Constructs and uses thereof for efficient and specific genome editing

Abstract

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%.