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 method of modifying a target locus of interest comprising delivering to said locus:
a) a CRISPR associated (Cas) polypeptide or a nucleic acid encoding the Cas polypeptide; and
b) a guide nucleic acid or DNA molecule encoding the guide nucleic acid, the guide nucleic acid comprising:
i) a first sequence that is capable of being bound by the Cas polypeptide, and
ii) a second sequence that hybridizes to a target sequence of a target nucleic acid, wherein the target sequence is a eukaryotic sequence,
wherein the target sequence is adjacent to a PAM of 5′-TTTN-3′, wherein T is thymine and N is any nucleotide.
88 . The method of claim 87 , wherein the Cas polypeptide recognizes a PAM of 5′-TTTR-3′, wherein T is thymine and R is selected from guanine and adenine.
89 . The method of claim 87 , wherein the Cas polypeptide is a variant of a polypeptide according to SEQ ID NO: 3 that has reduced nucleic acid cleavage activity relative to the polypeptide according to SEQ ID NO: 3.
90 . The method of claim 87 , wherein the Cas polypeptide is fused to a heterologous protein, and wherein the heterologous protein has an enzymatic activity selected from: DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.
91 . The method of claim 89 , wherein the Cas polypeptide is fused to a heterologous protein, and wherein modifying the target locus comprises introducing, removing, or altering an epigenetic modification of the target locus.
92 . The method of claim 89 , wherein the Cas polypeptide is linked to a methyltransferase.
93 . The method of claim 89 , wherein the Cas polypeptide comprises at least one amino acid substitution in a RuvC domain.
94 . The method of claim 87 , wherein the Cas polypeptide is catalytically inactive.
95 . The method of claim 87 , wherein the Cas polypeptide comprises at least one amino acid substitution at an aspartic acid residue or a glutamic acid residue relative to a protein that is 100% identical to SEQ ID NO: 3.
96 . The method of claim 95 , wherein the at least one amino acid substitution is located in a RuvC domain.
97 . The method of claim 87 , wherein the Cas polypeptide comprises at least one amino acid substitution that is selected from an amino acid corresponding to D405, E586 and D684 of SEQ ID NO: 39, and any combination thereof, wherein the amino acid sequence of the engineered polypeptide is aligned with SEQ ID NO: 39 for greatest percent identity.
98 . The method of claim 97 , comprising modifying the target locus of interest in a cell.
99 . The method of claim 98 , wherein the cell is a eukaryotic cell.
100 . The method of claim 99 , wherein the eukaryotic cell is selected from a stem cell, a germ cell, a fibroblast, an oligodendrocyte, a glial cell, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell, a myofibroblast, a cardiac myoblast, a skeletal myoblast, and a T cell.
101 . The method of claim 87 , comprising modifying the target locus of interest in a human subject.
102 . The method of claim 101 , comprising delivering the nucleic acid encoding the Cas polypeptide and the guide nucleic acid to the human subject via a lipid nanoparticle.
103 . The method of claim 102 , wherein the nucleic acid encoding the Cas polypeptide comprises a messenger RNA.
104 . The method of claim 103 , comprising delivering the nucleic acid encoding the Cas polypeptide and the DNA molecule encoding the guide nucleic acid to the human subject via an adeno-associated viral vector.
105 . The method of claim 87 , comprising delivering a donor nucleic acid to said locus.
106 . A composition comprising:
a) a Cas polypeptide or a nucleic acid encoding the Cas polypeptide; and
b) a guide nucleic acid or DNA molecule encoding the guide nucleic acid, the guide nucleic acid comprising:
i) a first sequence that is capable of being bound by the polypeptide, and
ii) a second sequence that hybridizes to a target sequence of a target nucleic acid, wherein the target sequence is a eukaryotic sequence,
wherein the target sequence is adjacent to a PAM of 5′-TTTN-3′, wherein T is thymine and N is any nucleotide.