24, 2016, and WO 2016 / 186946, published November 24, 2016.

[0176] Cas endonucleases can include modified forms of Cas polypeptides. Modified forms of Cas polypeptides can include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the naturally occurring nuclease activity of the Cas protein. For example, in some instances, modified forms of Cas proteins have less than 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the nuclease activity of the corresponding wild-type Cas polypeptide (SEQ ID NO: 48; U.S. Patent Application Publication No. 2014 / 0068797, published March 6, 2014). In some instances, modified forms of Cas polypeptides have substantially no nuclease activity and are referred to as catalytically "inactivated Cas" or "deactivated Cas (dCas)." Inactivated / deactivated Cas includes deactivated Cas endonuclease (dCas). Catalytically inactive Cas effector proteins can be fused to heterologous sequences to induce or alter activity.

[0177] The Cas endonuclease may be part of a fusion protein that includes one or more heterologous protein domains (e.g., one, two, three, or more domains in addition to the Cas protein). Such a fusion protein may include any additional protein sequences and, optionally, a linker sequence between any two domains (e.g., between the Cas and the first heterologous domain). Examples of protein domains that can be fused to the Cas proteins herein include, but are not limited to, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]), and domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activator activity (e.g., VP16 or VP64), transcriptional repressor activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also be fused to proteins that bind to DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.

[0178] Catalytically active and / or inactive Cas endonucleases can be fused to heterologous sequences (U.S. Patent Application Publication No. 2014 / 0068797, published March 6, 2014). Suitable fusion partners include, but are not limited to, polypeptides that provide activities that indirectly increase transcription by acting directly on target DNA or polypeptides associated with target DNA (e.g., histones or other DNA-binding proteins). Additional suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylation activity, acetyltransferase activity, deacetylation activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitination activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Still other suitable fusion partners include, but are not limited to, polypeptides that directly cause increased transcription of a target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that induce transcriptional activators, small molecule / drug-responsive transcriptional regulators, etc.). Partially active or catalytically inactive Cas-endonucleases can also be fused to another protein or domain (e.g., Clo51 nuclease or FokI nuclease) to generate a double-stranded break (Guilinger et al. Nature biotechnology, volume 32, number 6, June 2014).

[0179] Catalytically active or inactive Cas proteins, such as the Cas endonuclease proteins described herein, can also be fused to molecules that direct the editing of single or multiple bases in a polynucleotide sequence (e.g., site-specific deaminases that can change the identity of a nucleotide, for example, from C·G to T·A or from A·T to G·C) (Gaudelli et al. 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 double-stranded DNA cleavage." Nature 533(7603)(2016):420-4). Base editing fusion proteins can include, for example, active (double-strand breaking), partially active (nickase), or inactivated (catalytically inactive) Cas endonucleases and deaminases (e.g., but not limited to, cytidine deaminase, adenine deaminase, APOBEC1, APOBEC3A, BE2, BE3, BE4, ABE, etc.). Base editing repair inhibitors and glycosylase inhibitors (e.g., uracil glycosylase inhibitors (which prevent the removal of uracil)) are contemplated as other components of the base editing system in some embodiments.

[0180] The Cas endonucleases described herein can be expressed and purified by methods known in the art, for example, the methods described in WO 2016 / 186953, published November 24, 2016.

[0181] Many Cas endonucleases have been described that can recognize specific PAM sequences (WO 2016 / 186953 published November 24, 2016; WO 2016 / 186946 published November 24, 2016; and Zetsche B et al. 2015. Cell 163, 1013) and cleave target DNA at specific locations. Based on the methods and embodiments described herein that use the novel inducible Cas system, one skilled in the art will understand that these methods can be adapted so that they can be used with any inducible endonuclease system.

[0182] The Cas effector protein may comprise a heterologous nuclear localization sequence (NLS). The heterologous NLS amino acid sequence herein may be strong enough to drive the accumulation of detectable amounts of the Cas protein in the nucleus of, for example, a yeast cell herein. The NLS may comprise a short sequence (e.g., 2-20 residues) of one (monoclavicular) or multiple (e.g., biclavicular) basic, positively charged residues (e.g., lysine and / or arginine) and may be positioned anywhere within the Cas amino acid sequence, but exposed on the surface of the protein. The NLS may be operably linked, for example, to the N-terminus or C-terminus of the Cas protein herein. For example, two or more NLS sequences may be linked to the Cas protein, for example, to both the N-terminus and C-terminus of the Cas protein. The Cas endonuclease gene may be operably linked to an SV40 nuclear targeting signal upstream of the Cas codon region and a bipartite VirD2 nuclear localization signal (Tinland et al. (1992) Proc. Natl. Acad. Sci. USA 89:7442-6) downstream of the Cas codon region. Non-limiting examples of suitable NLS sequences herein include those disclosed in U.S. Patent Nos. 6,660,830 and 7,309,576.

[0183] Guide polynucleotide The guide polynucleotide allows for target recognition, target binding, and optional target cleavage by a Cas endonuclease, and can be a single molecule or a double molecule. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequence). Optionally, the guide polynucleotide can include at least one nucleotide, phosphodiester bond, or linkage modification, including but not limited to, locked nucleic acid (LNA), 5-methyl dC, 2,6-diaminopurine, 2'-fluoro A, 2'-fluoro U, 2'-O-methyl RNA, phosphorothioate bond, linkage to a cholesterol molecule, linkage to a polyethylene glycol molecule, linkage to a spacer 18 (hexaethylene glycol chain) molecule, or a 5' to 3' covalent linkage resulting in cyclization. A guide polynucleotide that contains only ribonucleic acid is also called a "guide RNA" or "gRNA" (U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015). Guide polynucleotides can be engineered or synthetic.

[0184] Guide polynucleotides include chimeric non-natural guide RNAs that contain regions that are not found together in nature (i.e., they are heterologous to each other). For example, in a chimeric non-natural guide RNA that contains a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in a target DNA linked to a second nucleotide sequence that can recognize a Cas endonuclease, the first nucleotide sequence and the second nucleotide sequence are not found linked together in nature.

[0185] The guide polynucleotide can be a duplex molecule (also called a double-stranded guide polynucleotide) that includes a cr nucleotide sequence (e.g., crRNA) and a tracr nucleotide sequence (e.g., tracrRNA). In some instances, a linker polynucleotide is present that links the crRNA and tracrRNA to form a single guide (e.g., sgRNA).

[0186] The cr nucleotide comprises a first nucleotide sequence domain (called a variable targeting domain or VT domain) capable of hybridizing to a nucleotide sequence in the target DNA and a second nucleotide sequence (also called a tracr mate sequence) that is part of the Cas endonuclease recognition (CER) domain. The tracr mate sequence hybridizes to the tracr nucleotide along a complementary region, and together they can form a Cas endonuclease recognition domain, i.e., a CER domain. The CER domain can interact with a Cas endonuclease polypeptide. The cr nucleotide and tracr nucleotide of the double-stranded guide polynucleotide can be RNA, DNA, and / or RNA-DNA combination sequences. In some embodiments, the cr nucleotide molecule of the double-stranded guide polynucleotide is referred to as "crDNA" (when composed of a continuous stretch of DNA nucleotides), "crRNA" (when composed of a continuous stretch of RNA nucleotides), or "crDNA-RNA" (when composed of a combination of DNA nucleotides and RNA nucleotides). The cr nucleotide can comprise a fragment of a crRNA naturally occurring in bacteria and archaea. The size of the fragments of naturally occurring crRNA in bacteria and archaea that can be present in the cr nucleotides disclosed herein can range from, but is not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides in length.

[0187] In some embodiments, tracr nucleotides are referred to as "tracrRNA" (when composed of a continuous stretch of RNA nucleotides), "tracrDNA" (when composed of a continuous stretch of DNA nucleotides), or "tracrDNA-RNA" (when composed of a combination of DNA and RNA nucleotides). In one embodiment, the RNA that guides the RNA / Cas9 endonuclease complex is a double-stranded RNA comprising a double-stranded crRNA-tracrRNA. tracrRNA (trans-activating CRISPR RNA) contains, from 5' to 3', (i) a sequence that anneals to the repeat region of CRISPR type II crRNA and (ii) a stem-loop-containing portion (Deltcheva et al. Nature 471:602-607). A double-stranded guide polynucleotide can form a complex with a Cas endonuclease, and the guide polynucleotide / Cas endonuclease complex (also called a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, allowing the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single- or double-strand break) at the target site (U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015).

[0188] In one aspect, the guide polynucleotide is a guide polynucleotide capable of forming a PGEN as described herein, wherein said guide polynucleotide comprises a first nucleotide sequence domain that is complementary to a nucleotide sequence of a target DNA and a second nucleotide sequence domain that interacts with a Cas polypeptide as disclosed herein.

[0189] In one aspect, the guide polynucleotide is a guide polynucleotide described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain are selected from the group consisting of a DNA sequence, an RNA sequence, and a combination thereof.

[0190] In one aspect, the guide polynucleotide is a guide polynucleotide described herein, wherein the first nucleotide sequence and the second nucleotide sequence domain are selected from the group consisting of stability-enhancing RNA backbone modifications, stability-enhancing DNA backbone modifications, and combinations thereof (see Kanasty et al. 2013, Common RNA-Backbone Modifications, Nature Materials 12:976-977;; U.S. Patent Application Publication No. 2015 / 0082478 published March 19, 2015 and U.S. Patent Application Publication No. 2015 / 0059010 published February 26, 2015).

[0191] The guide RNA includes a duplex molecule containing a chimeric non-natural crRNA linked to at least one tracrRNA. Chimeric non-natural crRNAs include crRNAs that contain regions that are not found together in nature (i.e., they are heterologous to each other). For example, a crRNA that contains a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in target DNA linked to a second nucleotide sequence (also called a tracr mate sequence) (such that the first sequence and the second sequence are not found linked together in nature).

[0192] A guide polynucleotide can also be a single molecule (also called a single guide polynucleotide) that includes a cr nucleotide sequence linked to a tracr nucleotide sequence. A single guide polynucleotide includes a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence within the target DNA and a Cas endonuclease recognition domain (CER domain) that interacts with a Cas endonuclease polypeptide.

[0193] The VT and / or CER domains of a single guide polynucleotide can comprise RNA, DNA, or combined RNA-DNA sequences. A single guide polynucleotide composed of sequences derived from cr and tracr nucleotides can be referred to as a "single guide RNA" (when composed of a contiguous stretch of RNA nucleotides), a "single guide DNA" (when composed of a contiguous stretch of DNA nucleotides), or a "single guide RNA-DNA" (when composed of a combination of RNA and DNA nucleotides). A single guide polynucleotide can form a complex with a Cas endonuclease, and the guide polynucleotide / Cas endonuclease complex (also referred to as a guide polynucleotide / Cas endonuclease system) can guide the Cas endonuclease to a genomic target site, allowing the Cas endonuclease to recognize, bind, and optionally nick or cleave (introduce a single- or double-strand break) the target site. (U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015).

[0194] Chimeric non-natural single guide RNAs (sgRNAs) include sgRNAs that contain regions that are not found together in nature (i.e., they are heterologous to each other), such as an sgRNA that contains a first nucleotide sequence domain (called a variable targeting domain or VT domain) that can hybridize to a nucleotide sequence in target DNA linked to a second nucleotide sequence (also called a target mate sequence) that are not found linked together in nature.

[0195] The nucleotide sequence linking the cr and tracr nucleotides of a single guide polynucleotide can comprise an RNA sequence, a DNA sequence, or a combined RNA-DNA sequence. In one embodiment, the nucleotide sequence linking the cr and tracr nucleotides of a single guide polynucleotide (also referred to as the "loop") is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 1 The length of the nucleotide sequence linking the cr and tracr nucleotides of a single guide polynucleotide can be 3, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 nucleotides. In another embodiment, the nucleotide sequence linking the cr and tracr nucleotides of a single guide polynucleotide can comprise a tetraloop sequence (such as, but not limited to, a GAAA tetraloop sequence).

[0196] The guide polynucleotide may be generated by any method known in the art, including chemically synthesizing the guide polynucleotide (such as, but not limited to, Hendel et al. 2015, Nature Biotechnology 33, 985-989), in vitro generation of the guide polynucleotide, and / or self-splicing of the guide RNA (such as, but not limited to, Xie et al. 2015, PNAS 112:3570-3575).

[0197] The guide polypeptide can be used in combination with any of the modified Cas polypeptides disclosed herein, including any one of the modified Cas polypeptides or endonucleases disclosed in the Summary of the Invention section of this specification at paragraphs

[0007] -

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

[0198] Protospacer adjacent motif (PAM) As used herein, a "protospacer adjacent motif" (PAM) refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that can be recognized (targeted) by the guide polynucleotide / Cas endonuclease system. The Cas endonuclease may not be able to successfully recognize a target DNA sequence unless the target DNA sequence is followed by a PAM sequence. The sequence and length of the PAM herein may vary depending on the Cas protein or Cas protein complex used. The PAM sequence 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.

[0199] The terms "randomized PAM" and "randomized protospacer adjacent motif" are used interchangeably herein and refer to a random DNA sequence adjacent to the target sequence (protospacer) recognized (targeted) by the 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 one of the nucleotides A, C, G, or T.

[0200] Guide polynucleotide / Cas endonuclease complex The guide polynucleotide / Cas endonuclease complexes described herein are capable of recognizing, binding to, and optionally nicking, unnicking, or cleaving all or part of a target sequence.

[0201] A guide polynucleotide / Cas endonuclease complex capable of cleaving both strands of a DNA target sequence typically contains a Cas protein having all of its endonuclease domains in a functional state (e.g., a wild-type endonuclease domain, or a variant thereof that retains the activity of part or all of each endonuclease domain). Thus, a wild-type Cas protein (e.g., a Cas protein disclosed herein) or a variant thereof that retains the activity of part or all of each endonuclease domain of the Cas protein are suitable examples of Cas proteins that can cleave both strands of a DNA target sequence.

[0202] A guide polynucleotide / Cas endonuclease complex capable of cleaving a single strand of a DNA target sequence may be characterized herein as having nickase activity (e.g., partial cleavage ability). Cas nickases typically contain one functional endonuclease domain that enables Cas to cleave (i.e., nick) only one strand of a DNA target sequence. For example, a Cas9 nickase may contain (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, a Cas9 nickase may contain (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a mutated, dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Application Publication No. 2014 / 0189896, published July 3, 2014. A pair of Cas nickases can be used to increase the specificity of DNA targeting. Generally, this can be achieved by providing two Cas nickases that are linked to RNA components with different guide sequences to target and nick nearby DNA sequences on opposite strands within the desired targeting region. Such nearby cleavage of each DNA strand creates a double-strand break (i.e., a DSB with a single-strand overhang), which is then recognized as a substrate for non-homologous end-joining (NHEJ) (prone to imperfect repair leading to mutations) or homologous recombination (HR). In these embodiments, the nicks can be separated from one another by, for example, at least about 5, 5-10, at least 10, 10-15, at least 15, 15-20, at least 20, 20-30, at least 30, 30-40, at least 40, 40-50, at least 50, 50-60, at least 60, 60-70, at least 70, 70-80, at least 80, 80-90, at least 90, 90-100, or 100 or more (or any integer between 5 and 100) bases. One or two Cas nickase proteins herein can be used in a Cas nickase pair.For example, a Cas9 nickase with a mutated RuvC domain but a functional HNH domain (i.e., Cas9 HNH / RuvC) can be used (e.g., Streptococcus pyogenes Cas9 HNH / RuvC). Each Cas9 nickase (e.g., Cas9 HNH / RuvC) can be directed to specific DNA sites close together (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences that target each nickase to its specific DNA site.

[0203] In certain embodiments, the guide polynucleotide / Cas endonuclease complex can bind to a DNA target site sequence but not cleave any strand at the target site sequence. Such a complex can include a Cas protein whose nuclease domains are all mutated and dysfunctional. For example, a Cas9 protein that can bind to a DNA target site sequence but not cleave any strand at the target site sequence can include both a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. Cas proteins herein that bind to but do not cleave a target DNA sequence can be used to regulate gene expression; for example, in this case, the Cas protein can be fused to a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein).

[0204] In one aspect, a guide polynucleotide / Cas endonuclease complex (PGEN) described herein is a PGEN, wherein said Cas endonuclease is optionally covalently or non-covalently linked to or associated with at least one protein subunit or functional fragment thereof.

