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US 12,312,614 B2Granted
CRISPR-Cas effector polypeptides and methods of use thereof
AssigneeThe Regents of the University of California
InventorsJennifer A. Doudna, Jillian F. Banfield, Basem Al-Shayeb
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Abstract
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.
Claims16
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.
4 more elements
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.
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.
4 more elements
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.
3 more elements
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.
2 more elements
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.
1 more element
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.
3 more elements
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.
23 more elements
Dependencies are inferred from the claim text.