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 pharmaceutical composition comprising:
a) a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 120;
b) a recombinant guide RNA comprising:
i) a constant region comprising a first nucleotide sequence comprising at least 15 contiguous nucleotides of SEQ ID NO: 181; and
ii) a targeting region comprising a second nucleotide sequence that is complementary to a target sequence on a target strand (TS) of a double-stranded DNA molecule, wherein:
1) the double-stranded DNA molecule comprises a 5′-NTTN-3′ protospacer adjacent motif (PAM) sequence positioned immediately 5′ of the target sequence on a non-target strand (NTS) of the double stranded DNA molecule, wherein T is thymine and N is any nucleotide, and
2) a 3′ end of the first nucleotide sequence is linked to a 5′ end of the second nucleotide sequence; and
c) a pharmaceutically acceptable excipient.
151 . The pharmaceutical composition of claim 150 , comprising a lipid nanoparticle (LNP).
152 . The pharmaceutical composition of claim 151 , wherein the nucleic acid, recombinant guide RNA, or combination thereof is encapsulated in the LNP.
153 . The pharmaceutical composition of claim 150 , comprising a donor nucleic acid.
154 . The pharmaceutical composition of claim 150 , comprising an adeno associated viral (AAV) vector, wherein the AAV vector comprises a donor nucleic acid.
155 . The pharmaceutical composition of claim 150 , wherein the constant region comprises at least 20 contiguous nucleotides of SEQ ID NO: 181.
156 . The pharmaceutical composition of claim 150 , wherein the recombinant guide RNA comprises a nucleic acid modification.
157 . The pharmaceutical composition of claim 156 , wherein the nucleic acid modification improves the stability of the recombinant guide RNA in a biological fluid relative to the recombinant guide RNA without the nucleic acid modification.
158 . The pharmaceutical composition of claim 156 , wherein the nucleic acid modification is selected from: a 2′-O-methyl modified nucleotide, a 2′-Fluoro modified nucleotide, a locked nucleic acid (LNA) modified nucleotide, a peptide nucleic acid (PNA) modified nucleotide, a phosphorothioate linkage, and a 5′ cap.
159 . The pharmaceutical composition of claim 156 , wherein the nucleic acid modification comprises a phosphorothioate linkage between the last 3 to 5 nucleotides at the 5′- or 3′-end of the recombinant guide RNA.
160 . The pharmaceutical composition of claim 156 , comprising a nucleobase modification.
161 . The pharmaceutical composition, wherein the nucleic acid is a messenger RNA (mRNA).
162 . The pharmaceutical composition of claim 150 , wherein the nucleic acid encodes a fusion partner protein linked to the amino acid sequence that is at least 95% identical to SEQ ID NO: 120.
163 . The pharmaceutical composition of claim 162 , wherein the fusion partner protein comprises a deaminase.
164 . The pharmaceutical composition of claim 162 , wherein the fusion partner protein comprises a reverse transcriptase.
165 . The pharmaceutical composition of claim 162 , wherein the fusion partner protein has an enzymatic activity selected from: nuclease activity, methyltransferase activity, demethylase activity, 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.
166 . The pharmaceutical composition of claim 150 , wherein the nucleic acid encodes at least one nuclear localization signal (NLS).
167 . The pharmaceutical composition of claim 166 , wherein the nucleic acid encodes a first NLS at an N terminus of the polypeptide and a second NLS at the C terminus of the polypeptide.
168 . A method of administering the pharmaceutical composition of claim 150 to a subject, the method comprising administering the pharmaceutical composition to the subject.
169 . The method of claim 168 , comprising administering an AAV vector, wherein the AAV vector comprises a donor nucleic acid.