[0205] In one embodiment of the present disclosure, a 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 functional fragment thereof, the guide polynucleotide is a chimeric non-natural guide polynucleotide, and the guide polynucleotide / Cas endonuclease complex can recognize, bind to, and optionally nick, unwind, or cleave all or a portion of a target sequence.

[0206] The Cas effector protein can be a Cas endonuclease effector protein disclosed herein.

[0207] 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 can recognize, bind to, and optionally nick, unwind, or cleave all or a portion of a target sequence.

[0208] The PGEN can 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 functional fragment thereof. In some embodiments, the protein subunit is selected from the group consisting of a Cas1 protein subunit, a Cas2 protein subunit, a Cas4 protein subunit, and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex, wherein the Cas effector protein further comprises at least two different protein subunits selected from the group consisting of Cas1, Cas2, and Cas4.

[0209] 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 one additional Cas protein including Cas4.

[0210] In one aspect, the guide polynucleotide / Cas effector protein complex (PGEN) described herein is a PGEN in which the Cas effector protein is covalently or non-covalently linked to at least one protein subunit or functional fragment thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex in which the Cas effector protein polypeptide is covalently or non-covalently linked to or associated with one copy or multiple copies of at least one protein subunit or functional fragment thereof selected from the group consisting of a Cas1 protein subunit, a Cas2 protein subunit, optionally one additional Cas protein subunit, including a Cas4 protein subunit, and any combination thereof. The PGEN can be a guide polynucleotide / Cas effector protein complex in which the Cas effector protein is covalently or non-covalently linked to or associated with at least two different protein subunits selected from the group consisting of a Cas1 protein subunit, a Cas2 protein subunit, and optionally one additional Cas protein, including a Cas4 protein subunit. The PGEN may 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 selected from the group consisting of Cas1, Cas2, and optionally one additional Cas protein, including Cas4, and any combination thereof.

[0211] Any component of the guide polynucleotide / Cas effector protein complex, the guide polynucleotide / Cas effector protein complex itself, and the polynucleotide modification template and / or donor DNA may be introduced into a heterologous cell or organism by any method known in the art.

[0212] Recombinant constructs for cell transformation The guide polynucleotides, Cas polypeptides or Cas endonucleases of the present disclosure, optionally further comprising one or more polynucleotides of interest, polynucleotide modification templates, donor DNA, guide polynucleotide / Cas endonuclease systems disclosed herein, and any one combination thereof, may be introduced into cells, including, but not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein.

[0213] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in more detail in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described below.

[0214] Vectors and constructs include circular plasmids and linear polynucleotides, which contain a polynucleotide of interest and optionally other components, including linkers, adapters, regulatory elements, or analytical elements. In some examples, recognition and / or target sites may be contained within introns, coding sequences, 5'UTRs, 3'UTRs, and / or regulatory regions.

[0215] Components for the expression and utilization of novel CRISPR-Cas systems in prokaryotic and eukaryotic cells The present invention further provides expression constructs for expressing guide RNA / Cas systems in prokaryotic or eukaryotic cells / organisms that are capable of recognizing, binding to, and optionally nicking, unnicking, or cleaving all or part of a target sequence.

[0216] In one embodiment, an expression construct of the present disclosure comprises a promoter operably linked to a nucleotide sequence encoding a Cas gene (or an optimized plant comprising a Cas endonuclease gene as described herein) and a promoter operably linked to a guide RNA of the present disclosure. The promoter can drive expression of the operably linked nucleotide sequence in a prokaryotic or eukaryotic cell / organism.

[0217] Nucleotide sequence modifications of the guide polynucleotide, VT domain, and / or CER domain may be selected from the group consisting of, but are not limited to, a 5' cap, a 3' polyadenylation tail, a riboswitch sequence, a stable regulatory sequence, a sequence that forms a dsRNA duplex, a modification or sequence that targets the guide polynucleotide to a subcellular location, a modification or sequence that provides tracking, a modification or sequence that provides a binding site for a protein, locked nucleic acid (LNA), 5-methyl dC nucleotides, 2,6-diaminopurine nucleotides, 2'-fluoro A nucleotides, 2'-fluoro U nucleotides; 2'-O-methyl RNA nucleotides, phosphorothioate linkages, linkages to cholesterol molecules, linkages to polyethylene glycol molecules, linkages to spacer 18 molecules, 5' to 3' covalent linkages, or any combination thereof. These modifications may result in at least one additional advantageous characteristic selected from the group of modified or modulated stability, intracellular targeting, tracking, fluorescent labeling, binding sites for proteins or protein complexes, modified binding affinity for complementary target sequences, modified resistance to cellular degradation, and increased cell permeability.

[0218] A method for expressing RNA components, such as gRNAs, in eukaryotic cells for Cas9-mediated DNA targeting is to use an RNA polymerase III (Pol III) promoter, which allows transcription of RNAs with precisely defined, unmodified 5' and 3' ends (DiCarlo et al. 2013, Nucleic Acids Res. 41(7):4336-4343; Ma et al. 2014, Mol. Ther. Nucleic Acids 3:e161). This strategy has been successfully applied to cells of several different species, including maize and soybean (U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015). A method for expressing RNA components without a 5' cap has been described (WO 2016 / 025131, published February 18, 2016).

[0219] Various methods and compositions can be used to obtain cells or organisms with a polynucleotide of interest inserted into a target site for the Cas endonuclease. Such methods can use homologous recombination (HR) to integrate the polynucleotide of interest into the target site. In one method described herein, the polynucleotide of interest is introduced into the cells of the organism via a donor DNA construct.

[0220] The donor DNA construct further comprises first and second regions of homology flanking the polynucleotide of interest, the first and second regions of homology of the donor DNA having homology to first and second genomic regions, respectively, present in or flanking the target site in the genome of the cell or organism.

[0221] Donor DNA can be linked to a guide polynucleotide, which can enable co-localization of target and donor DNA, useful for genome editing, gene insertion, and target genome regulation, and can also be useful for targeting postmitotic cells, which are thought to have significantly reduced function of the endogenous HR machinery (Mali et al. 2013, Nature Methods Vol. 10:957-963).

[0222] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and can be between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-600 bp, 550-750 bp, 600-800 bp, 700-800 bp, 800-900 bp, 900-1000 bp, 1000-1200 bp, 1200-1400 bp, 1400-1600 bp The ranges include lengths and / or regions having integer values within the ranges of 0 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or the full length of the target site. These ranges include all integers within the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bp. The amount of homology may also be described in terms of percent sequence identity over the fully aligned length of two polynucleotides, including percent sequence identities of at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98% to 99%, 99%, 99% to 100%, or 100%. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of consecutive nucleotides or local percent sequence identity; for example, sufficient homology may be described as a 75-150 bp region having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be described by the predictive ability of two polynucleotides to specifically hybridize under high stringency conditions; see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al. Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0223] The structural similarity between a given genomic region and the corresponding homologous region found on the donor DNA can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of homology or sequence identity shared by a "homologous region" of the donor DNA and a "genomic region" of the organism's genome can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, such that the sequences undergo homologous recombination.

[0224] The homologous region on the donor DNA may have homology to any sequence adjacent to the target site. In some cases, the homologous region shares substantial sequence homology with the genomic sequence directly adjacent to the target site, but it is recognized that the homologous region can be designed to have sufficient homology to a region that may be further 5' or 3' to the target site. The homologous region can also have homology to a fragment of the target site in addition to downstream genomic regions.

[0225] In one embodiment, the first homologous region further comprises a first fragment of the target site and the second homologous region comprises a second fragment of the target site, wherein the first fragment and the second fragment are different.

[0226] Polynucleotide of interest Polynucleotides of interest are further described herein, including polynucleotides that reflect commercial markets and commercial market interests related to crop development. Crops and markets of interest will change, and as developing countries expand global markets, additional novel crops and technologies will also emerge. Furthermore, as our understanding of agronomic traits and characteristics such as yield and heterosis increases, gene selection for genetic modification will change accordingly.

[0227] General categories of polynucleotides of interest include, for example, genes of interest involved in signaling such as zinc fingers, genes of interest involved in signal transduction such as kinases, and genes of interest involved in housekeeping such as heat shock proteins. More specific polynucleotides of interest include, but are not limited to, genes involved in agronomic traits including, but not limited to, those affecting crop yield, grain quality, crop nutrient content, starch and carbohydrate quality and quantity, and kernel size, sucrose loading, protein quality and quantity, nitrogen fixation and / or utilization, fatty acid and oil composition; genes encoding proteins that confer resistance to abiotic stress (e.g., those that confer resistance to drought, nitrogen, temperature, salinity, toxic metals or trace elements, or toxins such as pesticides and herbicides); and genes encoding proteins that confer resistance to biotic stress (e.g., attack by fungi, viruses, bacteria, insects, and nematodes, and the development of diseases associated with these organisms).

[0228] Agronomically important traits such as oil, starch, and protein content can be genetically altered in addition to using traditional breeding methods. Alterations include increasing oleic acid, saturated and unsaturated oil content, increasing lysine and sulfur concentrations, providing essential amino acids, and modifying starch. Modifications of the phoridothionin protein are described in U.S. Patent Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0229] The polynucleotide sequence of interest may encode a protein involved in conferring disease resistance or pest resistance. "Disease resistance" or "pest resistance" refers to the avoidance of harmful symptoms in plants resulting from interactions between the plant and a pathogen. Pest resistance genes may encode resistance to pests that inhibit high yields, such as cutworms, armyworms, and the European corn borer. Disease and insect resistance genes, such as lysozyme or cecropin for antibacterial protection; proteins, such as defensins, glucanases, or chitinases for antifungal protection; or Bacillus thuringiensis endotoxin, protease inhibitors, collagenases, lectins, or glucosidases for controlling nematodes or insects, are all useful examples of gene products. Genes encoding disease resistance traits include antidote genes such as those for fumonisins (U.S. Pat. No. 5,792,931), avirulence (avr) genes, and disease resistance (R) genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; and Mindrinos et al. (1994) Cell 78:1089). Insect resistance genes can encode resistance to pests that inhibit high yields, such as cutworms, armyworms, and European corn borers. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48:109).

[0230] Proteins obtained by expression of "herbicide tolerance proteins" or "nucleic acid molecules encoding herbicide tolerance" include proteins that confer the ability of cells to tolerate higher concentrations of herbicide than cells that do not express the protein, or to tolerate a particular concentration of herbicide for a longer period of time than cells that do not express the protein. Herbicide tolerance traits can be introduced into plants by genes encoding tolerance to herbicides that act by inhibiting the activity of acetolactate synthase (ALS, also known as acetohydroxyacid synthase, AHAS), particularly sulfonylurea (UK: sulfonylurea)-type herbicides, genes encoding tolerance to herbicides that act by inhibiting the activity of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), genes encoding tolerance to glyphosate (e.g., the EPSP synthase gene and the GAT gene), genes encoding tolerance to HPPD inhibitors (e.g., the HPPD gene), or other such genes known in the art. See, e.g., U.S. Patent Nos. 7,626,077, 5,310,667, 5,866,775, 6,225,114, 6,248,876, 7,169,970, 6,867,293, and 9,187,762. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene mutant encodes resistance to the herbicide chlorsulfuron.

[0231] Furthermore, the polynucleotide of interest may also contain an antisense sequence complementary to at least a portion of the messenger RNA (mRNA) for the target gene sequence of interest. The antisense nucleotide is constructed to hybridize with the corresponding mRNA. Modifications of the antisense sequence may be made as long as the sequence hybridizes with the corresponding mRNA and disrupts the expression of the corresponding mRNA. In this manner, antisense constructs having 70%, 80%, or 85% sequence identity to the corresponding antisense sequence may be used. Furthermore, portions of the antisense nucleotide may be used to disrupt the expression of the target gene. Generally, sequences of at least 50, 100, 200, or more nucleotides may be used.

[0232] In addition, a polynucleotide of interest can also be used in the sense orientation to suppress the expression of an endogenous gene in a plant. Methods for suppressing gene expression in a plant using a polynucleotide in the sense orientation are known in the art. This method generally involves transforming a plant with a DNA construct containing a promoter that drives expression in the plant, operably linked to at least a portion of a nucleotide sequence corresponding to the transcription of the endogenous gene. Typically, such a nucleotide sequence has considerable sequence identity to the transcribed sequence of the endogenous gene, generally greater than about 65% sequence identity, greater than about 85% sequence identity, or greater than about 95% sequence identity. See U.S. Pat. Nos. 5,283,184 and 5,034,323.

[0233] The polynucleotide of interest can also be a phenotypic marker. Phenotypic markers are screenable or selectable markers, including visual markers and selectable markers, whether positively or negatively selectable. Any phenotypic marker can be used. Specifically, selectable or screenable markers often contain DNA segments that allow for the identification of a molecule or cells containing the molecule, or for the selection of favorable or unfavorable molecules or cells under specific conditions. These markers can encode activities such as, but not limited to, the production of RNA, peptides, or proteins, or can provide binding sites for RNA, peptides, proteins, inorganic and organic compounds or compositions, and the like.

[0234] Examples of selectable markers include, but are not limited to, DNA segments containing restriction enzyme sites; DNA segments encoding products that provide resistance to otherwise toxic compounds, including antibiotics such as spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), and hygromycin phosphotransferase (HPT); DNA segments encoding products that are otherwise deficient in recipient cells (e.g., tRNA genes, auxotrophic markers); DNA segments encoding products that can be 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); generation of novel primer sites for PCR (e.g., juxtaposition of two DNA sequences not previously juxtaposed), inclusion of DNA sequences that are unaffected or have been acted upon by restriction endonucleases or other DNA-modifying enzymes, chemicals, etc., and inclusion of DNA sequences required for specific modifications (e.g., methylation) that allow their identification.

[0235] Additional selectable markers include genes that confer resistance to herbicidal compounds such as sulfonylureas, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). For example, acetolactate synthase (ALS) for resistance to sulfonylureas, imidazolinones, triazolopyrimidine sulfonamides, pyrimidinyl salicylates, and sulfonylaminocarbonyltriazolinones (Shaner and Singh, 1997, Herbicide Activity: Toxicol Biochem Mol Biol 69-110); 5-enolpyruvylshikimate-3-phosphate (EPSPS) for glyphosate resistance (Saroha et al. 1998, J. Plant Biochemistry & Biotechnology Vol 7:65-72);

[0236] Polynucleotides of interest include genes that can be stacked or used in combination with other traits (such as, but not limited to, herbicide tolerance, or any other trait described herein). Polynucleotides and / or traits of interest can be stacked together in complex trait loci, such as those described in U.S. Patent Application Publication No. 2013 / 0263324, published October 3, 2013, and WO 2013 / 112686, published August 1, 2013.

[0237] A polypeptide of interest includes any protein or polypeptide encoded by a polynucleotide of interest described herein.

[0238] Also provided is a method for identifying at least one plant cell containing a polynucleotide of interest integrated into its genome at a target site. Various methods can be used to identify plant cells with insertion at or near the target site in the genome. Such methods may be considered direct analysis of the target sequence to detect alterations in the target sequence, including, but not limited to, PCR, sequencing, nuclease digestion, Southern blotting, and any combination thereof. See, for example, U.S. Patent Application Publication No. 20090133152, published May 21, 2009. The method also includes recovering a plant from the plant cell containing the polynucleotide of interest integrated into its genome. The plant can be sterile or fertile. Any polynucleotide of interest can be provided and confirmed to be capable of being integrated into the target site in the plant's genome and expressed in the plant.

[0239] Optimization of sequences for expression in plants Methods for synthesizing plant-preferred genes are available in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391 and Murray et al. (1989) Nucleic Acids Res. 17:477-498. Additional sequence modifications are known to enhance gene expression in plant hosts. These modifications include, for example, the removal of one or more sequences encoding spurious polyadenylation signals, the removal of one or more exon-intron splice site signals, the removal of one or more transposon-like repeats, and the removal of other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to an average level for a given plant host (calculated with reference to known genes expressed in this host plant cell). Where possible, the sequence is modified to avoid one or more predicted hairpin secondary structures in mRNA. Therefore, the "plant-optimized nucleotide sequence" of the present disclosure includes one or more such sequence modifications.

[0240] Expression elements Any polynucleotide encoding a variant Cas polypeptide or other CRISPR system component disclosed herein can be operably linked to heterologous expression elements to facilitate transcription or regulation in a host cell. Such expression elements include, but are not limited to, promoters, leaders, introns, and terminators. Expression elements can be "minimal," meaning short sequences derived from natural sources that still function as expression regulators or modifiers. Alternatively, expression elements can be "optimized," meaning that the polynucleotide sequence has been altered from its native state to function with more desirable characteristics in a particular host cell (e.g., but not limited to, a bacterial promoter can be "corn-optimized" to improve expression in corn plants). Alternatively, expression elements can be "synthetic," meaning that the expression element is designed in silico and synthesized for use in a host cell. Synthetic expression elements can be entirely synthetic or partially synthetic (including fragments of naturally occurring polynucleotide sequences).

