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. The present disclosure provides methods of modulating transcription of a target nucleic acid.
1. A system for modifying a target double-stranded DNA, the system comprising:
a) a nucleic acid encoding a polypeptide, wherein the amino acid sequence of the polypeptide is at least 95% identical to SEQ ID NO: 120;
b) a guide RNA, or a DNA molecule encoding the guide RNA, comprising:
i) a first region that may be bound by the polypeptide; and
ii) a second region comprising a guide sequence,
wherein the first region is heterologous to the second region and wherein the first region is 5′ to the second region,
wherein the guide sequence hybridizes to a target sequence that is immediately adjacent to the 3′ end of a protospacer adjacent motif (PAM) of 5′-NTTN-3′, wherein T is thymine and N is any nucleotide
wherein the target double-stranded DNA comprises the target sequence and the PAM, wherein the PAM is on the non-target strand of the target double-stranded DNA.
2. The system of claim 1 , wherein the nucleic acid encoding the polypeptide is an expression vector.
3. The system of claim 2 , wherein the expression vector is an adeno-associated virus (AAV) vector.
4. The system of claim 1 , comprising the DNA molecule encoding the guide RNA, wherein the nucleic acid encoding the polypeptide and the DNA molecule are located on a single expression vector.
5. The system of claim 4 , wherein the single expression vector is an AAV vector.
6. The system of claim 1 , comprising a lipid nanoparticle.
7. The system of claim 6 , wherein the nucleic acid encoding the polypeptide is a messenger RNA (mRNA).
8. The system of claim 7 , wherein the lipid nanoparticle comprises the mRNA and the guide RNA.
9. The system of claim 8 , comprising an AAV vector, wherein the AAV vector comprises a donor template.
10. The system of claim 1 , comprising the DNA molecule encoding the guide RNA, wherein the nucleic acid encoding the polypeptide is located on a first expression vector and the DNA molecule is located on a second expression vector.
11. The system of claim 10 , wherein the first expression vector and the second expression vector are AAV vectors.
12. The system of claim 1 , comprising a donor nucleic acid.
13. The system of claim 1 , comprising an expression vector comprising a donor nucleic acid.
14. The system of claim 13 , wherein the expression vector is an AAV vector.
15. A method of editing a target sequence of a target nucleic acid with the system of claim 1 .
16. The method of claim 13 , wherein the target sequence is a eukaryotic sequence.
17. The method of claim 15 , wherein editing occurs in a cell.
18. An adeno-associated virus comprising the system of claim 5 , wherein the system comprises the nucleic acid encoding the polypeptide and the DNA molecule encoding the guide RNA.