Casz compositions and methods of use

Abstract

Provided are compositions and methods that include one or more of: (1) a “CasZ” protein (also referred to as a CasZ polypeptide), a nucleic acid encoding the CasZ protein, and/or a modified host cell comprising the CasZ protein (and/or a nucleic acid encoding the same); (2) a CasZ guide RNA that binds to and provides sequence specificity to the CasZ protein, a nucleic acid encoding the CasZ guide RNA, and/or a modified host cell comprising the CasZ guide RNA (and/or a nucleic acid encoding the same); and (3) a CasZ transactivating noncoding RNA (trancRNA) (referred to herein as a “CasZ trancRNA”), a nucleic acid encoding the CasZ trancRNA, and/or a modified host cell comprising the CasZ trancRNA (and/or a nucleic acid encoding the same).

1 .- 86 . (canceled) 
     
     
         87 . A composition comprising an engineered polypeptide, or a nucleic acid encoding said engineered polypeptide, wherein the engineered polypeptide is at least 95% identical to SEQ ID NO: 3 and comprises reduced nucleic acid cleavage activity relative to a polypeptide comprising an amino acid sequence that is 100% identical to SEQ ID NO: 3. 
     
     
         88 . The composition of  claim 87 , wherein the engineered polypeptide comprises a substituted amino acid relative to the amino acid sequence of SEQ ID NO: 3 at a respective position of SEQ ID NO: 3, wherein the respective position of SEQ ID NO: 3 is selected from D 326 , E 422 , and D 510 . 
     
     
         89 . The composition of  claim 88 , wherein the substituted amino acid is selected from glycine, alanine, valine, leucine and isoleucine. 
     
     
         90 . The composition of  claim 88 , wherein the substituted amino acid is alanine. 
     
     
         91 . The composition of  claim 87 , wherein said reduced nucleic acid cleavage activity is detected by a reduction in a signal from a ssDNA molecule following cleavage of the single stranded DNA by said engineered polypeptide as compared to the polypeptide comprising the amino acid sequence that is 100% identical to SEQ ID NO: 3. 
     
     
         92 . The composition of  claim 91 , wherein such detection includes one or more of gold nanoparticle based detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, semiconductor-based sensing, phosphatase-generated pH change and detection of a labeled detector ssDNA. 
     
     
         93 . The composition of  claim 87 , comprising a guide RNA, or a nucleic acid encoding said guide RNA, wherein said guide RNA comprises a first region that interacts with the engineered polypeptide and a second region that is complementary to a target sequence of a target nucleic acid. 
     
     
         94 . The composition of  claim 93 , wherein the target sequence is a eukaryotic sequence. 
     
     
         95 . The composition of  claim 93 , wherein the guide RNA is a single guide RNA. 
     
     
         96 . The composition of  claim 93 , wherein the nucleic acid encoding said guide RNA is the nucleic acid encoding the engineered polypeptide. 
     
     
         97 . The composition of  claim 87 , wherein the composition comprises the nucleic acid encoding the engineered polypeptide. 
     
     
         98 . The composition of  claim 97 , wherein the nucleic acid encoding the engineered polypeptide is a nucleic acid expression vector. 
     
     
         99 . The composition of  claim 98 , wherein the nucleic acid expression vector is an AAV vector. 
     
     
         100 . The composition of  claim 99 , wherein the AAV vector encodes a guide nucleic acid. 
     
     
         101 . The composition of  claim 87 , wherein the nucleic acid encoding the engineered polypeptide is an RNA molecule. 
     
     
         102 . The composition of  claim 101 , comprising a lipid nanoparticle. 
     
     
         103 . The composition of  claim 87 , wherein the engineered polypeptide does not have nuclease activity. 
     
     
         104 . The composition of  claim 87 , wherein the nucleic acid cleavage activity is selected from nuclease activity and nickase activity. 
     
     
         105 . The composition of  claim 87 , wherein the engineered polypeptide is conjugated to a heterologous polypeptide or the nucleic acid encoding the engineered polypeptide encodes a fusion polypeptide comprising the engineered polypeptide and a heterologous polypeptide. 
     
     
         106 . A method of cleaving a target nucleic acid in a cell, the method comprising contacting the cell with the composition of  claim 97 .