[0241] Certain promoters have been shown to be capable of inducing RNA synthesis at higher rates than others. These are called "strong promoters." Certain other promoters have been shown to induce high levels of RNA synthesis only in specific cell or tissue types; if a promoter preferentially induces RNA synthesis in certain tissues and at lower levels in other tissues, it is often called a "tissue-specific promoter" or "tissue-predominant promoter."

[0242] Plant promoters include promoters that can initiate transcription in plant cells. For a general 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.

[0243] Constitutive promoters include, for example, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-2); rice actin (McElroy et al. (1990) Plant Cell 2:163-71); ubiquitin (Christensen et al. (1989) Plant Mol Biol 12:619-32); and the ALS promoter (U.S. Pat. No. 5,659,026).

[0244] Tissue-preferred promoters can be used to enhance expression in specific plant tissues. Examples of tissue-preferred promoters include those described in International Publication No. 2013 / 103367 published on July 11, 2013, Kawamata et al. (1997) Plant Cell Physiol 38:792-803; Hansen et al. (1997) Mol Gen Genet 254:337-43; Russell et al. (1997) Transgenic Res 6:157-68; Rinehart et al. (1996) Plant Physiol 112:1331-41; Van Camp et al. (1996) Plant Physiol 112:525-35; Canevascini et al. (1996) Plant Physiol 112:513-524; Lam, (1994) Results Probabilistic Cell Differentiation 20:181-96; and Guevara-Garcia et al. (1993) Plant J 4:495-505. Examples of leaf-preferred promoters include those described in 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 4:2723-9; Timko et al. (1988) Nature 318:57-8.Root-preferred promoters include, for example, Hire et al. (1992) Plant Mol Biol 20:207-18 (soybean root-specific glutamine synthase gene); Miao et al. (1991) Plant Cell 3:11-22 (cytosolic glutamine synthase (GS)); Keller and Baumgartner, (1991) Plant Cell 3:1051-61 (root-specific regulatory element in the French bean GRP1.8 gene); Sanger et al. (1990) Plant Mol Biol 14:433-43 (root-specific promoter of A. tumefaciens mannopine synthase (MAS)); Bogusz et al. (1990) Plant Cell 2:633-41 (Parasponia andersonii) andersonii and Trema tomentosa; Leach and Aoyagi, (1991) Plant Sci 79:69-76 (A. rhizogenes rolC and rolD root-inducible genes); Teeri et al. (1989) EMBO J 8:343-50 (Agrobacterium wound-inducible TR1' and TR2' genes); VfENOD-3 gene promoter (Kuster et al. (1995) Plant Mol Biol 29:759-72); and rolB promoter (Capana et al. (1994) Plant Mol Biol 25:681-91); phaseolin gene (Murai et al. (1983) Science 23:476-82; Sengopta-Gopalen et al. (1988) Proc. Natl. Acad. Sci. USA 82:3320-4). See also U.S. Patent Nos. 5,837,876, 5,750,386, 5,633,363, 5,459,252, 5,401,836, 5,110,732, and 5,023,179.

[0245] Seed-preferred promoters include both seed-specific promoters active during seed development and seed germination promoters active during seed germination. See Thompson et al. (1989) BioEssays 10:108. Seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-inducible message); cZ19B1 (maize 19 kDa zein); and milps (myo-inositol-1-phosphate synthase); and those disclosed, for example, in International Publication WO 2000 / 011177 published March 2, 2000, and U.S. Patent No. 6,225,529. Seed-preferred promoters for dicotyledonous plants include, but are not limited to, bean-β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. Seed-preferred promoters for monocotyledonous plants include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa gamma zein, waxy, shrunken 1, shrunken 2, globulin 1, oleosin, and nuc1. See also WO 2000 / 012733, published March 9, 2000, which discloses seed-preferred promoters from the END1 and END2 genes.

[0246] Chemically inducible (regulated) promoters can be used to regulate gene expression in prokaryotic and eukaryotic cells or organisms by the application of exogenous chemical regulators. The promoter can be a chemically inducible promoter, in which application of a chemical induces gene expression, or a chemically repressible promoter, in which application of a chemical represses gene expression. Chemically inducible promoters include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners (De Veylder et al. (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-II-27, WO 1993 / 001294, published January 21, 1993), which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1a promoter (Ono et al. (2004) Biosci Biotechnol Biochem 68:803-7). Other chemically regulated promoters include steroid-responsive promoters (e.g., glucocorticoid-inducible promoters (Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-5; McNellis et al. (1998) Plant J 14:247-257); tetracycline-inducible 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).

[0247] Pathogen-inducible promoters that are induced after infection with a pathogen include, but are not limited to, those that regulate the expression of PR proteins, SA proteins, beta-1,3-glucanase, chitinase, and the like.

[0248] Stress-inducible promoters include the RD29A promoter (Kasuga et al. (1999) Nature Biotechnol. 17:287-91). Those skilled in the art are familiar with protocols for simulating stress conditions such as drought, osmotic stress, salt stress, and temperature stress, as well as protocols for evaluating the stress tolerance of plants subjected to simulated or naturally occurring stress conditions.

[0249] Another example of an inducible promoter useful in plant cells is the ZmCAS1 promoter, described in U.S. Patent Application Publication No. 2013 / 0312137, published November 21, 2013.

[0250] New promoters of various types useful in plant cells are constantly being discovered, and many examples can be found in Okamuro and Goldberg (1989), The Biochemistry of Plants, Vol. 115, Stumpf and Conn, eds. (New York, NY: Academic Press), pp. 1-82.

[0251] Genome modification with novel CRISPR-Cas system components As described herein, the induced Cas endonuclease can recognize and bind to DNA target sequences and introduce single-strand (nicks) or double-strand breaks. When single- or double-strand breaks are introduced into DNA, the cell's DNA repair machinery is activated to repair the break. Error-prone DNA repair mechanisms can create mutations at the double-strand break site. The most common repair mechanism for joining broken ends together is the non-homologous end joining (NHEJ) pathway (Bleuyard et al. (2006) DNA Repair 5:1-12). The structural integrity of chromosomes is typically maintained by repair, but deletions, insertions, or other rearrangements (such as chromosomal translocations) are also possible (Siebert and Puchta, 2002, Plant Cell 14:1121-31; Pacher et al. 2007, Genetics 175:21-9).

[0252] DNA double-strand breaks appear to be effective agents for stimulating the homologous recombination pathway (Puchta et al. (1995) Plant Mol Biol 28:281-92; Tzfira and White, (2005) Trends Biotechnol 23:567-9; Puchta, (2005) J Exp Bot 56:1-14). Using DNA-cleaving agents, a 2- to 9-fold increase in homologous recombination was observed between artificially constructed homologous DNA repeats in plants (Puchta et al. (1995) Plant Mol Biol 28:281-92). Experiments using linear DNA molecules in maize protoplasts demonstrated enhanced homologous recombination between plasmids (Lyznik et al. (1991) Mol Gen Genet 230:209-18).

[0253] Homologous recombination repair (HDR) is a mechanism for repairing double-stranded and single-stranded DNA breaks in cells. Homologous recombination repair includes homologous recombination (HR) and single-stranded annealing (SSA) (Lieber. 2010 Annu. Rev. Biochem. 79:181-211). The most common form of HDR is called homologous recombination (HR), which requires the longest sequence homology between the donor DNA and the acceptor DNA. Other forms of HDR include single-stranded annealing (SSA) and break-induced replication, which require shorter sequence homology than HR. Homologous recombination repair at nicks (single-stranded breaks) may occur via a different mechanism than HDR for double-stranded breaks (Davis and Maizels. PNAS (0027-8424), 111(10), p. E924-E932).

[0254] Altering the genomes of prokaryotic and eukaryotic living cells or organisms, for example, by homologous recombination (HR), is a powerful tool in genetic engineering. Homologous recombination has been demonstrated in plants (Halfter et al. (1992) Mol Gen Genet 231:186-93) and insects (Dray and Gloor, 1997, Genetics 147:689-99). Homologous recombination has also been achieved in other organisms. For example, at least 150-200 bp of homology was required for homologous recombination in the protozoan parasite Leishmania (Papadopoulou and Dumas, (1997) Nucleic Acids Res 25:4278-86). In the filamentous fungus Aspergillus nidulans, gene replacement has been achieved using as little as 50 bp of contiguous homology (Chaveroche et al. (2000) Nucleic Acids Res 28:e97). Targeted gene replacement has also been demonstrated in the ciliate Tetrahymena thermophila (Gaertig et al. (1994) Nucleic Acids Res 22:5391-8). In mammals, homologous recombination has been most successful in mice using pluripotent embryonic stem cell (ES) lines that can be grown in culture, transformed, selected, and introduced into mouse embryos (Watson et al. 1992, Recombinant DNA, 2nd Ed., Scientific American Books (distributed by W.H. Freeman & Co.)).

[0255] gene targeting The guide polynucleotide / Cas system described herein can be used for gene targeting.

[0256] In general, DNA targeting can be achieved by cleaving one or both strands at a specific polynucleotide sequence in a cell with the modified Cas polypeptides disclosed herein in association with a suitable polynucleotide component. Upon induction of a single- or double-strand break in the DNA, the cell's DNA repair machinery can be activated to repair the break by non-homologous end joining (NHEJ) or homology-directed repair (HDR) processes, thereby modifying the target site.

[0257] The length of the DNA sequence of the target site can vary, including, for example, target sites that are at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. The target site can be palindromic, i.e., it is even possible for a sequence on one strand to read the same in the opposite direction on the complementary strand. The cleavage site can be within the target sequence, or the cleavage site can be outside the target sequence. In another variation, cleavage can occur at nucleotide positions directly opposite each other, resulting in a blunt-end cleavage, or in other cases, the incision can be offset, resulting in a single-stranded overhang (which can be a 5' overhang or a 3' overhang), also known as a "sticky end." Active variants of genomic target sites can also be used. Such active variants can comprise at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a given target site, and the active variant retains biological activity and thus can be recognized and cleaved by a Cas endonuclease.

[0258] Assays for measuring single- or double-stranded cleavage of target sites by endonucleases are known in the art and generally measure the overall activity and specificity of the agent towards a DNA substrate containing the recognition site.

[0259] The targeting method herein can be implemented, for example, so that the method targets two or more DNA target sites. Such a method can optionally be characterized as a multiplex method. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sites can be simultaneously targeted. A multiplex method is typically implemented by the targeting method herein, in which multiple different RNA components are provided, each of which is designed to guide the guide polynucleotide / Cas endonuclease complex to a unique DNA target site.

[0260] Gene editing The process of editing a genome sequence by combining a DSB with a modified template generally involves introducing into a host cell a DSB inducer or a nucleic acid encoding the DSB inducer that can recognize a target sequence in a chromosomal sequence and introduce a DSB into the genome sequence, and at least one polynucleotide modified template that contains at least one nucleotide change compared to the nucleotide sequence to be edited. The polynucleotide modified template may further include a nucleotide sequence flanking the at least one nucleotide change. This flanking sequence is substantially homologous to the chromosomal region adjacent to the DSB. Genome editing using DSB inducers (e.g., Cas-gRNA complexes) is described, for example, in U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015; WO 2015 / 026886, published February 26, 2015; WO 2016 / 007347, published January 14, 2016; and WO 2016 / 025131, published February 18, 2016.

[0261] Several uses of the guide RNA / Cas endonuclease system have been described (see, e.g., U.S. Patent Application Publication No. 2015 / 0082478A1, published March 19, 2015; WO 2015 / 026886, published February 26, 2015; and U.S. Patent Application Publication No. 2015 / 0059010, published February 26, 2015), including, but not limited to, altering or substituting a nucleotide sequence of interest (e.g., a regulatory element), inserting a polynucleotide of interest, gene knockout, gene knockin, altering a splice site and / or introducing an alternative splice site, altering a nucleotide sequence encoding a protein of interest, fusion of amino acids and / or proteins, and gene silencing by expressing an inverted repeat in a gene of interest.

[0262] Proteins can be modified in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known. For example, amino acid sequence variants of proteins can be prepared by mutations in DNA. Methods for mutagenesis and nucleotide sequence 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. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance regarding amino acid substitutions unlikely to affect the biological activity of a protein of interest can be found in the model Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, DC). Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be preferred. Conservative deletions, insertions, and amino acid substitutions are not expected to cause radical changes in the properties of the protein, and the effects of substitutions, deletions, insertions, or combinations thereof can be evaluated in routine screening assays. Assays for double-strand break-inducing activity are known and generally measure the overall activity and specificity of an agent on a DNA substrate containing a target site.

[0263] Described herein are genome editing methods using Cas endonucleases and complexes comprising the Cas endonucleases and guide polynucleotides. Following characterization of the guide RNA and PAM sequences, the endonucleases and associated CRISPR RNA (crRNA) components can be utilized to modify chromosomal DNA in other organisms, including plants. To facilitate optimal expression and nuclear localization (in eukaryotic cells), the genes comprising this complex can be optimized as described in International Publication No. WO 2016 / 186953, published November 24, 2016, and then delivered to cells as DNA expression cassettes using methods known in the art. The components necessary to comprise an active complex can also be delivered as RNA, i.e., capped or uncapped mRNA, with or without modifications that protect the RNA from degradation (Zhang, Y. et al. 2016, Nat. Commun. 7:12617), or as a Cas protein-guided polynucleotide complex (WO 2017 / 070032, published April 27, 2017), or any combination thereof. In addition, one or more parts of the complex and the crRNA can be expressed from a DNA construct, while the other components can be delivered as RNA, i.e., capped or uncapped mRNA, with or without modifications that protect the RNA from degradation (Zhang et al. 2016 Nat. Commun. 7:12617), or as a Cas protein-guided polynucleotide complex (WO 2017 / 070032, published April 27, 2017), or any combination thereof. To generate crRNA in vivo, tRNA-derived elements may also be used to recruit endogenous RNAses to cleave the crRNA transcript into a mature form capable of directing the complex to its DNA target site, as described, for example, in WO 2017 / 105991, published June 22, 2017. Nickase complexes may be used individually or in concert to generate single or multiple DNA nicks in one or both DNA strands.Furthermore, the cleavage activity of Cas endonucleases can be inactivated by altering key catalytic residues within their cleavage domains (Sinkunas, T. et al. 2013, EMBO J. 32:385-394), resulting in the generation of RNA-guided helicases that can be used to enhance homology-directed repair, induce transcriptional activation, or remodel local DNA structure. Furthermore, the activities of the Cas cleavage and helicase domains can both be knocked out and used in combination with other DNA cleavage, DNA nicking, DNA binding, transcriptional activation, transcriptional repression, DNA remodeling, DNA deamination, DNA unwinding, DNA recombination enhancement, DNA integration, DNA inversion, and DNA repair agents.

[0264] The direction of transcription of the tracrRNA (if present) and other components of the CRISPR-Cas system (e.g., variable targeting domain, crRNA repeats, loops, anti-repeats) can be deduced as described in WO 2016 / 186946, published November 24, 2016, and WO 2016 / 186953, published November 24, 2016.

[0265] Once appropriate guide RNA requirements are established, as described herein, the PAM preferences for each novel system disclosed herein can be investigated. If the cleavage complex results in degradation of the randomized PAM library, the complex can be converted into a nickase by disabling the ATPase-dependent helicase activity through mutagenesis of key residues or by assembling the reaction in the absence of ATP, as previously described (Sinkunas, T. et al., 2013, EMBO J. 32:385-394). Two regions of PAM randomization separated by two protospacer targets can be used to generate double-stranded DNA breaks, which can be captured and sequenced to determine the PAM sequences that support cleavage by each complex.

[0266] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, comprising introducing into the cell at least one PGEN described herein 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) a replacement of at least one nucleotide; (ii) a deletion of at least one nucleotide; (iii) an insertion of at least one nucleotide; a chemical modification of at least one nucleotide; and (v) any combination of (i)-(iv).

[0267] The edited nucleotide can be located within or outside the target site recognized and cleaved by the Cas endonuclease. In one embodiment, the at least one nucleotide modification is not a modification at the target site recognized and cleaved by the Cas endonuclease. In another embodiment, there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 900, or 1000 nucleotides between the at least one edited nucleotide and the genomic target site.

