CASZ COMPOSITIONS AND METHODS OF USE
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 .