CONSTRUCTS AND USES THEREOF FOR EFFICIENT AND SPECIFIC GENOME EDITING
A composition comprising a CRISPR nuclease, where the nuclease includes a sequence with at least 60% sequence identity to any one of SEQ ID NOs: 143-177 and 229, optionally also including a guide nucleic acid (gNA), such as a guide ribonucleic acid (gRNA) and, in some cases, a donor sequence. Also include are methods of creating a strand break at or near a target sequence in a target polynucleotide by contacting the target polynucleotide with a nuclease including a sequence with at least 60% sequence identity to any one of SEQ ID NOs: 143-177 and 229, and a compatible guide nucleic acid (gNA), such as a guide ribonucleic acid (gRNA).
1 .- 46 . (canceled)
47 . A composition comprising:
(i) an engineered nucleic acid-guided nuclease comprising an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 143-177 and 229, or a polynucleotide or polynucleotides encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 143-177 and 229.
48 . The composition of claim 47 comprising an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 144, 153, and 229, or a polynucleotide or polynucleotides encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 144, 153, and 229.
49 . The composition of claim 47 comprising an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 144 or a polynucleotide or polynucleotides encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 144.
50 . The composition of claim 47 comprising an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 153 or a polynucleotide or polynucleotides encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 153.
51 . The composition of claim 47 comprising an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 229 or a polynucleotide or polynucleotides encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 229.
52 . The composition of claim 47 wherein the sequence identity is at least 95%.
53 . The composition of claim 47 wherein the sequence identity is at least 99%.
54 . The composition of claim 47 wherein the sequence identity is 100%.
55 . The composition of claim 47 wherein the engineered nuclease polypeptide does not contain the peptide motif YLFQIYNKDF (SEQ ID NO. 224) or a polynucleotide or polynucleotides encoding the engineered nuclease polypeptide that does not contain the peptide motif YLFQIYNKDF (SEQ ID NO. 224).
56 . The composition of claim 47 further comprising:
(ii) a compatible guide nucleic acid, wherein the nucleic acid-guided nuclease is complexed with guide nucleic acid to form a nucleic acid-guided nuclease complex.
57 . The composition of claim 56 wherein the guide nucleic acid is a gRNA and the complex is an RNP.
58 . The composition of claim 56 wherein the guide nucleic acid is a dual guide nucleic acid.
59 . The composition of claim 56 wherein the guide nucleic acid is an engineered guide nucleic acid.
60 . The composition of claim 56 wherein the guide nucleic acid comprises any one of SEQ ID NO: 291-325, or a portion thereof.
61 . The composition of claim 56 wherein the guide nucleic acid is a synthetic guide nucleic acid.
62 . The composition of claim 56 wherein the guide nucleic acid comprises one or more chemical modifications.
63 . A method of creating a strand break at or near a target sequence in a target polynucleotide comprising contacting the target polynucleotide with a nucleic acid-guided nuclease complex comprising
(i) an engineered nucleic acid-guided nuclease comprising an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 143-177 and 229, and
(ii) a compatible guide nucleic acid, wherein
(a) the nucleic acid-guided nuclease is complexed with guide nucleic acid to form a nucleic acid-guided nuclease complex, and
(b) wherein the compatible guide nucleic acid of the complex targets the target sequence;
and allowing the nucleic acid-guided nuclease complex to create the strand break.
64 . The method of claim 63 wherein the nucleic acid-guided nuclease complex comprises an engineered nuclease polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 144, 153, and 229.
65 . The method of claim 63 wherein the nucleic acid-guided nuclease complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 144.
66 . The method of claim 63 wherein the nucleic acid-guided nuclease complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 153.
67 . The method of claim 63 wherein the nucleic acid-guided nuclease complex comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 229.
68 . A composition comprising an engineered polynucleotide or polynucleotides comprising one or more polynucleotides comprising a sequence corresponding to a sequence with at least 90% sequence identity to any one of SEQ ID NO: 1-142 and 225-228.
69 . The composition of claim 68 comprising one or more polynucleotides comprising a sequence corresponding to a sequence with at least 90% sequence identity to any one of SEQ ID NO: 1, 5, 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, and 225.
70 . The composition of claim 68 wherein the polynucleotide codes for one or more additional amino acid sequences at either the N-terminus, the C-terminus or both, of a polypeptide encoded by the polynucleotide, wherein the additional amino acid sequences comprise at least one of
(i) one or more NLSs;
(ii) one or more purification tags;
(iii) one or cleavage sequences; and
(iv) FLAG or 3×FLAG.
71 . The composition of claim 68 wherein the polynucleotide or polynucleotides are codon optimized for E. coli.
72 . The composition of claim 68 wherein the polynucleotide or polynucleotides are codon optimized for S. cerevisiae.
73 . The composition of claim 68 wherein the polynucleotide or polynucleotides are codon optimized for human.
74 . The composition of claim 68 wherein the sequence identity is at least 95%.
75 . The composition of claim 68 wherein the sequence identity is at least 99%.
76 . The composition of claim 68 wherein the sequence identity is 100%.