[0268] Knockouts can occur by indels (insertion or deletion of nucleotide bases in the targeted DNA sequence by NHEJ) or by specific removal of sequences that reduce or completely abolish the function of sequences at or near the target site.

[0269] Guide polynucleotide / Cas endonuclease-induced targeted mutations can occur within nucleotide sequences located within or outside the genomic target site recognized and cleaved by the Cas endonuclease.

[0270] The method of editing a nucleotide sequence in the genome of a cell can be a method that does not use an exogenous selectable marker by restoring function to a non-functional gene product.

[0271] In one embodiment, the present invention describes a method for modifying a target site in the genome of a cell, comprising introducing into the cell at least one PGEN described herein and at least one donor DNA, wherein the donor DNA comprises a polynucleotide of interest; and optionally further comprising identifying at least one cell in which the polynucleotide of interest has been integrated at or near the target site.

[0272] In one aspect, the methods disclosed herein may use homologous recombination (HR) to provide for integration of a polynucleotide of interest at a target site.

[0273] Various methods and compositions can be used to generate cells or organisms having a polynucleotide of interest inserted into a target site through the activity of the CRISPR-Cas system components described herein. In one method described herein, the polynucleotide of interest is introduced into an organism cell via a donor DNA construct. As used herein, "donor DNA" refers to a DNA construct containing the polynucleotide of interest that is inserted into the target site of the Cas endonuclease. The donor DNA construct further comprises first and second homologous regions flanking the polynucleotide of interest. The first and second homologous regions of the donor DNA are homologous to first and second genomic regions, respectively, present in or flanking the target site in the genome of the cell or organism.

[0274] Donor DNA can be linked to a guide polynucleotide, which can enable co-localization of target and donor DNA, useful for genome editing, gene insertion, and target genome regulation, and can also be useful for targeting postmitotic cells, which are thought to have significantly reduced function of the endogenous HR machinery (Mali et al. 2013 Nature Methods Vol. 10:957-963).

[0275] The amount of homology or sequence identity shared by the target and donor polynucleotides can vary and can be between about 1-20 bp, 20-50 bp, 50-100 bp, 75-150 bp, 100-250 bp, 150-300 bp, 200-400 bp, 250-500 bp, 300-600 bp, 350-750 bp, 400-800 bp, 450-900 bp, 500-600 bp, 550-750 bp, 600-800 bp, 700-800 bp, 800-900 bp, 900-1000 bp, 1000-1200 bp, 1200-1400 bp, 1400-1600 bp The ranges include lengths and / or regions having integer values within the ranges of 0 bp, 500-1000 bp, 600-1250 bp, 700-1500 bp, 800-1750 bp, 900-2000 bp, 1-2.5 kb, 1.5-3 kb, 2-4 kb, 2.5-5 kb, 3-6 kb, 3.5-7 kb, 4-8 kb, 5-10 kb, or the full length of the target site. These ranges include all integers within the range, for example, a range of 1-20 bp includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 bp. The amount of homology may also be described in terms of percent sequence identity over the fully aligned length of two polynucleotides, including percent sequence identity of at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sufficient homology includes any combination of polynucleotide length, overall percent sequence identity, and optionally conserved regions of consecutive nucleotides or local percent sequence identity; for example, sufficient homology may be described as a 75-150 bp region having at least 80% sequence identity to a region of the target locus.Sufficient homology can also be described by the predictive ability of two polynucleotides to specifically hybridize under high stringency conditions; see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al. Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0276] Episomal DNA molecules can also be ligated into double-strand breaks, for example, by integrating T-DNA into chromosomal double-strand breaks (Chilton and Que, (2003) Plant Physiol 133:956-65; Salomon and Puchta, (1998) EMBO J 17:6086-95). When sequences near double-strand breaks are altered, for example, by exonuclease activity involved in double-strand break maturation, gene conversion pathways can be restored to their original structure if homologous sequences, such as homologous chromosomes in non-dividing somatic cells or sister chromatids after DNA replication, are available (Molinier et al. (2004) Plant Cell 16:342-52). Ectopic and / or epigenetic DNA sequences can also serve as DNA repair templates for homologous recombination (Puchta, (1999) Genetics 152:1173-81).

[0277] In one embodiment, the present disclosure provides a method for editing a nucleotide sequence in the genome of a cell, comprising introducing into the cell 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 optionally, further comprising selecting at least one cell comprising the edited nucleotide sequence.

[0278] The guide polynucleotide / Cas endonuclease system can be used in combination with at least one polynucleotide modification template to enable editing (modification) of a genomic nucleotide sequence of interest (see also U.S. Patent Application Publication No. 2015 / 0082478, published March 19, 2015, and International Publication No. WO 2015 / 026886, published February 26, 2015).

[0279] Polynucleotides and / or traits of interest can be stacked together at complex trait loci, such as those described in International Publication No. WO 2012 / 129373, published September 27, 2012, and International Publication No. WO 2013 / 112686, published August 1, 2013. The guide polynucleotide / Cas9 endonuclease system described herein provides a system for efficiently generating double-stranded breaks, allowing for the stacking of traits at complex trait loci.

[0280] The guide polynucleotide / Cas system described herein for mediating gene targeting can be used in methods for directing heterologous gene insertion and / or generating complex trait loci containing multiple heterologous genes in a manner similar to that disclosed in International Publication No. WO 2012 / 129373, published September 27, 2012, where the guide polynucleotide / Cas system disclosed herein replaces the use of double-strand break-inducing agents to introduce genes of interest. Independent transgenes can be inserted within 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 2, or 5 centimorgans (cM) of each other to propagate transgenes as a single locus (see, e.g., U.S. Patent Application Publication No. 2013 / 0263324, published October 3, 2013, or WO 2012 / 129373, published March 14, 2013). After plants containing the transgenes are selected, plants containing (at least) one transgene can be crossed to form an F1 containing both transgenes. Of the progeny from these F1s (F2 or BC1), 1 / 500 will have the two different transgenes recombined on the same chromosome. The complex locus can then be propagated with both transgenes as a single locus. This process can be repeated to accumulate any number of desired traits.

[0281] Further uses of guide RNA / Cas endonuclease systems have been described (e.g., U.S. Patent Application Publication No. 2015 / 0082478 published March 19, 2015; WO 2015 / 026886 published February 26, 2015; U.S. Patent Application Publication No. 2015 / 0059010 published February 26, 2015; WO 2016 / 007347 published January 14, 2016; and PCT application WO 2016 / 007347 published February 18, 2016). 025131), which include, but are not limited to, altering or substituting a nucleotide sequence of interest (e.g., a regulatory element), inserting a polynucleotide of interest, gene knockout, gene knockin, altering a splice site and / or introducing an alternative splice site, altering a nucleotide sequence encoding a protein of interest, amino acid and / or protein fusions, and gene silencing by expressing an inverted repeat in a gene of interest.

[0282] The characteristics obtained from the gene editing compositions and methods described herein can be evaluated. Chromosomal intervals correlated with the desired phenotype or trait can be identified. Various methods well known in the art can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers linked to genes that control the desired trait. In other words, chromosomal intervals are determined so that any marker present within the interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for a specific trait. In one embodiment, a chromosomal interval contains at least one QTL, and may actually contain multiple QTLs. When multiple QTLs are located very close to each other within the same interval, one marker may be linked to more than one QTL, making the association of a specific marker with a specific QTL unclear. Conversely, for example, when two closely spaced markers cosegregate with a desired phenotypic trait, it is often unclear whether these markers identify the same QTL or two different QTLs. The term "quantitative trait locus" or "QTL" refers to a region of DNA associated with differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., at least one breeding population. A QTL region encompasses or is closely linked to a gene that influences the trait in question. An "allele of a QTL" can include multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can refer to a haplotype within a specific window, which is a contiguous genomic region that can be defined and tracked by a set of one or more polymorphic markers. A haplotype can be defined by the unique fingerprint of the allele at each marker within a specific window.

[0283] Introduction of CRISPR-Cas system components into cells The methods and compositions described herein do not depend on a particular method for introducing a sequence into an organism or cell, but merely on the introduction of a polynucleotide or polypeptide into at least one cell of the organism. Introduction includes reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell, where the nucleic acid may be incorporated into the genome of the cell, and includes reference to the transient (direct) provision of a nucleic acid, protein, or polynucleotide-protein complex (PGEN, RGEN) to a cell. Methods for introducing polynucleotides or polypeptides or polynucleotide-protein complexes into cells or organisms are known in the art and include, but are not limited to, microinjection, electroporation, stable transformation, transient transformation, ballistic particle acceleration (particle bombardment), whisker-mediated transformation, Agrobacterium-mediated transformation, direct gene transfer, virus-mediated transfer, transfection, transduction, cell-penetrating peptides, mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, topical application, sexual crossing, sexual propagation, and any combination thereof.

[0284] For example, guide polynucleotides (guide RNA, cr nucleotides + tracr nucleotides, guide DNA and / or guide RNA-DNA molecules) can be directly (transiently) introduced into cells as single-stranded or double-stranded polynucleotide molecules. Guide RNA (or crRNA + tracrRNA) can also be indirectly introduced into cells by introducing a recombinant DNA molecule comprising a heterologous nucleic acid fragment encoding guide RNA (or crRNA + tracrRNA) operably linked to a specific promoter capable of transcribing guide RNA (crRNA + tracrRNA molecule) in said cells. A specific promoter can be, but is not limited to, an RNA polymerase III promoter, which allows transcription of RNA with strictly defined, unmodified 5' and 3' ends (Ma et al. 2014, Mol. Ther. Nucleic Acids 3:e161; DiCarlo et al. 2013, Nucleic Acids Res. 41:4336-4343; WO 2015 / 026887, published February 26, 2015). Any promoter capable of transcribing the guide RNA in a cell can be used, including heat shock / heat-inducible promoters operably linked to a nucleotide sequence encoding the guide RNA.

[0285] Plant cells differ from animal cells (eg, human cells), fungal cells (eg, yeast cells), and protoplasts, for example, in that plant cells contain a plant cell wall that can act as a barrier to the delivery of components.

[0286] Delivery of the Cas endonuclease, and / or guide RNA, and / or ribonucleoprotein complex, and / or polynucleotide encoding any one or more of the foregoing into plant cells may be achieved by methods known in the art, including, but not limited to, Rhizobiales-mediated transformation (e.g., Agrobacterium, Ochrobactrum), particle-mediated delivery (particle bombardment), polyethylene glycol (PEG)-mediated transfection (e.g., into protoplasts), electroporation, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery.

[0287] The Cas endonucleases described herein can be introduced into cells by directly introducing the Cas polypeptide itself (referred to as direct Cas endonuclease delivery), mRNA encoding the Cas protein, and / or the guide polynucleotide / Cas endonuclease complex itself using any method known in the art. The Cas endonuclease can also be introduced into cells indirectly by introducing a recombinant DNA molecule encoding the Cas endonuclease. The endonuclease can be transiently introduced into cells or integrated into the genome of the host cell using any method known in the art. Cellular uptake of the endonuclease and / or guide polynucleotide can be facilitated using a cell-penetrating peptide (CPP), such as those described in International Publication WO 2016 / 073433, published May 12, 2016. Any promoter capable of expressing the Cas endonuclease in cells can be used, including heat-shock / heat-inducible promoters operably linked to a nucleotide sequence encoding the Cas endonuclease.

[0288] 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 transfection into protoplasts, whisker-mediated transformation, electroporation, particle bombardment, cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct protein delivery, can be successfully used for delivery of polynucleotide-modified templates in eukaryotic cells, such as plant cells.

[0289] Donor DNA can be introduced by any means known in the art. Donor DNA can be provided by any transformation method known in the art, including, for example, Agrobacterium-mediated transformation or biolistic particle bombardment. Donor DNA can be transiently present in the cell, or it can be introduced by a viral replicon. In the presence of the Cas endonuclease and the target site, the donor DNA is inserted into the transformed plant genome.

[0290] Direct delivery of any one of the inducible Cas system components can be accompanied by direct delivery (co-delivery) of other mRNAs that can facilitate enrichment and / or visualization of cells that receive the guide polynucleotide / Cas endonuclease complex components. For example, direct co-delivery of the guide polynucleotide / Cas endonuclease components (and / or the guide polynucleotide / Cas endonuclease complex itself) with mRNAs encoding phenotypic markers (such as, but not limited to, transcriptional activators such as CRC (Bruce et al. 2000 The Plant Cell 12:65-79)) can restore function to a non-functional gene product, thereby enabling selection and enrichment of cells without the use of an exogenous selection marker, as described in International Publication WO 2017 / 070032, published April 27, 2017.

[0291] Introduction of the guide RNA / Cas endonuclease complex (representing the cleavage-ready complex described herein) into a cell includes introducing the individual components of the complex into the cell separately or together, as well as directly (direct delivery of the guide RNA and the Cas endonuclease protein and protein subunit, or functional fragments thereof) or via a recombinant construct expressing the components (guide RNA, Cas endonuclease, protein subunit, or functional fragments thereof). Introduction of the guide RNA / Cas endonuclease complex (RGEN) into a cell includes introducing the guide RNA / Cas endonuclease complex into the cell as a ribonucleotide-protein. The ribonucleotide-protein may be assembled before introduction into the cell, as described herein. The components that make up the guide RNA / Cas endonuclease ribonucleotide protein (at least one Cas endonuclease, at least one guide RNA, at least one protein subunit) can be assembled by any means known in the art, either in vitro or before being introduced into a cell (which will be targeted for genome modification as described herein).

[0292] Direct delivery of RGEN ribonucleoprotein allows genome editing at the target site of the cell genome, with the complex then rapidly degrading, making its presence in the cell transient. The transient presence of this RGEN complex can lead to reduced off-target effects. In contrast, delivery of RGEN components (guide RNA, Cas9 endonuclease) via plasmid DNA sequences results in sustained expression of RGEN from these plasmids, which can increase off-target effects (Cradick, TJ et al. (2013) Nucleic Acids Res 41:9584-9592; Fu, Y et al. (2014) Nat. Biotechnol. 31:822-826).

[0293] Direct delivery can be achieved by combining any one of the components of the guide RNA / Cas endonuclease complex (RGEN) (representing the cleavage-ready complex described herein) (e.g., at least one guide RNA, at least one Cas protein, and optionally one additional protein) with a delivery matrix comprising microparticles (e.g., but not limited to, gold particles, tungsten particles, and silicon carbide whisker particles) (see also International Publication No. 2017 / 070032, published April 27, 2017). The delivery matrix can include any one of the components, such as the Cas endonuclease, attached to a solid matrix (e.g., a bombardment particle).

[0294] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein that form the guide RNA / Cas endonuclease complex are introduced into a cell as RNA and protein, respectively.

[0295] In one aspect, a guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and Cas endonuclease protein that form the guide RNA / Cas endonuclease complex and at least one protein subunit of the complex are introduced into a cell as RNA and protein, respectively.

[0296] In one aspect, the guide polynucleotide / Cas endonuclease complex is a complex in which the guide RNA and the Cas endonuclease protein that form the guide RNA and Cas endonuclease complex (cleavage-ready complex) and at least one protein subunit of the complex are pre-assembled in vitro and introduced into a cell as a ribonucleotide-protein complex.

[0297] Protocols for introducing polynucleotides, polypeptides, or polynucleotide-protein complexes (PGENs, RGENs) into eukaryotic cells, such as plants or plant cells, are known and include microinjection (Crossway et al. (1986) Biotechniques 4:320-34 and U.S. Pat. No. 6,300,543), meristem transformation (U.S. Pat. No. 5,736,369), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-6, Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), whisker-mediated transformation (Ainley et al. 2013, Plant Biotechnology Journal 11:1126-1134; Shaheen A. and M. Arshad 2011 Properties and Applications of Silicon Carbide (2011), 345-358 Editor(s): Gerhardt, Rosario. Publisher: InTech, Rijeka, Croatia. CODEN: 69PQBP; ISBN: 978-953-307-201-2), direct gene transfer (Paszkowski et al. (1984) EMBO J 3:2717-22), and ballistic particle acceleration (U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment" in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg 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) Bio / Technology 87:671-4 (soybean), Finer and McMullen (1991) In Vitro Cell Dev. Biol 27P:175-82 (soybean); Singh et al. (1998) Theor Appl Genet 96:319-24 (soybean); Datta et al. (1990) Biotechnology 8:736-40 (rice), Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-9 (corn), Klein et al. (1988) Biotechnology 6:559-63 (corn); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Cell 91:440-4 (corn); Fromm et al. (1990) Biotechnology 8:833-9 (corn); Hooykaas-Van Slogteren et al. (1984) Nature 311:763-4; U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-9 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Rep 9:415-8, and Kaeppler et al. (1992) Theor Appl Genet 84:560-6 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-505 (electroporation); Li et al.(1993) Plant Cell Rep 12:250-5; Christou and Ford (1995) Annals Botany 75:407-13 (rice) and Osjoda et al. (1996) Nat Biotechnol 14:745-50 (maize with Agrobacterium tumefaciens).

