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 CRISPR-Cas effector polypeptide, or a nucleic acid molecule encoding the CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96; and
b) a CRISPR-Cas effector guide RNA, or one or more DNA molecules encoding the CRISPR-Cas effector guide RNA, wherein the CRISPR-Cas effector guide RNA comprises:
i) a first region that binds to the CRISPR-Cas effector polypeptide; and
ii) a second region that hybridizes to a target nucleic acid, wherein the first region is heterologous to the second region.
2. The composition of claim 1 , wherein the first region of the CRISPR-Cas effector guide RNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs:98-116 with T(s) substituted with U(s).
3. The composition of claim 1 , wherein the CRISPR-Cas effector polypeptide is fused to a nuclear localization signal (NLS).
4. The composition of claim 1 :
a) wherein the composition comprises a lipid;
b) wherein a) and b) are within a liposome;
c) wherein a) and b) are within a particle; or
d) wherein the composition comprises one or more of a buffer, a nuclease inhibitor, and a protease inhibitor.
5. The composition of claim 1 , further comprising a DNA donor template.
6. A CRISPR-Cas effector fusion polypeptide comprising: a CRISPR-Cas effector polypeptide fused to a heterologous polypeptide, wherein the CRISPR-Cas effector polypeptide comprises an amino acid sequence having the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96.
7. A nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector fusion polypeptide of claim 6 .
8. One or more nucleic acids comprising:
(a) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA, wherein the CRISPR-Cas effector guide RNA comprises:
i) a first region that binds to the CRISPR-Cas effector polypeptide; and
ii) a second region that hybridizes to a target nucleic acid, wherein first region is heterologous to the second region; and
(b) a nucleotide sequence encoding a CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96.
9. A eukaryotic cell comprising one or more of:
a) a CRISPR-Cas effector polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96;
b) a CRISPR-Cas effector fusion polypeptide, or a nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector fusion polypeptide, wherein the CRISPR-Cas effector polypeptide present in the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96; and
c) a CRISPR-Cas effector guide RNA, or a nucleic acid comprising a nucleotide sequence encoding the CRISPR-Cas effector guide RNA.
10. A method of modifying a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) a CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96; and
b) a CRISPR-Cas effector guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid,
wherein said contacting results in modification of the target nucleic acid by the CRISPR-Cas effector polypeptide.
11. A method of modulating transcription from a target DNA, modifying a target nucleic acid, or modifying a protein associated with a target nucleic acid, the method comprising contacting the target nucleic acid with:
a) a CRISPR-Cas effector fusion polypeptide comprising a CRISPR-Cas effector polypeptide fused to a heterologous polypeptide, wherein the CRISPR-Cas effector polypeptide present in the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96; and
b) a CRISPR-Cas effector guide RNA comprising a guide sequence that hybridizes to a target sequence of the target nucleic acid.
12. A transgenic, multicellular, non-human organism whose genome comprises a transgene comprising a nucleotide sequence encoding one or more of:
a) a CRISPR-Cas effector polypeptide, wherein the CRISPR-Cas effector polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96;
b) a CRISPR-Cas effector fusion polypeptide, wherein the CRISPR-Cas effector polypeptide present in the fusion polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96; and
c) a CRISPR-Cas effector guide RNA.
13. A system comprising one or more of:
a) a CRISPR-Cas effector polypeptide and a CRISPR-Cas effector guide RNA;
b) a CRISPR-Cas effector polypeptide, a CRISPR-Cas effector guide RNA, and a DNA donor template;
c) a CRISPR-Cas effector fusion polypeptide and a CRISPR-Cas effector guide RNA;
d) a CRISPR-Cas effector fusion polypeptide, a CRISPR-Cas effector guide RNA, and a DNA donor template;
e) an mRNA encoding a CRISPR-Cas effector polypeptide, and a CRISPR-Cas effector guide RNA;
f) an mRNA encoding a CRISPR-Cas effector polypeptide; a CRISPR-Cas effector guide RNA, and a DNA donor template;
g) an mRNA encoding a CRISPR-Cas effector fusion polypeptide, and a CRISPR-Cas effector guide RNA;
h) an mRNA encoding a CRISPR-Cas effector fusion polypeptide, a CRISPR-Cas effector guide RNA, and a DNA donor template;
i) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a CRISPR-Cas effector polypeptide; and ii) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA;
j) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a CRISPR-Cas effector polypeptide; ii) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA;
and iii) a DNA donor template;
k) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide; and ii) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; and
l) one or more recombinant expression vectors comprising: i) a nucleotide sequence encoding a CRISPR-Cas effector fusion polypeptide; ii) a nucleotide sequence encoding a CRISPR-Cas effector guide RNA; and a DNA donor template,
wherein, in any of (a)-(I), the CRISPR-Cas effector polypeptide, or the CRISPR-Cas effector polypeptide present in the fusion polypeptide, comprises an amino acid sequence the amino acid sequence set forth in any one of SEQ ID NOs: 51, 53, 55, 57, 59-62, 64, 66, 68, 70, 72-78, 80, 82, 84, 86, 88, 90, 92, 94, and 96.
14. A sterile container comprising the CRISPR-Cas effector system of claim 13 .
15. The sterile container of claim 14 , wherein the container is a syringe.
16. An implantable device comprising the CRISPR-Cas effector system of claim 13 .