CRISPR RNA targeting enzymes and systems and uses thereof
The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of a nucleic acid.
1. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated (Cas) system comprising:
(a) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence; and
(b) a CRISPR-Cas effector protein or a nucleic acid encoding the CRISPR-Cas effector protein, wherein the CRISPR-Cas effector protein comprises the amino acid sequence set forth in SEQ ID NO: 2,
wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to a target nucleic acid.
2. The system of claim 1 , wherein the direct repeat sequence comprises a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 72 or SEQ ID NO: 153.
3. The system of claim 2 , wherein the direct repeat sequence comprises the nucleotide sequence set forth in SEQ ID NO: 72 or SEQ ID NO: 153.
4. The system of claim 1 , wherein the spacer sequence comprises between 15 and 42 nucleotides in length.
5. The system of claim 1 , wherein the target nucleic acid is an RNA molecule.
6. The system of claim 1 , wherein the target nucleic acid comprises a sequence complementary to a nucleotide sequence in the spacer sequence.
7. The system of claim 1 , wherein the CRISPR-Cas effector protein cleaves the target nucleic acid.
8. The system of claim 1 , wherein the system further comprises an accessory protein.
9. The system of claim 8 , wherein the accessory protein comprises at least one WYL domain.
10. The system of claim 8 , wherein the accessory protein comprises at least one DNA binding domain.
11. The system of claim 8 , wherein the accessory protein comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 81.
12. The system of claim 11 , wherein the accessory protein comprises the amino acid sequence set forth in SEQ ID NO: 81.
13. The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES.
14. The system of claim 1 , wherein the CRISPR-Cas effector protein further comprises a peptide tag, a fluorescent protein, a base-editing domain, an RNA methyltransferase, an RNA demethylase, a splicing modifier, a localization factor, or a translation modification factor.
15. The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is codon-optimized for expression in a cell.
16. The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is operably linked to a promoter.
17. The system of claim 1 , wherein the nucleic acid encoding the CRISPR-Cas effector protein is in a vector.
18. The system of claim 17 , wherein the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.
19. The system of claim 1 , wherein the system is present in a delivery system comprising a nanoparticle, a liposome, an exosome, a microvesicle, or a gene-gun.
20. A cell comprising the system of claim 1 .
21. A method of binding the system of claim 1 to the target nucleic acid in a cell comprising:
(a) providing the system; and
(b) delivering the system to the cell,
wherein the cell comprises the target nucleic acid, wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to the target nucleic acid.