[0298] Alternatively, polynucleotides can be introduced into plants or plant cells by contacting the cells or organisms with a virus or viral nucleic acid. Generally, such methods involve incorporating the polynucleotide into a viral DNA or RNA molecule. In some embodiments, a polypeptide of interest may first be synthesized as part of a viral polyprotein, which may then be proteolytically processed in vivo or in vitro to produce the desired recombinant protein. Methods for introducing polynucleotides, including viral DNA or RNA molecules, into plants and expressing the encoded proteins therein are known; see, e.g., U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931.

[0299] For Zea mays, 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 protoplast-based approaches (Sardesai and Subramanyam (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. 12, 1037-1040; Rhodes et al. (1988) Science. 240, 204-207; and Golovkin et al. (1993) Plant. In this example, Cas-alpha 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 as a single transfer DNA fragment into maize immature embryos using Agrobacterium as previously described (Lowet et al. (2018) In vitro Cellular & Developmental Biology-Plant. 54, 240-252).

[0300] Several methods can be used for transformation of Saccharomyces cerevisiae, including lithium acetate, polyethylene glycol (PEG), heat shock, electroporation, and biolistic transformation (Kawai et al. (2010) Bioengineered Bugs. 1:395-403).

[0301] Polynucleotides or recombinant DNA constructs can be provided to or introduced into prokaryotic and eukaryotic cells or organisms using a variety of transient transformation methods, including, but not limited to, direct introduction of polynucleotide constructs into cells.

[0302] Nucleic acids and proteins can be provided to cells in any manner, including using molecules (e.g., cell-penetrating peptides and nanocarriers) that facilitate uptake of any or all components (proteins and / or nucleic acids) of the inducible Cas system. See also U.S. Patent Application Publication No. 2011 / 0035836, published February 10, 2011, and European Patent Application Publication No. 2821486A1, published January 7, 2015.

[0303] Other methods for introducing polynucleotides into prokaryotic and eukaryotic cells or organisms or plant parts may be used, such as plastid transformation methods and methods for introducing polynucleotides into tissues from seedlings or mature seeds.

[0304] Stable transformation is intended to mean that a nucleotide construct introduced into an organism is integrated into the genome of the organism and can be inherited by its progeny. Transient transformation is intended to mean that a polynucleotide is introduced into an organism but is not integrated into the genome of this organism, or that a polypeptide is introduced into an organism. Transient transformation indicates that the introduced composition is only expressed or present temporarily in the organism.

[0305] A variety of methods may be utilized to identify cells with altered genomes at or near a target site without the use of a screening marker phenotype, which may be considered to be processes that directly analyze a target sequence to detect any alterations within the target sequence, including, but not limited to, PCR, sequencing, nuclease digestion, Southern blotting, and any combination thereof.

[0306] Cells and plants The polynucleotides and polypeptides of the present disclosure can be introduced into cells, including, but not limited to, human, non-human, animal, mammalian, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells, as well as plants and seeds produced by the methods described herein. Any plant, including monocotyledonous and dicotyledonous plants and plant elements, can be used with the compositions and methods described herein.

[0307] Examples of monocotyledonous plants that can be used include, but are not limited to, maize (Zea mays), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), barnyard millet (e.g., pearl millet, Pennisetum glaucum), common millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana), wheat (Triticum species, e.g., Triticum aestivum), and the like. aestivum, Triticum monococcum), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), switchgrass (Panicum virgatum), pineapple (Ananas comosus), banana (Musa spp.), palms, ornamentals, turfgrass, and other grasses.

[0308] Examples of dicotyledonous plants that can be used include, but are not limited to, soybean (Glycine max), Brassica species (e.g., but not limited to, rapeseed or canola) (Brassica napus, B. campestris, Brassica rapa, Brassica juncea), alfalfa (Medicago sativa), tobacco (Nicotiana tabacum), Arabidopsis (Arabidopsis thaliana), sunflower (Helianthus annuus), cotton (Gossypium arboreum), and the like. arboreum, Gossypium barbadense), and peanut (Arachis hypogaea), tomato (Solanum lycopersicum), and potato (Solanum tuberosum).

[0309] Additional plants that can be used include safflower (Carthamus tinctorius), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea plants (Camellia sinensis), bananas (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), and others. guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), vegetables, ornamental plants and conifers.

[0310] Vegetables that can be used include tomato (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis, such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and muskmelon (C. melo). Ornamental plants include azaleas (Rhododendron spp.), hydrangeas (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnations (Dianthus caryophyllus), poinsettias (Euphorbia pulcherrima), and chrysanthemums.

[0311] Conifers that can be used include pines, such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); Redwood (Sequoia sempervirens); and Norway spruce (Abies amabilis). fir trees, such as Japanese cedar (Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis); and cedar trees, such as Japanese red cedar (Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis).

[0312] In certain embodiments of the present disclosure, a fertile plant is one that produces viable male and female gametes and is a self-fertile plant. Such a self-fertile plant can produce progeny plants without the contribution of the gametes and genetic material contained therein of another plant. Other embodiments of the present disclosure may involve the use of plants that are not self-fertile, because the plant does not produce viable or otherwise fertilizable male gametes or female gametes, or both.

[0313] The present disclosure finds use in breeding plants that contain one or more introduced traits or edited genomes.

[0314] A non-limiting example of how two traits can be stacked in the genome, for example, at a genetic distance of 5 cM from each other, is described as follows: A first plant containing a first transgenic target site integrated at a first DSB target site within a genomic window and not having a first genomic locus of interest is crossed to a second transgenic plant containing a genomic locus of interest at a different genomic insertion site within the genomic window, the second plant not containing the first transgenic target site. Approximately 5% of the plant progeny from this cross will have both the first transgenic target site integrated at the first DSB target site and the first genomic locus of interest integrated at a different genomic insertion site within the genomic window. Progeny plants having both sites within the defined genomic window can be further crossed with a third transgenic plant that contains a second transgenic target site integrated at the second DSB target site and / or a second genomic locus of interest within the defined genomic window and lacks the first transgenic target site and the first genomic locus of interest, after which progeny having the first transgenic target site, the first genomic locus of interest, and the second genomic locus of interest integrated at a different genomic insertion site within the genomic window are selected. Such methods can be used to create transgenic plants containing complex trait loci with 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 at the DSB target site and / or genomic loci of interest integrated at different sites within the genomic window. In this manner, a variety of complex trait loci can be generated.

[0315] Cells and animals The polynucleotides and polypeptides of the present disclosure may be introduced into animal cells. Animal cells include, but are not limited to, organisms from the phylum Chordata, Arthropods, Molluscs, Annelids, Cnidarians, or Echinoderms; or from the class Mammals, Insects, Birds, Amphibians, Reptiles, or Fish. In some embodiments, the animal is a human, mouse, C. elegans, rat, fruit fly (Drosophila species), zebrafish, chicken, dog, cat, guinea pig, hamster, chicken, rooster, Japanese killifish, sea lamprey, pufferfish, tree frog (e.g., Xenopus species), 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, bone cells, germ 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 may be used.

[0316] The disclosed novel modified Cas polypeptides can be used to edit the genome of an animal cell in a variety of ways. In one aspect, it may be desirable to delete one or more nucleotides. In another aspect, it may be desirable to insert one or more nucleotides. In one aspect, it may be desirable to substitute one or more nucleotides. In another aspect, it may be desirable to modify one or more nucleotides by covalent or non-covalent interaction with another atom or molecule.

[0317] Genome modification with the disclosed engineered Cas polypeptides can be used to effect genotypic and / or phenotypic changes in target organisms. Such changes are preferably related to the improvement of a desired phenotype or physiologically significant characteristic, the correction of an endogenous defect, or the expression of some type of expression marker. In some embodiments, the desired phenotype or physiologically significant characteristic is related to the overall health, fitness, or reproductive potential of the animal, the animal's ecological fitness, or the relationship or interaction of this animal with other organisms in its environment. In some embodiments, the phenotype or physiologically significant trait of interest is selected from the group consisting of: improvement in overall health, reversal of disease, amelioration of disease, stabilization of disease, prevention of disease, treatment of parasitic infection, treatment of viral infection, treatment of retroviral infection, treatment of bacterial infection, treatment of neurological disorders (e.g., but not limited to, multiple sclerosis), correction of an intrinsic genetic defect (e.g., but not limited to, metabolic disorders, achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, Barth syndrome, breast cancer, Charcot-Marie-Tooth disease, colon cancer, cricket syndrome, Crohn's disease, cystic fibrosis, fibrosis), Dercum's disease, Down's syndrome, Duane's syndrome, Duchenne muscular dystrophy, Factor V Leiden thrombophilia, familial hypercholesterolemia, familial Mediterranean fever, fragile X syndrome, Gaucher's disease, hemochromatosis, hemophilia, holoprosencephaly, Huntington's disease, Klinefelter's syndrome, Marfan's syndrome, myotonic dystrophy, neurofibromatosis, Noonan's syndrome, osteogenesis imperfecta, Parkinson's disease, phenylketonuria, Poland's syndrome, porphyria, progeria, prostate cancer, retinitis pigmentosa and treatment of diseases including rare or "orphan" conditions for which there are no other effective treatment options (e.g., encephalopathy, severe combined immunodeficiency (SCID), sickle cell disease, skin cancer, spinal muscular atrophy, Tay-Sachs disease, thalassemia, trimethylaminuria, Turner syndrome, palatocardiofacial syndrome, WAGR syndrome, and Wilson's disease), treatment of congenital immune disorders (e.g., but not limited to, immunoglobulin subclass deficiencies), treatment of acquired immune disorders (e.g., but not limited to, AIDS and other HIV-related disorders), treatment of cancer, and treatment of diseases including rare or "orphan" conditions for which there are no other effective treatment options.

[0318] Cells genetically engineered using the compositions or methods disclosed herein can be transplanted into a subject for purposes such as gene therapy, for example to treat disease or as antiviral, antipathogen or anticancer therapeutics, for the production of genetically engineered organisms in agriculture, or for biological research.

[0319] In vitro detection, binding, and modification of polynucleotides In some embodiments, the compositions disclosed herein may further be used as compositions for in vitro methods involving isolated polynucleotide sequences. The isolated polynucleotide sequences may contain one or more target sequences for modification. In some embodiments, the isolated polynucleotide sequences may be genomic DNA, PCR products, or synthesized oligonucleotides.

[0320] composition The modification of the target sequence can be in the form of nucleotide insertion, nucleotide deletion, nucleotide substitution, addition of an atomic molecule to an existing nucleotide, nucleotide modification, or attachment of a heterologous polynucleotide or polypeptide to the target sequence. The insertion of one or more nucleotides can be achieved by including a donor polynucleotide in the reaction mixture, which is inserted into the double-stranded break generated by the modified Cas endonuclease disclosed herein. This insertion can be performed by non-homologous end joining or homologous recombination.

[0321] In one embodiment, the sequence of the target polynucleotide is known prior to modification and is compared to the sequence of the polynucleotide resulting from treatment with the modified Cas endonuclease. In one embodiment, the sequence of the target polynucleotide is unknown prior to modification and treatment with the modified Cas endonuclease is used as part of a method for determining the sequence of said target polynucleotide.

[0322] Polynucleotide modification with modified Cas polypeptides can be achieved by using full-length polypeptides identified from the Cas locus, or can be achieved from fragments, modifications, or variants of polypeptides identified from the Cas locus. In some aspects, the Cas polypeptide variant is a polypeptide sharing at least 80% identity with any of SEQ ID NOs: 15-47. In some aspects, the Cas polypeptide variant is a functional variant of any of SEQ ID NOs: 15-47. In some aspects, the Cas polypeptide variant is a functional fragment of any of SEQ ID NOs: 15-47. In some aspects, the Cas polypeptide is a Cas endonuclease polypeptide that recognizes the PAM sequence N(T>W>C)TTC. In some aspects, the Cas polypeptide variant is provided by the Cas polypeptide polynucleotide.

[0323] In some aspects, the modified Cas polypeptide may be selected from the group consisting of an engineered or modified wild-type Cas endonuclease ortholog, a functional Cas endonuclease ortholog variant, a functional modified Cas polypeptide fragment, a fusion protein comprising an active or inactivated modified Cas polypeptide variant, a modified Cas polypeptide further comprising one or more nuclear localization sequences (NLS) at the C-terminus or N-terminus or both the N-terminus and C-terminus, a biotinylated modified Cas polypeptide, a modified Cas endonuclease nickase, a modified Cas polypeptide orthologous endonuclease, a modified Cas polypeptide further comprising a histidine tag, and a mixture of any two or more thereof.

[0324] 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 the cell from which the target polynucleotide sequence is obtained or derived.

[0325] In some aspects, multiple engineered Cas polypeptides may be desirable. In some aspects, the multiple engineered Cas polypeptides may include engineered Cas polypeptides from different biological sources or from different loci within the same organism. In some aspects, the multiple engineered Cas polypeptides may include engineered Cas polypeptides with different binding specificities for a target polynucleotide. In some aspects, the multiple engineered Cas polypeptides may include engineered Cas endonucleases with different cleavage efficiencies. In some aspects, the multiple engineered Cas polypeptides may include engineered Cas polypeptides with different PAM specificities. In some aspects, the multiple engineered Cas polypeptides may include engineered Cas polypeptides that differ in molecular composition (i.e., polynucleotides encoding the engineered Cas polypeptides and polypeptides that are engineered Cas polypeptides).

[0326] The modified Cas polypeptides disclosed herein can be used in conjunction 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.

[0327] Storage conditions for modified Cas polypeptides and / or guide polynucleotides include temperature, state of matter, and time parameters. In some embodiments, the Cas polypeptides and / or guide polynucleotides are stored at about -80°C, about -20°C, about 4°C, about 20-25°C, or about 37°C. In some embodiments, the Cas polypeptides and / or guide polynucleotides are stored as a liquid, frozen liquid, or lyophilized powder. In some embodiments, the Cas polypeptides and / or guide polynucleotides are stable for at least one day, at least one week, at least one month, at least one year, or more than one year.

[0328] Any or all of the possible polynucleotide components of a reaction (e.g., guide polynucleotide, donor polynucleotide, polynucleotide encoding optional Cas polypeptide) can be provided as part of a vector, construct, linear or circular plasmid, or as part of a chimeric molecule. Each component can be provided separately or together in the reaction mixture. In some aspects, one or more polynucleotide components are operably linked to heterologous non-coding regulatory elements that regulate their expression.

[0329] Methods for modification of a target polynucleotide involve combining the minimal elements into a reaction mixture comprising an engineered Cas polypeptide (or a variant, fragment, or other related molecule as described above), a guide polynucleotide comprising a sequence that is substantially complementary to or selectively hybridizes to a target polynucleotide sequence of the 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.

[0330] The storage buffer or reaction mixture of any one of the components may be optimized for stability, efficacy, or other parameters. Additional components of the storage buffer or reaction mixture may include a 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, proteinase, and / or viscosity agent. In some embodiments, the storage buffer or reaction mixture further comprises a buffer comprising at least one of the following components: HEPES, MgCl, NaCl, EDTA, proteinase, proteinase K, glycerol, nuclease-free water.

[0331] Incubation conditions vary according to the desired results. The temperature is preferably at least 10°C, 10-15°C, at least 15°C, 15-17°C, at least 17°C, 17-20°C, at least 20°C, 20-22°C, at least 22°C, 22-25°C, at least 25°C, 25-27°C, at least 27°C, 27-30°C, at least 30°C, 30-32°C, at least 32°C, 32-35°C, at least 35°C, at least 36°C, at least 37°C, at least 38°C, at least 39°C, at least 40°C, or greater than 40°C. The incubation time is at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, or greater than 10 minutes.

[0332] The sequence of the polynucleotide in the reaction mixture before, during, or after incubation may be determined by any method known in the art. In one aspect, modification of the target polynucleotide may be confirmed by comparing the sequence of a polynucleotide purified from the reaction mixture with the sequence of the target polynucleotide prior to combination with a modified Cas polypeptide disclosed herein.

