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.
1 . A composition comprising:
a) a Cas12L polypeptide, or a nucleic acid molecule encoding the Cas12L polypeptide, wherein the Cas12L polypeptide comprises an amino acid sequence having 99% or more amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 102; and
b) a Cas12L guide RNA, or one or more DNA molecules encoding the Cas12L guide RNA, wherein the guide RNA is engineered such that the guide RNA's guide sequence is heterologous with the guide RNA's scaffold.
2 . The composition of claim 1 , wherein the Cas12L guide RNA comprises a nucleotide sequence having 80% or more nucleotide sequence identity with the crRNA sequence set forth as SEQ ID NO.
3 . The composition of claim 1 , wherein the Cas12L polypeptide is fused to a nuclear localization signal (NLS).
4 . The composition of claim 1 , wherein:
i) the composition comprises a lipid;
ii) a) and b) are within a liposome;
iii) a) and b) are within a particle; or
iv) the composition comprises one or more of: a buffer, a nuclease inhibitor, and a protease inhibitor.
5 . The composition of claim 1 , wherein the Cas12L polypeptide is a nickase that can cleave only one strand of a double-stranded target nucleic acid molecule.
6 . The composition of claim 1 , wherein the Cas12L polypeptide is a catalytically inactive Cas12L polypeptide (dCas12L).
7 . The composition of claim 1 , further comprising a DNA donor template.
8 . The composition of claim 1 , wherein the nucleic acid molecule encoding the Cas12L polypeptide is codon optimized such that the nucleotide sequence encoding the Cas12L polypeptide is non-naturally occurring.
9 . The composition of claim 1 , wherein the guide RNA comprises a guide sequence that is 100% complementary, over 17 or more continuous nucleotides, to a target sequence of a eukaryotic DNA.
10 . The composition of claim 1 , wherein the guide RNA comprises one or more base modifications, one or more backbone modifications, one or more modified sugar moieties, one or more non-natural internucleoside linkages, one or more polynucleotide mimetics, or any combination thereof.
11 . The composition of claim 1 , comprising the nucleic acid molecule encoding the Cas12L polypeptide, and/or the one or more DNA molecules encoding the Cas12L guide RNA.
12 . The composition of claim 11 , wherein the nucleic acid molecule encoding the Cas12L polypeptide comprises a codon optimized nucleotide sequence encoding the Cas12L polypeptide.
13 . One or more nucleic acids comprising a nucleotide sequence encoding the Cas12L guide RNA and a nucleotide sequence encoding the Cas12L polypeptide of claim 1 .
14 . The one or more nucleic acids of claim 13 , wherein the nucleotide sequence encoding the Cas12L polypeptide is non-naturally occurring because it is codon optimized.
15 . The one or more nucleic acids of claim 13 , wherein the nucleotide sequence encoding the Cas12L guide RNA is operably linked to a promoter that is functional in a eukaryotic cell, and/or the nucleotide sequence encoding the Cas12L polypeptide is operably linked to a promoter that is functional in a eukaryotic.
16 . The one or more nucleic acids of claim 13 , wherein the one or more nucleic acids is one or more recombinant expression vectors.
17 . The one or more nucleic acids of claim 16 , wherein the one or more recombinant expression vectors are one or more adenoassociated viral vectors, one or more recombinant retroviral vectors, or one or more recombinant lentiviral vectors.
18 . A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) a Cas12L polypeptide that comprises an amino acid sequence having 99% or more amino acid sequence identity with the amino acid sequence set forth in SEQ ID NO: 102; and
b) a Cas12L guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid, wherein the guide RNA is engineered such that the guide RNA's guide sequence is heterologous with the guide RNA's scaffold,
wherein said contacting results in modification of the target nucleic acid by the Cas12L polypeptide.
19 . The method of claim 18 , wherein said contacting comprises introducing a nucleic acid molecule encoding the Cas12L polypeptide and/or a DNA molecule encoding the Cas12L guide RNA into a cell.
20 . The method of claim 18 , wherein said contacting comprises introducing the Cas12L polypeptide and the Cas12L guide RNA into the cell as a ribonucleoprotein (RNP) that comprises the Cas12L polypeptide and the Cas12L guide RNA.