CRISPR-CAS EFFECTOR POLYPEPTIDES AND METHODS OF USE THEREOF
The present disclosure provides RNA-guided CRISPR-Cas effector proteins, nucleic acids encoding same, and compositions comprising same. The present disclosure provides ribonucleoprotein complexes comprising: an RNA-guided CRISPR-Cas effector protein of the present disclosure; and a guide RNA. The present disclosure provides methods of modifying a target nucleic acid, using an RNA-guided CRISPR-Cas effector protein of the present disclosure and a guide RNA. The present disclosure provides methods of modulating transcription of a target nucleic acid.
1 .- 149 . (canceled)
150 . A composition comprising:
a) a nuclease comprising a single RuvC active site capable of both cleaving DNA and binding crRNA and wherein the amino terminus (N terminus) of the nuclease does not begin with the amino acid sequence “MIS”; and
b) a recombinant guide RNA.
151 . The composition of claim 150 , wherein the N terminus of the nuclease does not begin with the amino acid sequence “MISK”.
152 . The composition of claim 150 , wherein the nuclease comprises an amino acid sequence that is at least 50% identical to SEQ ID NO: 120.
153 . The composition of claim 150 , wherein the recombinant guide RNA comprises a nucleobase sequence that is at least 80% identical to SEQ ID NO: 15 or 139.
154 . The composition of claim 150 , wherein the recombinant guide RNA comprises a nucleobase sequence that is at least 95% identical to SEQ ID NO: 15 or 139.
155 . The composition of claim 150 , comprising a nuclear localization signal (NLS) that is fused to the N terminus of the nuclease, the C terminus of the nuclease, or both termini of the nuclease.
156 . The composition of claim 155 , wherein the NLS is fused directly to the N terminus of the nuclease via an amide bond, and wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 49.
157 . The composition of claim 155 , wherein the NLS is fused to the carboxyl terminus (C terminus) of the nuclease, and wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 50.
158 . The composition of claim 155 , wherein a first NLS comprising the amino acid sequence set forth in SEQ ID NO: 49 is fused to the N terminus of the nuclease via an amide bond and wherein a second NLS comprising the amino acid sequence set forth in SEQ ID NO: 50 is fused to the C terminus of the nuclease.
159 . The composition of claim 150 , wherein the nuclease is a nickase or a catalytically inactive nuclease.
160 . The composition of claim 150 , comprising a DNA donor template, an additional recombinant guide RNA, or a combination thereof.
161 . The composition of claim 150 , wherein the nuclease recognizes a TTN protospacer adjacent motif, wherein T is thymine and N is any nucleotide.
162 . The composition of claim 150 , comprising a lipid, a liposome, a vector, or a particle.
163 . The composition of claim 150 , comprising one or more of: a buffer, a nuclease inhibitor, and a protease inhibitor.
164 . A pharmaceutical composition comprising the composition of claim 150 and a pharmaceutically acceptable excipient.
165 . The composition of claim 150 , comprising a detectable label.
166 . A method of editing a gene comprising contacting a cell comprising the gene with the composition of claim 150 .
167 . A population of cells modified by the composition of claim 150 .
168 . A method of producing a recombinant protein, comprising culturing the population of cells of claim 167 .
169 . A cell comprising the composition of claim 150 .