[0333] Any one or more of the compositions disclosed herein useful for detecting, binding, and / or modifying polynucleotides in vitro or in vivo can be included in a kit. The kit comprises a modified Cas polypeptide (e.g., a modified Cas endonuclease) disclosed herein, or a polynucleotide encoding the modified Cas polypeptide or endonuclease, and optionally further comprises buffer components that allow for efficient storage and one or more additional compositions that allow for the introduction of the modified Cas polypeptide into a cell or system containing a heterologous polynucleotide, where the variable Cas polypeptide can result in the modification, addition, deletion, or substitution of at least one nucleotide of the heterologous polynucleotide. In a further aspect, the modified Cas polypeptides disclosed herein can be used for enrichment of one or more polynucleotide target sequences from a mixed pool. In a further aspect, the modified Cas polypeptides disclosed herein can be immobilized on a matrix for use in detecting, binding, and / or modifying target polynucleotides in vitro.

[0334] The engineered Cas endonuclease may be attached, associated with, or attached to a solid matrix for purposes of storage, purification, and / or characterization. Examples of solid matrices include, but are not limited to, filters, chromatography resins, assay plates, test tubes, cryogenic vials, etc. The Cas endonuclease may be substantially purified and stored in an appropriate buffer solution or lyophilized.

[0335] Detection Method Methods for detecting the engineered Cas polypeptide:guide polynucleotide complex bound to the target polynucleotide can 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.

[0336] The DNA electrophoretic mobility shift assay (EMSA) examines protein binding to a known DNA oligonucleotide probe to assess the specificity of the interaction. This technique is based on the principle that protein-DNA complexes migrate more slowly than free DNA molecules when subjected to electrophoresis in polyacrylamide or agarose gels. Because DNA migration slows upon binding to a protein, this assay is also called a gel retardation assay. Addition of a protein-specific antibody to the binding component generates a larger complex (antibody-protein-DNA) that migrates even more slowly during electrophoresis, a phenomenon known as a supershift, which can be used to confirm the identity of the protein.

[0337] DNA pull-down assays use DNA probes labeled with a high-affinity tag, such as biotin, that allows for recovery or immobilization of the probe. The DNA probe can be complexed with proteins from cell lysates in a reaction similar to that used in EMSA, which can then be used to purify the complex using agarose or magnetic beads. The protein is then eluted from the DNA and detected by Western blot or identified by mass spectrometry. Alternatively, the protein can be labeled with an affinity tag, or the DNA-protein complex can be isolated using an antibody against the protein of interest (similar to a supershift assay). In this case, the unknown DNA sequence bound to the protein is detected by Southern blot or PCR analysis.

[0338] Reporter assays provide an in vivo real-time readout of the translational activity of a promoter of interest. A reporter gene is a fusion of a target promoter DNA sequence with a reporter gene DNA sequence customized by the researcher. The DNA sequence encodes a protein with a detectable property, such as firefly / renilla luciferase or alkaline phosphatase. These genes produce an enzyme only when the promoter of interest is activated. The enzyme then catalyzes a substrate, resulting in a light or color change that can be detected by spectroscopy. The signal from the reporter gene is used as an indirect determinant of the translation of an endogenous protein driven from the same promoter.

[0339] Microplate capture and detection assays use immobilized DNA probes to capture specific protein-DNA interactions and confirm protein identity and relative abundance with target-specific antibodies. Typically, DNA probes are immobilized on the surface of a streptavidin-coated 96- or 384-well microplate. Cell extracts are prepared and added to allow binding proteins to bind to the oligonucleotides. The extract is then removed, and each well is washed several times to remove nonspecifically bound proteins. Finally, proteins are detected using a labeled specific antibody. This method can be extremely sensitive, detecting less than 0.2 pg of target protein per well. This method can also be utilized with oligonucleotides labeled with other tags, such as primary amines, which can be immobilized on microplates coated with amine-reactive surface chemistry.

[0340] DNA footprinting is one of the most widely used methods for obtaining detailed information about individual nucleotides in protein-DNA complexes, even in 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 site from modification or digestion. This can then be visualized by denaturing gel electrophoresis, in which unprotected DNA is cleaved more or less randomly. Therefore, it appears as a "ladder" of bands; sites protected by the protein have no corresponding bands and appear as footprints in the band pattern. This footprint therefore identifies the specific nucleoside at the protein-DNA binding site.

[0341] Microscopy techniques include optical microscopy, fluorescence microscopy, electron microscopy, and atomic force microscopy (AFM).

[0342] Chromatin immunoprecipitation analysis (ChIP) allows proteins to be covalently bound to this DNA target, after which they can be unbound and characterized separately.

[0343] The SELEX method exposes a target protein to a random library of oligonucleotides. Those that bind are isolated and amplified by PCR.

[0344] While the present invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the relevant art will understand that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. For example, while the specific examples below may illustrate the methods and embodiments described herein using particular target sites or target organisms, the principles in these examples may be applied to any target site or target organism. Accordingly, it will be understood that the scope of the present invention encompasses the embodiments of the present invention listed herein, rather than the specific examples exemplified below. All cited patents, applications, and publications referred to in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each were individually and specifically incorporated by reference, except for any definitions, subject matter disclaimers, or disclaimers, and except to the extent that the incorporated material contradicts an explicit disclosure herein, in which case the language of the present disclosure shall control. [Example]

[0345] Below are examples of specific embodiments of some aspects of the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0346] Example 1: Cas-alpha nuclease and guide RNA expression cassettes This example describes methods to generate Cas-alpha endonuclease and guide RNA expression cassettes for use in Saccharomyces cerevisiae, Zea mays, and Homo sapiens cells.

[0347] In S. cerevisiae, the gene encoding the Cas-alpha endonuclease was yeast codon-optimized. To facilitate nuclear localization of this optimized Cas-alpha endonuclease protein, a nucleotide sequence encoding the Simian virus 40 (SV40) monopartite nuclear localization signal (NLS) (PKKKRKV (SEQ ID NO: 13)) was added to the 5' and / or 3' end. The nucleotide sequences of the optimized Cas-alpha endonuclease gene and NLS variants were then synthesized and operably cloned into low-copy yeast plasmid DNA (GenScript) containing a CEN6 origin of replication (ORI) between the ROX3 promoter and CYC1 terminator. An example of a yeast-optimized Cas-alpha nuclease expression cassette, and references to the sequences described herein, can be found in Figure 1.

[0348] Cas-alpha endonucleases are guided by small RNAs (referred to herein as guide RNAs) to cleave double-stranded DNA in the presence of a 5' protospacer adjacent motif (PAM) (Bigelyte et al. (2021), Nature Communications. 12:6191; Karvelis et al. (2020), Nucleic Acids Research. 48:5016-5023; U.S. Patent Application Publication No. US2020 / 0190494A1; and WO2022 / 082179). These guide RNAs contain a sequence that assists recognition by Cas-alpha (referred to as the Cas-alpha recognition domain) and a sequence that serves to direct Cas-alpha cleavage by base-pairing with one strand of the DNA target site (the Cas-alpha variable targeting domain). To transcribe the small RNAs required to induce Cas-alpha endonuclease cleavage activity in S. cerevisiae cells, a DNA sequence encoding a hepatitis delta virus ribozyme was first added to the 3' end of a DNA sequence encoding a Cas-alpha single guide RNA (sgRNA) with a variable targeting domain capable of targeting the yeast ADE2 or CAN1 gene. The SNR52 promoter and Sup4 terminator were then operably linked to the ribozyme and the end of the cas-alpha-encoding sgRNA, which incorporated a G bp at the 3' end of SNR52, to drive transcription of the Cas-alpha sgRNA. DNA fragments were then synthesized and cloned into the S. cerevisiae CEN6 vector (GenScript) containing the cas-alpha gene. A schematic diagram of the yeast-optimized Cas-alpha guide RNA expression cassette, with references to the sequences described herein, is shown in Figure 1.

[0349] A Zea mays Cas-alpha nuclease expression cassette was constructed essentially as described above, except that the nuclease-encoding sequence was codon-optimized using the Zea mays codon table, GC content was adjusted using standard techniques, and gene-destabilizing features such as repeat sequences and miniature inverted repeat transposable elements (MITEs) were removed. The resulting gene was then synthesized (GenScript) and cloned by restriction enzyme digestion and ligation into a Gateway-compatible plasmid DNA containing the Polymerase II promoter and terminator. For expression in maize, this includes the Zea mays ubiquitin (UBI) promoter and terminator. To further enhance expression, 5' untranslated regions (UTRs) (e.g., but not limited to, the maize UBI 5'UTR) and additional introns (e.g., the UBI Zea mays intron 1 and the potato ST-LS1 intron 2) are used, although other introns are functional. A schematic diagram of the maize-optimized Cas-alpha endonuclease expression construct is shown in Figure 8 (top panel).

[0350] Cas-alpha sgRNA expression cassettes were constructed generally as described above, except that they utilized the maize U6 promoter and terminator and incorporated a variable targeting domain capable of directing the Cas-alpha endonuclease to Zea mays MS26 (SEQ ID NO: 303), CR36 (SEQ ID NO: 304), or D8 (SEQ ID NO: 305) target sites. A schematic diagram of the maize-optimized Cas-alpha sgRNA expression cassette is shown in Figure 8 (bottom panel).

[0351] For testing in human cells, Cas-alpha endonuclease expression cassettes for SEQ ID NOs: 14, 60, 157, and 362 were similarly constructed generally as described above, except that the nuclease-encoding sequences were human codon-optimized, a ribosome-skipping T2A peptide (Ahier et al. (2014) Genetics. 196:605-613) and green fluorescent protein (GFP) were fused in-frame to the 3' end of the Cas-alpha endonuclease open reading frame, expression was driven using a human beta-herpesvirus promoter and enhancer, and transcription was terminated using sequences from a human gamma-herpesvirus. A schematic diagram of the human-optimized Cas-alpha nuclease expression cassette is shown in Figure 6 (top panel).

[0352] Cas-alpha sgRNA expression cassettes were then constructed as described above, except that they utilized the human U6 promoter and terminator and incorporated a variable targeting domain capable of directing the Cas-alpha endonuclease to human Runx (SEQ ID NO: 306), WTAP (SEQ ID NO: 307), or DNMT (SEQ ID NO: 308) target sites. A schematic of the human-optimized Cas-alpha sgRNA expression cassette is shown in Figure 6 (bottom panel).

[0353] Example 2: Cas-alpha nuclease and guide RNA expression cassette transformation This example describes methods for transforming Cas-alpha endonuclease and guide RNA expression cassettes into Saccharomyces cerevisiae, Zea mays, and Homo sapiens cells.

[0354] For Saccharomyces cerevisiae, we used an approach similar to the lithium cation-based method using the Frozen-EZ yeast Transformation II kit (Zymo Research). Competent S. cerevisiae cells were generated by growing S. cerevisiae (BY4742 (1998) Yeast. 14:115-132) (ATCC)) in yeast extract-peptone-dextrose (YPD) broth (Gibco) according to the manufacturer's instructions to mid-logarithmic growth, corresponding to an OD600nm of 0.8-1.0. The cells were then pelleted by centrifugation (500 × g, 4 min), the medium was decanted, and the pellet was gently washed with 10 ml of EZ 1 solution. The cells were again centrifuged and the wash was then removed. The cells were then resuspended in 1 ml of EZ 2 solution, aliquoted, and stored at -70°C or used in the next step. Transformation was then performed by adding 0.5–1 μg (<5 μl) of CEN6 yeast plasmid DNA containing the Cas-alpha nuclease and single-guide RNA expression cassette to 50 μl of competent cells. Optionally, a homologous double-stranded DNA repair template flanking the predicted Cas-alpha double-strand break site was also included (0.5 μl at 50 μM). After gentle mixing in the DNA, 500 μl of EZ 3 solution was added. The cells were then incubated at 30°C for 60–90 minutes, flicking or vortexing the cells 3–4 times during this incubation. After transformation, cells were grown in YPD broth for approximately 3 hours, pelleted, washed once with 1 ml of sterile water, resuspended in 1 ml of sterile water, and then approximately 200 μl was plated onto selective media (such as, but not limited to, 6.7 g / L Yeast Nitrogen Base 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 optimal culture conditions for activity, cells were either 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.

[0355] For particle-mediated biolistic transformation of Zea mays, DNA expression cassettes were co-precipitated onto 0.6 μm (average size) gold particles using TransIT-2020. The DNA-coated gold particles were pelleted by centrifugation, washed with absolute ethanol, and redispersed by sonication. Following sonication, 10 μl of the DNA-coated gold particles were loaded onto a macrocarrier and air-dried. Biolistic transformation was performed using a PDS-1000 / He Gun (Bio-Rad) equipped with a 425 lb / in² rupture disk. To promote cell division, the BabyBoom (BBM) and Wuschel2 (WUS2) genes, expressed from non-constitutive promoters, maize phospholipid transferase protein, and maize auxin-inducible gene expression cassettes, respectively, were also co-delivered with the Cas-alpha endonuclease and guide RNA expression cassettes (Lowe et al. (2018) In vitro cellular & developmental biology-Plant. 54:240-252). A visual marker DNA expression cassette encoding a fluorescent protein, such as yellow fluorescent protein, can also be co-delivered to aid in the selection of uniformly transformed tissue. Furthermore, a chemical selection marker (e.g., but not limited to, neomycin phosphotransferase II) can also be delivered with the aforementioned plasmids to select for transformed cells. To examine Cas-alpha nuclease-guide RNA activity, transformed tissues can be incubated at 28°C, the standard for particle gun transformation, or at a range of temperatures below or above 28°C.

[0356] For Agrobacterium delivery, the Cas-alpha nuclease and associated sgRNA expression cassettes were Gateway cloned into a T-DNA that already contained genes encoding the morphogenetic transcription factors Bbm and Wus2 operably linked to the Zm-PLTP and Axig1 promoters, respectively. The resulting T-DNA was then placed into Agrobacterium tumefaciens LBA4404, a thymine auxotrophic strain, containing a separate plasmid, Vir9, encoding the Bo542 virulence genes (U.S. Patent Application Publication No. 2017 / 0121722 A1 and WO 2017 / 078836). The resulting Agrobacterium strain was then used to transform 9-10-day-old immature maize embryos (approximately 2 mm in size) by immersion for 5 minutes in 700A liquid medium containing the strain at an optical density (OD) of 0.4-2.0 at 550 nm. The embryos were then removed from the medium and placed on solid co-cultivation medium overnight at 21°C in the dark. After T-DNA delivery, the embryos were transferred to static medium 13266R and grown in the dark at 28°C for 5-7 days. Next, the cotyledons were removed and the embryos were transferred to selective medium and maintained in the dark at 28°C for 11-16 days. They were then transferred to maturation medium and incubated in the dark at 28°C for 14-25 days, followed by exposure to light for 2-5 days. They were then transferred to rooting medium and incubated in the light at 26-28°C for 14-28 days, with medium changes as needed. To examine Cas-alpha nuclease-guide RNA activity, transformed tissues were incubated at 28°C, the standard for Agrobacterium transformation, or at a range of temperatures below or above 28°C.

[0357] 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 μg / ml) at 37°C in 5% CO. Cells were then seeded into 96-well plates at a density of 1.8 × 10 cells / well. After growing the cells for approximately 1 day, they were transfected with plasmids encoding nuclease 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-alpha nuclease and 0.099 μg of sgRNA expression cassette) was diluted with room temperature Opti-MEM (Gibco) to a final volume of 20 μL, mixed, and 1.35 μL of FuGENE HD was added directly to the solution (ensuring that the undiluted transfection reagent did not contact the sides or bottom of the tube or plate) to achieve a transfection reagent-to-plasmid DNA ratio of 3:1. The resulting mixture was then incubated at room temperature for 10 minutes, and 20 μL was added to each well seeded with HEK293T cells and mixed gently by pipetting or using a shaker. The transfected cells were then grown at 37°C in 5% CO2 for 4 days, and GFP expression was monitored by fluorescence microscopy. Experiments were performed in 3 to 12 independent replicates, and transfections omitting the Cas-alpha10 endonuclease and / or sgRNA plasmid DNA expression cassettes were performed as negative controls.

[0358] Example 3: Detection of Cas-alpha and guide RNA variants with improved cellular DNA target cleavage This example describes methods to detect Cas-alpha endonuclease and guide RNA variants with improved double-stranded DNA target cleavage.

