Genetically engineered cells
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 eukaryotic cell comprising:
a) a nuclease comprising an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 115;
b) a target nucleic acid comprising a target strand (TS) and a non-target strand (NTS); and
c) a recombinant guide nucleic acid comprising in a 5′ to 3′ orientation:
i) a first region that binds to the nuclease; and
ii) a second region that is complementary to a portion of the TS, wherein the target nucleic acid comprises a protospacer adjacent motif (PAM), wherein the PAM is on the NTS of the target nucleic acid, and wherein the PAM comprises the sequence 5′-VTTN-3′, wherein T is thymine, N is any nucleotide, and V is selected from adenine, guanine, and cytosine.
2. The eukaryotic cell of claim 1 , wherein the nuclease comprises an amino acid sequence that is at least 100% identical to the amino acid sequence of SEQ ID NO: 115.
3. The eukaryotic cell of claim 1 , wherein the first region of the recombinant guide nucleic acid comprises a nucleotide sequence that is at least 80% identical to SEQ ID NO: 175 and wherein the nuclease comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 115.
4. The eukaryotic cell of claim 1 , wherein the first region of the recombinant guide nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 175 and wherein the nuclease comprises an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 115.
5. The eukaryotic cell of claim 1 , wherein a nuclear localization signal (NLS) is fused via an amide bond to the N terminus of the nuclease, the C-terminus of the nuclease, or both termini of the nuclease.
6. The eukaryotic cell of claim 5 , wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO:50.
7. The eukaryotic cell of claim 1 , wherein said eukaryotic cell comprises a DNA donor template.
8. The eukaryotic cell of claim 1 , wherein the nuclease is fused to a heterologous polypeptide.
9. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a human cell, a mammalian cell, a hematopoietic stem cell, a T cell or a plant cell.
10. A population of cells comprising the eukaryotic cell of claim 1 .
11. The eukaryotic cell of claim 1 , wherein the second region is 14-20 nucleotides.
12. The eukaryotic cell of claim 1 , wherein the recombinant guide nucleic acid comprises at least one modified sugar moiety.
13. The eukaryotic cell of claim 12 , wherein the at least one modified sugar moiety is 2′-O-Methyl.
14. The eukaryotic cell of claim 1 , wherein the recombinant guide nucleic acid comprises a phosphorothioate linkage.
15. A eukaryotic cell comprising
A) a target nucleic acid comprising a target strand (TS) and a non-target strand (NTS),
wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) comprising the sequence 5′-VTTN-3′, wherein T is thymine, N is any nucleotide, and V is selected from adenine, guanine, and cytosine on the NTS; and
B) at least one nucleic acid molecule encoding:
a) a nuclease comprising an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 115; and
b) a recombinant guide nucleic acid comprising in a 5′ to 3′ orientation:
i) a first region that binds to the nuclease; and
ii) a second region that is complementary to a portion of the TS.
16. The eukaryotic cell of claim 15 wherein the second region is 14-20 nucleotides in length.
17. The eukaryotic cell of claim 15 , wherein the nucleic acid molecule is an expression vector.
18. The eukaryotic cell of claim 17 , wherein the expression vector is an adeno-associated viral vector.
19. The eukaryotic cell of claim 15 , wherein the eukaryotic cell comprises a DNA donor template.
20. The eukaryotic cell of claim 15 , wherein the nucleic acid molecule is associated with a lipid or liposome.
21. The eukaryotic cell of claim 15 , wherein the nuclease is fused to a heterologous polypeptide.
22. The eukaryotic cell of claim 15 , wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, a human cell, a T cell, a hematopoietic stem cell and a plant cell.
23. A population of cells comprising the eukaryotic cell of claim 15 .