Class 2 CRISPR/Cas compositions and methods of use
Provided are compositions and methods that include one or more of: (1) a Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the effector protein, and/or a modified host cell comprising the effector protein (and/or a nucleic acid encoding the same); (2) a CRISPR/Cas guide RNA that binds to and provides sequence specificity to the Class 2 CRISPR/Cas effector protein, a nucleic acid encoding the CRISPR/Cas guide RNA, and/or a modified host cell comprising the CRISPR/Cas guide RNA (and/or a nucleic acid encoding the same); and (3) a CRISPR/Cas transactivating noncoding RNA (trancRNA), a nucleic acid encoding the CRISPR/Cas trancRNA, and/or a modified host cell comprising the CRISPR/Cas trancRNA (and/or a nucleic acid encoding the same).
1. A method of guiding a CasY protein to a target sequence of a target nucleic acid,
the method comprising contacting the target nucleic acid with an engineered and/or non-naturally occurring complex comprising:
(a) a CasY protein;
(b) a CasY guide RNA that comprises a guide sequence that hybridizes to a target sequence of the target nucleic acid, and comprises a region that binds to the CasY protein; and
(c) a CasY transactivating noncoding RNA (CasYtrancRNA);
wherein:
(i) the CasY protein comprises an amino acid sequence having 95% or more identity with the CasYI sequence of SEQ ID NO: 1, and the CasY trancRNA comprises a nucleotide sequence having 98% or more identity with the CasYI trancRNA sequence of SEQ ID NO: 245; or
(ii) the CasY protein comprises an amino acid sequence having 95% or more identity with the CasY2 sequence of SEQ ID NO: 2, and the CasYtrancRNA comprises a nucleotide sequence having 98% or more identity with the CasY2 trancRNA sequence of SEQ ID NO: 246; or
(iii) the CasY protein comprises an amino acid sequence having 95% or more identity with the CasY4 sequence of SEQ ID NO: 4, and the CasYtrancRNA comprises a nucleotide sequence having 98% or more identity with the CasY4 trancRNA sequence of SEQ ID NO: 247.
2. The method of claim 1 , wherein the method results in modification of the target nucleic acid, modulation of transcription from the target nucleic acid, or modification of a polypeptide associated with a target nucleic acid.
3. The method of claim 2 , wherein the target nucleic acid is modified by being cleaved.
4. The method of claim 1 , wherein the target nucleic acid is selected from: double stranded DNA, single stranded DNA, RNA, genomic DNA, and extrachromosomal DNA.
5. The method of claim 1 , wherein the guide sequence and the region that binds to the CasY protein are heterologous to one another.
6. The method of claim 1 , wherein said contacting results in genome editing.
7. The method of claim 1 , wherein said contacting takes place outside of a bacterial cell and outside of an archaeal cell.
8. The method of claim 1 , wherein said contacting takes place in vitro outside of a cell.
9. The method of claim 1 , wherein said contacting takes place inside of a target cell.
10. The method of claim 9 , wherein said contacting comprises: introducing into the target cell at least one of:
(a) the CasY protein, or a nucleic acid encoding the CasY protein;
(b) the CasY guide RNA, or a nucleic acid encoding the CasY guide RNA; and
(c) the CasY trancRNA, or a nucleic acid encoding the CasY trancRNA.
11. The method of claim 10 , wherein the nucleic acid encoding the protein is a non-naturally sequence that is codon optimized for expression in the target cell.
12. The method of claim 9 , wherein the target cell is a eukaryotic cell.
13. The method of claim 9 , wherein the target cell is in culture in vitro.
14. The method of claim 9 , wherein the target cell is in vivo.
15. The method of claim 9 , wherein the target cell is ex vivo.
16. The method of claim 12 , wherein the eukaryotic cell is a plant cell, a fungal cell, a single cell eukaryotic organism, a mammalian cell, a reptile cell, an insect cell, an avian cell, a fish cell, a parasite cell, an arthropod cell, a cell of an invertebrate, a cell of a vertebrate, a rodent cell, a mouse cell, a rat cell, a primate cell, a non-human primate cell, or a human cell.
17. The method of claim 1 , wherein said contacting further comprises: introducing a DNA donor template into the target cell.
18. The method of claim 1 , wherein
(i) the CasY protein comprises an amino acid sequence having the CasYI sequence of SEQ ID NO: 1, and the CasY trancRNA comprises a nucleotide sequence having the CasYI trancRNA sequence of SEQ ID NO: 245;
(ii) the CasY protein comprises an amino acid sequence having the CasY2 sequence of SEQ ID NO: 2, and the CasY trancRNA comprises a nucleotide sequence having the CasY2 trancRNA sequence of SEQ ID NO: 246; or
(iii) the CasY protein comprises an amino acid sequence having the CasY4 sequence of SEQ ID NO: 4, and the CasYtrancRNA comprises a nucleotide sequence having the CasY4 trancRNA sequence of SEQ ID NO: 247.