[0359] In one method, Cas-alpha nuclease variants and associated single-guide RNAs (sgRNAs) were encoded in low-copy (2–5 copies per cell) Saccharomyces cerevisiae CEN6 self-replicating yeast plasmid DNA (Karim et al. (2013) FEMS Yeast Research. 13:107-116) and expressed using the ROX3 promoter, previously demonstrated to support weak gene expression (Generoso et al. (2016) Journal of Microbiological Methods. 127:203-205). Variants with improved cell-targeting DSB activity were then selected by targeting either the ADE2 and / or CAN1 genes in S. cerevisiae (BY4742, genotype - MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0). See Figures 2 and 3.

[0360] When targeting the ADE2 gene, selection for Cas-alpha nuclease and guide RNA target cleavage was performed by monitoring the shift from a white to a red (pink) cellular phenotype resulting from the formation of a nonfunctional ade2 gene (Figure 2). Depending on when colonies were considered and how quickly the ADE2 gene was disrupted, the red coloration varied from completely red to the observation of small red sectors within otherwise white colonies. These phenotypic patterns were also used to quantify activity differences between variants. Colonies scored as completely red, those containing several sectors, or those containing only one red sector were counted and divided by the total number of colonies present. In some cases, instead of counting colonies, images of yeast colonies were taken with a Nikon Digital Sight Ds-Fi1 camera (Nikon Corporation, Japan) and NIS-Elements BR software (version 4.00.07) (Nikon Corporation, Japan). The total yeast area (as pixels) was first determined, and then analyzed using a custom script to calculate the total percentage of red color.

[0361] In the CAN1 experiment, Cas-alpha nuclease variants with improved targeted DSB activity were selected by plating on medium containing L-canavanine, a toxic analog of arginine. In this assay, targeted cleavage and nonfunctional repair of the CAN1 gene (Figure 3) provides resistance to L-canavanine by disrupting its transport into the cell (Whelan et al. (1979) Genetics. 91:35-51). Thus, if the CAN1 gene is cleaved and a nonfunctional repair is made, L-canavanine does not cross the cell membrane, and cells survive; in contrast, if the CAN1 gene is not cleaved, L-canavanine enters and leads to cell death (Figure 3). To adjust the selection pressure, growth time in YPD medium was varied between 2 and 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 thermal preference of the Cas-alpha nuclease and the desired selection pressure.

[0362] In some examples, both the ADE2 and CAN1 genes in S. cerevisiae were targeted for Cas-alpha endonuclease targeted cleavage, where both the disrupted ade2 red cell phenotype and resistance to L-canavanine were used to select for Cas-alpha nuclease variants with improved targeted DSB activity.

[0363] Once an improved Cas-alpha variant was identified, it was transferred to 5 ml of histidine-free yeast broth (e.g., but not limited to, 6.7 g / L amino acid-free yeast nitrogen base (Becton Dickinson), 20 g / L glucose (Phytotechnology Labs), and 1.92 g / L yeast histidine dropout medium (MP Biomedicals)) and incubated overnight at 30°C with shaking. The CEN6 plasmid encoding the variant was then isolated using a Yeast Plasmid Miniprep 96 kit (Zymo Research). After purification, the plasmids were then transformed into TransforMax EPI300 (Lucigen) E. coli competent cells according to the manufacturer's instructions and plated on selective media (e.g., but not limited to, 6.7 g / L Yeast Nitrogen Base 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 demonstrate the feasibility of transforming and maintaining multiple CEN6 vectors in a single yeast cell, 4–6 colonies were selected from each E. coli transformation and each was used to inoculate 2 ml of 2X YT medium (Sigma-Aldrich) or equivalent containing kanamycin. Cultures were grown overnight at 37°C with shaking, and then half of the cultures were subjected to rolling circle amplification and Sanger sequencing (Eurofins Scientific) using primers specific for regions immediately adjacent to or within the cas-alpha gene. After sequencing, plasmid DNA was isolated from the remaining half of the E. coli cultures known to contain the various variants using a Qiagen Spin Miniprep kit (Qiagen). Finally, approximately 1 μg of each plasmid was retransformed into S. cerevisiae and assessed for a red cell phenotype and / or L-canavanine resistance to confirm improvement.To compare the improved variants with the Cas-alpha endonucleases that underwent directed evolution, additional yeast transformations were typically performed with all variants and their ability to recognize and cleave the ADE2 and / or CAN1 target sites was reassessed.

[0364] In the second assay, Cas-alpha nuclease and sgRNA target cleavage efficiency was assessed by measuring the frequency of small insertion or deletion (indel) mutations resulting from cellular repair of chromosomal DNA target cleavage in Zea mays or human cells. To this end, experiments were performed using methods similar to those described in Svitashev et al., 2015, Karvelis et al., 2015, and Bigelyte et al., 2021.

[0365] For Zea mays, two assays were used. In the first assay, transformed immature maize embryos were harvested 2–10 days after transformation, genomic DNA was extracted, and the Cas-alpha target was examined by Ampli-seq for the presence of mutations indicative of RNA-guided Cas-alpha editing. Briefly, 20–30 immature embryos that were most uniformly transformed based on fluorescence were collected for each experiment. Next, total genomic DNA was extracted, and the region surrounding the intended target site was PCR amplified using Phusion® High Fidelity PCR Master Mix (New England Biolabs, M0531L) and Illumina sequencing using two rounds of PCR "tailed" primers, which add the necessary sequences for amplicon-specific barcodes. The resulting reads were then examined for the presence of mutations at the predicted cleavage site by comparing them with a control experiment in which the small RNA transcription cassette was omitted from the transformation. Sequence reads containing putative indels were further confirmed as true mutations by confirming their absence in the control dataset.

[0366] In the second Zea mays assay, regenerated plants were sampled and DNA targets were examined for evidence of editing using Ampli-Seq. This was performed as described above, but for each plant, the frequency of reads from the edited and wild-type sequences was also calculated to assess the likelihood of inheritance. Because maize is diploid, plants with approximately 50% and 100% mutant reads were assumed to be heterozygous and homozygous for the targeted mutation, respectively (Zhang et al., (2014) Plant Biotechnology Journal. 12:797-807).

[0367] For human cells, 2–4 days after transfection, cells were washed twice with PBS and lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 0.2% Tween 20, 0.2 mg / ml proteinase K). RUNX and WTAP targets were then assessed for the presence of targeted mutations using T7 endonuclease I (Guschin, DY 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.

[0368] Example 4: Library design and generation This example describes methods to design and generate Cas endonuclease variants for improved double-stranded DNA target cleavage.

[0369] In one approach, saturation mutagenesis was performed, introducing every other amino acid (19 total) at every position in the entire amino acid sequence of the Cas-alpha10 variant (SEQ ID NO: 14 (Figure 4)). In a second approach, the beneficial changes identified herein were incorporated (alone or in combination) into variants already containing one or more beneficial changes, and their combined effects were assayed. In a third approach, in variants containing two or more changes, the amino acid substitutions were reverted to wild-type residues and assayed for improvement in cleavage activity; if two or more beneficial reversions were identified, they were combined and re-assayed for improvement.

[0370] For the first library method, codons within the cas-alpha nuclease gene (SEQ ID NO:2) in the yeast expression plasmid shown in Figure 1 were altered to encode various amino acids using GenPlus gene synthesis technology (GenScript). For library methods 2 and 3, the Cas-alpha nuclease genes encoding amino acid substitutions and / or reversions were generated synthetically (GenScript) and / or introduced using site-directed mutagenesis as previously described (Kunkel (1985) Proc Natl Acad Sci USA 82, 488-492).

[0371] Example 5: Cas-alpha endonuclease variants with improved cellular activity This example describes Cas-alpha endonuclease variants with improved double-stranded DNA cleavage activity. All variants generated in Example 4 were tested. These included variants with all possible amino acid substitutions across the entire length of the protein (positions 1-497 relative to SEQ ID NO: 14), variants containing combinations of beneficial changes, and variants composed of combinations of beneficial changes with one or more amino acids reverted to wild-type residues. Assays were performed to identify variants with the desired improved activity.

[0372] In Zea mays, the amino acid substitutions A40G, E81G, A87K, T190K, T217H, K298S, H306F, T335R, and I405N in the Cas-alpha10 variant SEQ ID NO:14 were individually reverted to wild-type residues (see corresponding positions in SEQ ID NO:157) to assess their effect on targeted mutagenesis. In these experiments, immature embryos were maintained at 28°C until 1 day after particle-mediated transformation, after which they were incubated at 33°C for 72 hours and harvested for analysis. Additionally, plasmids encoding the guide RNA recognition domain from the Cas-alpha10 sgRNA SEQ ID NO:61 targeting the MS26 or CR36 sites were co-delivered with the plasmids encoding the respective Cas-alpha10 nuclease variants. As shown in Table 2, several amino acids (G81E, S298K, F306H, and N405I) increased in frequency with targeted mutagenesis when reverted to the wild-type residue.

[0373] [Table 10]

[0374] The foregoing results identify a series of amino acid substitutions that, when reverted to wild-type residues, can be used to design Cas-alpha polypeptide variants that exhibit significantly improved DNA cleavage activity and targeting efficiency, including, but not limited to, SEQ ID NOS: 48, 313-323, and 362.

[0375] Next, in yeast, the Cas-alpha10 nuclease and guide RNA (the recognition domain of the Cas-alpha10 original sgRNA (SEQ ID NO: 61)) were encoded in a low-copy CEN6 yeast plasmid DNA, and the Cas nuclease was expressed from a weak ROX3 promoter. All possible amino acid substitutions (positions 1-497 relative to SEQ ID NO: 14) across the entire length of the protein were screened for those that improved double-stranded DNA target cleavage activity. Initial selection was applied by targeting both the ADE2 and CAN1 S. cerevisiae genes, applying a growth temperature regime of 30°C for 1 hour followed by 37°C for 2 hours or 30°C for 1 hour followed by 37°C for 4 hours. After growth, cells were plated on medium containing L-canavanine, placed at 37°C overnight, and then grown at 30°C until colony growth was visible. Variants that survived L-canavanine and exhibited the ade2 red cell phenotype were selected and sequenced. To visualize activity against the starting Cas-alpha nuclease variants (Figure 4, SEQ ID NO: 14), an experiment was next set up using only the ADE2 screen. To do this, cells were plated on adenine-deficient medium immediately after transformation, placed at 37°C overnight, and then grown at 30°C until colony growth appeared (approximately 2 days). Photographs of S. cerevisiae colonies were taken, and the total percentage of ade2 red phenotype was calculated by image analysis using a custom script. The fold improvement in target DNA cleavage was calculated by dividing the total percentage of red S. cerevisiae generated with each variant by that observed from the original protein that underwent directed evolution (Figure 4, SEQ ID NO: 14) (Table 3).

[0376] [Table 11]

[0377] Next, combinations of modifications listed in Table 3 were tested to identify those that provided further improvement using the ADE2 screen, incubated overnight at 37°C as described above. Certain combinations also incorporated the G81E reversion (SEQ ID NO: 48) identified in the Zea mays experiments. Based on visual assessment of the ade2 red phenotype, several combinations were shown to further improve targeted DNA cleavage activity (Table 4). To further differentiate each variant, the overnight incubation at 37°C was omitted, and cells were incubated at 25°C or 30°C until colonies were visible immediately after transformation. As shown in Table 4, all combinations improved activity at 30°C, with M45S+F54D+G81E+K85Q+G470D (SEQ ID NO: 50) providing the highest activity increase (1.52-fold at 37°C, 29.05-fold at 30°C, and 29.58-fold at 25°C) compared to the original protein (Figure 4, SEQ ID NO: 14).

[0378] Table 4 lists the amino acid substitutions that, in combination, further improved double-stranded DNA target cleavage activity. S. cerevisiae experiments were performed as described above, and the fold improvement in target DNA cleavage over the original protein was calculated as described above.

[0379] [Table 12]

[0380] K85Q was also predicted by AlphaFold to be involved in PAM recognition. Therefore, it was converted back to a K residue in M45S+F54D+G81E+K85Q+G470D (SEQ ID NO: 50) and T34E+M45S+G470D+G81E+K85Q+F54D (SEQ ID NO: 54) and retested using the two ADE2 target sites. Tests were performed as described above, and the fold improvement in target DNA cleavage relative to the original protein was calculated as described above. The results, shown in Table 5, indicate that altering the K residue had little effect on activity at the original ADE2 target with 5'-ATTC-3'PAM (ADE2-CR2, SEQ ID NO: 57). However, when used with a second ADE2 target site with 5'-GTTC-3'PAM (ADE2-CR1, SEQ ID NO: 58), the restored K residues substantially improved activity, as shown in Table 5, with T34E+M45S+F54D+G81E+G470D (SEQ ID NO: 60) (Figure 5) showing the greatest fold improvement against both targets.

[0381] [Table 13]

[0382] Furthermore, F54D was predicted by AlphaFold to potentially alter the oligomerization state of Cas-alpha10, and was reverted to phenylalanine. The targeted mutagenesis efficiency of the novel variant, T34E+M45S+G81E+G470D (SEQ ID NO: 362), was then compared with that of T34E+M45S+F54D+G81E+G470D (SEQ ID NO: 60) in human cells (37°C). As shown in Table 6, T34E+M45S+G81E+G470D (SEQ ID NO: 362) enhanced targeted mutagenesis at DNMT and WTAP sites and performed comparably to T34E+M45S+G81E+G470D (SEQ ID NO: 362) at the Runx target.

[0383] [Table 14]

[0384] 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 biochemically evaluated as described in Gasiunas et al. (2020) Nat Commun. 11:5512. Briefly, Cas-alpha10 protein was expressed and purified as described in Bigelyte et al., 2021, and sgRNA was generated using the HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB). The sgRNA was then dissolved in 1x reaction buffer (Tris, pH 7.5, 200 nM NaCl, 1 mM DTT, 1 mM EDTA, 10 mM MgCl2, and 0.01 mM ZnCl2), heat-denatured at 90°C for 3 minutes, and then slowly cooled to 12°C. Next, the sgRNA and purified Cas-alpha 10 nuclease, pre-diluted in 1x reaction buffer, were combined and incubated at room temperature for 5-10 minutes, followed by a 2-3 minute transfer to the desired reaction temperature before being mixed with the 5'-FAM-labeled double-stranded DNA substrate. The reaction was run in 10 µL of 1x reaction buffer with final concentrations of sgRNA, Cas-alpha nuclease, and DNA substrate of 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 µg / µL proteinase K (final concentration)). After dilution to 4x with nuclease-free water, the extent of target cleavage was assayed using capillary electrophoresis.

[0385] The effect of temperature on nuclease activity was first assessed. As shown in Figure 9, wild-type Cas-alpha10 nuclease (SEQ ID NO:157) robustly cleaved its double-stranded DNA substrate at temperatures ranging from 42 to 58°C (fraction of cleaved target >0.6). This contrasts with SEQ ID NO:14, SEQ ID NO:48, and SEQ ID NO:362, which have a lower requirement for high temperature (Figures 9-11). Furthermore, as a result of the modifications described herein, the temperature range over which SEQ ID NO:48 efficiently cleaved its target was expanded to 30-48°C compared to SEQ ID NO:14, which has a narrower preferred temperature of 37-43°C (Figure 10). Finally, the thermal profile of SEQ ID NO:362 was further shifted to lower temperatures, demonstrating efficient substrate cleavage at temperatures as low as 25°C (Figure 11).

[0386] The foregoing results identify a series of amino acid changes that can be used to design engineered Cas-alpha polypeptide variants that significantly improve DNA cleavage activity, targeting efficiency, and / or PAM preference. The improved function of the disclosed variants is notable compared to the starting Cas-alpha variant (Figure 4, SEQ ID NO: 14). The results also demonstrate that the disclosed variants and amino acid changes can broaden the range of applicability and organisms in which such engineered Cas-alpha polypeptide variants can be used as Cas effectors, e.g., endonucleases, including targeted genome editing.

[0387] Example 6: Cas-alpha sgRNA variants with improved cellular activity This example describes CasCas-alpha sgRNA variants with improved double-stranded DNA cleavage activity.

[0388] In one method, three nucleotides of the Cas-alpha10 sgRNA recognition domain (SEQ ID NO: 61) were systematically deleted at overlapping intervals of one nucleotide, and their ability to cleave the ADE2 gene was assessed as described in Example 5 above, except that two yeast plasmids were used. The first contained the CEN6 ORI and a Cas-alpha10 endonuclease (SEQ ID NO: 14) expression cassette, and the second comprised a 2 micron ORI and an sgRNA expression cassette. DNA sequences encoding the sgRNA variants were generated by inverse PCR and seamless cloning using the 2 micron plasmid as a template for S. cerevisiae, encoding an sgRNA (SEQ ID NO: 62) capable of directing Cas-alpha10 endonuclease cleavage at the ADE2-CR2 target site. Experiments were performed at 37°C, and the percentage of red cell phenotype resulting from ADE2-CR2 target cleavage was normalized to the original Cas-alpha sgRNA (SEQ ID NO: 62). A value of about 1.00 indicates activity equivalent to the original sgRNA, while values above or below about 1.00 correspond to increased or decreased ADE2-CR2 target cleavage, respectively. The results of deleting three different nucleotide regions of the Cas-alpha10 sgRNA recognition domain are shown in Table 7.

[0389] [Table 15]

[0390] [Table 16]

[0391] The aforementioned results (percentages of red yeast) in Table 7 show that deletion of three nucleotides was tolerated 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. In particular, deletion of 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 resulted in increased targeted cleavage efficiency at the ADE2-CR2 site.

[0392] Next, we tested the improvement in human cells by combining deletions of sgRNA recognition domains that either improved or had little effect on DNA target cleavage activity. These deletions were introduced into the recognition domains of four sgRNA designs listed in Tables 8 and 9: Cas-alpha10 original sgRNA (SEQ ID NO: 61), Cas-alpha10 sgRNA MS10 (SEQ ID NO: 324), Cas-alpha10 sgRNA MS13 (SEQ ID NO: 325), and Cas-alpha10 sgRNA design 5 (SEQ ID NO: 310 in WO 2022 / 082179A2) (SEQ ID NO: 326). Cas-alpha10 sgRNA MS10, Cas-alpha10 sgRNA MS13, and Cas-alpha10 sgRNA Design 5 already contained deletions at positions 1-3, 3-5, 5-7, 7-9, 147-149, 149-151, and 151-153, so it was not necessary to remove these nucleotides (Tables 8 and 9). Furthermore, deletions at positions 157-159 and 167-169 were not tested in Cas-alpha10 sgRNA MS13 because there were no regions encompassing these deletions (Tables 8 and 9). Furthermore, in some cases, the MS13 solution was further modified by replacing the sequence linking the tracrRNA and crRNA portions or the sgRNA with a 5'-GAAA-3' tetraloop (SEQ ID NO: 327) (Tables 8 and 9).

[0393] For the RUNX target, new sgRNA solutions derived from Design 5 sgRNA had the greatest effect on enhancing target indel frequency in combination with the Cas-alpha10 endonuclease variants, SEQ ID NO:14 and SEQ ID NO:362, with the highest mutagenesis frequencies obtained using Design 5_157-159 (Table 8).

[0394] [Table 17]

[0395] [Table 18]

[0396] At the WTAP site, design 5sgRNA produced the highest targeted mutation frequency when paired with Cas-alpha10 endonuclease SEQ ID NO: 14 and 3362 (Table 8). When used in combination with Cas-alpha10 nuclease SEQ ID NO: 157, design 5_157-159 produced the highest rate of targeted mutagenesis (Table 9). [Table 19] [Table 20]

[0397] In the second study, an RNA polypeptide was added to the Cas-alpha sgRNA. For example, a sequence that formed a hairpin-like secondary structure, including the hairpin-like RNA recognition sequence of the MS2 bacteriophage coat protein (SEQ ID NO: 208), was incorporated. The secondary structure of the Cas-alpha sgRNA was predicted using Vienna fold (Gruber et al. (2008) "Nucleic Acids Res. 36:W70-74). Next, MS2 recognition sequences were incorporated into the 5' end, the apex of the stem-loop, and the 5'-GAAA-3' linker connecting the tracrRNA and crRNA of the sgRNA, as shown in Figure 7. To facilitate proper folding of the aptamer, additional sequences (e.g., G or C nucleotides on either side of the MS2 recognition sequence positioned to base-pair with each other) were incorporated at each additional base. Using the evolved Cas-alpha10 nuclease SEQ ID NO:14, solutions were tested in human cells for their ability to induce Cas-alpha endonuclease target recognition and cleavage at a single target in a DNMT gene. Three independent transfections were performed for each sgRNA variant; negative controls included experiments in which the Cas-alpha10 endonuclease and sgRNA plasmid DNA expression cassette were omitted.

[0398] As shown in Table 10, sgRNAs incorporating the MS2 aptamer were tolerated at positions 1, 3, 5, 7, 10, 11, and 12. Improved targeted mutagenesis was observed at position 1 compared to the original sgRNA. To confirm that the results were not sequence-dependent, the hairpin-forming RNA recognition sequence of the Cas6 protein from Pseudomonas aeruginosa (SEQ ID NO: 209) (Sternberg et al. (2012) RNA. 18:661-672) was also incorporated into the Cas-alpha sgRNA at the same position and tested. Similar trends in targeted mutagenesis were observed, with positions 1, 3, 4, 5, 6, 7, 9, 10, and 11 demonstrating comparable or superior targeted mutagenesis frequencies compared to the original sgRNA solution (Table 10).

[0399] [Table 21]

[0400] In a third study, the efficiency of targeted mutagenesis of Cas-alpha10 endonuclease variants, SEQ ID NOs: 14, 48, 60, and 157, was compared with that of two sgRNA recognition domains, SEQ ID NOs: 61 and 327, tested at two target sites, MS26 and D8. Experiments were performed in Zea mays using Agrobacterium (Agro) transformation, as described in Example 2. Two temperature regimes were performed. First, Agro-transformed embryos (once daily) were subjected to 45°C for 4 hours on days 3, 4, and 5 post-infection (3 x 4 hours at 45°C). Second, embryos were continuously incubated at 37°C from days 1 to 3 post-infection (days 1 to 3 at 37°C). In both regimes, embryos were placed at 28°C before and after temperature treatment. In the first study, embryos were inoculated with Agro (OD550: 0.4) and incubated at 45°C for 4 hours on days 3, 4, and 5 post-infection (OD550: 0.4). 550 Immature embryos were harvested 7 days after 0.4) and assayed for the percentage of targeted mutations as described in Example 3.

[0401] The Cas-alpha10 endonuclease SEQ ID NO:48 in combination with the sgRNA recognition domain SEQ ID NO:327 showed the highest target mutagenesis frequency at both target sites and under both temperature treatments (Table 11). This resulted in an 11.50-fold and 12.61-fold improvement over the wild-type nuclease (SEQ ID NO:157) in combination with the sgRNA recognition domain SEQ ID NO:61 at the MS26 and D8 targets, respectively, using a 45°C temperature treatment (3 x 4 hours at 45°C). Under the 37°C regimen (1-3 days at 37°C), a 1053.00-fold and 13.15-fold enhancement was observed at the MS26 and D8 sites, respectively.

[0402] [Table 22]

[0403] To confirm the findings in Table 11, T0 plants were regenerated in some experiments and the percentage of plants with the predicted germline mutations was calculated as described in Example 3. The results confirmed that the Cas-alpha10 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).

[0404] [Table 23]

Claims

1. A modified Cas polypeptide having a sequence with 90% amino acid sequence identity to SEQ ID NO: 14, and comprising one or more of the following amino acids at positions relative to the alignment with SEQ ID NO: 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, which can site-specifically bind to a target site of a polynucleotide.

2. A modified Cas polypeptide, (a) C-terminal triple-split RuvC domain and three zinc finger motifs; and (b) The following amino acids are present in positions relative to the alignment with SEQ ID NO: 34-34, 36-36, 45-45, 54-54, 77-77, 81-81, or 85-85; The modified Cas polypeptide is a modified Cas polypeptide that does not contain at least one of the following amino acids at the position relative to the alignment with SEQ ID NO: phenylalanine at position 38, alanine at position 40, histidine at position 79, alanine at position 87, threonine at position 335, cysteine ​​at position 409, glutamic acid at position 421, lysine at position 467, or glutamic acid at position 468, and is capable of site-specifically binding to a target site of a polynucleotide.

3. The modified Cas polypeptide according to 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 to 56, or the group consisting of SEQ ID NOs: 59 and 60, or the group consisting of SEQ ID NOs: 309 to 323 and SEQ ID NOs:

362.

4. The following amino acids are positioned relative to the alignment with SEQ ID NO: 38th position aspartic acid or glutamic acid, 79th position aspartic acid, 120th position proline, 149th position aspartic acid, 226th position glycine, 230th position glycine, 293rd position histidine, 298th position serine, 306th position phenylalanine, 329th position glutamic acid, 313th position serine, 325th position asparagine, 327th position A modified Cas polypeptide according to claim 1 or 2, further comprising glutamic acid, 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. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is an endonuclease having higher activity than SEQ ID NO: 14 at one or more of the following temperatures: about 40°C, about 37°C, about 35°C, about 30°C, about 25°C, or about 20°C.

6. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide has higher polynucleotide target site cleavage activity than SEQ ID NO: 14 at temperatures of about 30°C, about 25°C, or about 20°C.

7. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide cleaves a target site on a polynucleotide with higher efficiency than SEQ ID NO: 14 at a temperature of about 30°C, about 25°C, or about 20°C.

8. The modified Cas polypeptide according to claim 7, wherein the modified Cas polypeptide cleaves the polynucleotide target site at a temperature of about 30°C, about 25°C, or about 20°C at least about 1.5 times more efficiently than SEQ ID NO:

14.

9. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide has a length of less than approximately 500 amino acids.

10. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is present in a complex, and the complex includes a target site on a double-stranded DNA polynucleotide.

11. A modified Cas polypeptide according to claim 1 or 2, further comprising a guide polynucleotide containing a variable targeting domain having a region complementary to the target site of the polynucleotide.

12. The modified Cas polypeptide according to claim 11, wherein the guide polynucleotide variable targeting domain comprises fewer than 20 nucleotides.

13. The modified Cas polypeptide according to claim 11, wherein the modified Cas polypeptide recognizes a PAM sequence on a target polynucleotide, and the guide polynucleotide and the Cas polypeptide form a complex that binds to the target site on a double-stranded DNA polynucleotide.

14. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is an endonuclease that cleaves double-stranded DNA polynucleotides.

15. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is catalytically inactive with respect to endonuclease activity.

16. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide recognizes a PAM sequence including N(T>W>C)TTC.

17. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is part of a fusion protein species.

18. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is part of a fusion protein, and the fusion protein further comprises a heterologous nuclease domain.

19. A modified Cas polypeptide according to claim 1 or 2, further comprising a deaminase.

20. A synthetic composition comprising a modified Cas polypeptide according to claim 1 or 2, further comprising a heterogeneous polynucleotide.

21. The synthetic composition according to claim 20, wherein the heterogeneous polynucleotide is an expression element, an introduced gene, a donor DNA molecule, or a polynucleotide modification template.

22. The synthetic composition according to claim 20, wherein the heterogeneous polynucleotide is a temperature-inducible promoter.

23. A synthetic composition, (a) The modified Cas polypeptide according to claim 1 or 2; (b) Target double-stranded DNA polynucleotides; and (c) Guide polynucleotide containing a variable targeting domain that includes a region complementary to the target double-stranded DNA polynucleotide Includes, The Cas polypeptide recognizes the PAM sequence on the target double-stranded DNA polynucleotide, and the guide polynucleotide and the Cas polypeptide form a complex that binds to the target double-stranded DNA polynucleotide. Synthetic composition.

24. A polynucleotide encoding the modified Cas polypeptide according to claim 1 or 2.

25. The polynucleotide according to claim 24, encoding the modified Cas polypeptide and at least one expression element.

26. The polynucleotide according to claim 24, wherein the polynucleotide encodes the modified Cas polypeptide and a gene.

27. The modified Cas polypeptide according to claim 1 or 2, wherein the modified Cas polypeptide is attached to a solid matrix, or the Cas polypeptide is complexed with a guide polynucleotide, and the Cas polypeptide / guide polynucleotide complex is attached to a solid matrix.

28. A eukaryotic cell comprising a modified Cas polypeptide according to claim 1 or 2, or a polynucleotide encoding the modified Cas polypeptide according to claim 1 or 2.

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

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

31. The eukaryotic cell according to claim 28, wherein the plant cell is derived from corn, soybean, cotton, wheat, canola, rapeseed, sorghum, rice, rye, barley, millet, oat, sugarcane, turfgrass, switchgrass, alfalfa, sunflower, tobacco, peanut, potato, Arabidopsis, safflower, or tomato.

32. The eukaryotic cell according to claim 28, wherein the eukaryotic cell exists at a temperature of approximately 40°C or lower, approximately 37°C or lower, approximately 35°C or lower, approximately 30°C or lower, approximately 25°C or lower, or approximately 20°C or lower.

33. A method for introducing targeted editing into a target polynucleotide, (a) to prepare a modified Cas polypeptide and a guide polynucleotide according to claim 11, wherein the Cas polypeptide / guide polynucleotide forms a complex that recognizes a PAM sequence on the target polynucleotide; and (b) Contacting the Cas polypeptide / guide polynucleotide complex with the target; and (c) Introducing targeted editing into the target polynucleotide. A method that includes this.

34. The target polynucleotide is a target genome sequence of a cell, and the method is (i) Delivering the Cas polypeptide / guide polynucleotide complex to the cells; (ii) Incubating the cells at a temperature of approximately 37°C or lower, approximately 35°C or lower, approximately 30°C or lower, approximately 25°C or lower, or approximately 20°C or lower; (iii) Modifying at least one nucleotide in the target genome sequence of the cell to generate a modified genome sequence compared to the target genome sequence of the cell before delivery of the Cas polypeptide / guide polynucleotide complex; and (iv) Producing an entire organism from the cells, wherein the organism includes the modified genome sequence. The method according to claim 33, including the method described in claim 33.

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

36. The method according to claim 35, wherein the eukaryotic cells are derived from or obtained from animals, fungi, or plants.

37. The method according to claim 36, wherein the eukaryotic cells are derived from a plant that is a monocotyledonous or dicotyledonous plant.

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

39. The method according to claim 33, wherein the guide polynucleotide variable targeting domain comprises fewer than 20 nucleotides.

40. The method according to claim 33, further comprising providing heterogeneous polynucleotides.

41. The method according to claim 40, wherein the heterogeneous polynucleotide is a donor DNA molecule.

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

43. The method according to claim 40, wherein the heterogeneous polynucleotide is an inducible promoter.

44. A kit comprising a modified Cas polypeptide according to claim 1 or 2, a polynucleotide encoding the modified Cas polypeptide according to claim 1 or 2, or a modified Cas endonuclease according to claim 27 and a solid matrix.

45. A method for modifying a target Cas polypeptide, comprising modifying the target Cas polypeptide so that, when aligned with SEQ ID NO: 14 and compared with the amino acid position numbers of SEQ ID NO: 14, the modified Cas polypeptide contains one or more amino acid changes selected from the group consisting 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, glutamic acid at position 81, or glutamine at position 85, wherein the modified polypeptide can site-specifically bind to a target site of a polynucleotide.

46. When aligned with Sequence ID No. 14 and compared with the amino acid position numbers of Sequence ID No. 14, the modified Cas polypeptide has 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, and asparagine at position 325. The method according to claim 45, further comprising modifying the Cas polypeptide to further contain 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 according to claim 45 or 46, comprising modifying the nucleic acid sequence encoding the target Cas polypeptide so that each sequence contains one or more modified codons at positions relative to the alignment with SEQ ID NO: glutamic acid at position 34, alanine at position 36, serine at position 45, aspartic acid at position 54, serine at position 77, or glutamine at position 85.

48. A system comprising a Cas polypeptide and a modified guide RNA (egRNA), wherein the egRNA is based on a template guide RNA sequence (sgRNA) containing a Cas polypeptide recognition domain, and the egRNA comprises (i) deletion, substitution or insertion at one or more 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 sequence numbers 62-156, 192-207, 260-284, 324-327, 346-355, or 362.

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

51. The system according to claim 48, wherein the egRNA includes deletions, substitutions, or insertions at positions 1-9, 6-63, 61-63, 146-153, 157-159, 158-170, 167-169, or combinations thereof, of the sgRNA Cas polypeptide recognition domain.

52. The system according to claim 48, wherein the egRNA includes sequence numbers 64, 65, 66, 92, 93, 94, 137, 138, 139, 141, 144, and 146.

53. A system comprising a Cas polypeptide and a modified guide RNA (egRNA), wherein the egRNA is based on a template guide RNA sequence (sgRNA), and 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 according to claim 48, wherein the Cas polypeptide is a modified Cas polypeptide according to claim 1 or 2.

55. A method for modifying a target site in a cell, comprising introducing the system described in claim 48 into a cell containing the target site of the modified guide RNA of the system.

Images