Modified cells comprising CRISPR systems
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 98% identical to the amino acid sequence set forth in SEQ ID NO:120;
b) a target nucleic acid comprising a target strand and a non-target strand; 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 target strand of the target nucleic acid,
wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) of 5′-NTTN-3′, wherein T is thymine and N is any nucleotide,
and wherein the PAM is on the non-target strand of the target nucleic acid.
2. The eukaryotic cell of claim 1 , wherein the nuclease comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 120.
3. The eukaryotic cell of claim 1 , wherein the nuclease comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 120.
4. The eukaryotic cell of claim 1 , wherein the nuclease comprises at least one amino acid sequence selected from:
a)
EEACKKFVREN
b)
GPAIANIIAKSREFTEW
c)
LNLIIKNAVN
d)
VKVDNKNKNNL
e)
RKNEIAKLNGEQE
f)
GYLLQKPSPNKSIYCY
g)
PGYVPKWQY
h)
CVFDMRGLLRT
i)
TVDVGQNNP
j)
GEKFNCLKCG,
and
k)
VAITAQSMPKP.
5. 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:181.
6. 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:181.
7. 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.
8. The eukaryotic cell of claim 7 , wherein the NLS comprises the amino acid sequence set forth in SEQ ID NO: 49 or SEQ ID NO:50.
9. The eukaryotic cell of claim 1 , wherein said eukaryotic cell comprises a DNA donor template.
10. The eukaryotic cell of claim 1 , 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.
11. The eukaryotic cell of claim 1 , wherein the nuclease is fused to a heterologous polypeptide.
12. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a human cell.
13. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a mammalian cell.
14. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a hematopoietic stem cell.
15. The eukaryotic cell of claim 1 , wherein the eukaryotic cell is a T cell.
16. A population of cells comprising the eukaryotic cell of claim 1 .
17. A eukaryotic cell comprising
A) a target nucleic acid comprising a target strand and a non-target strand,
wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) of 5′-NTTN-3′, wherein T is thymine and N is any nucleotide,
and wherein the PAM is on the non-target strand of the target nucleic acid; and
B) at least one nucleic acid molecule encoding:
a) a nuclease comprising an amino acid sequence that is at least 98% identical to the amino acid sequence set forth in SEQ ID NO:120; 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 target strand of the target nucleic acid.
18. The eukaryotic cell of claim 17 , wherein the nuclease comprises an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO: 120.
19. The eukaryotic cell of claim 17 , wherein the nuclease comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO: 120.
20. The eukaryotic cell of claim 17 , wherein the nuclease comprises at least one amino acid sequence selected from:
(a)
EEACKKFVREN
(b)
GPAIANIIAKSREFTEW
(c)
LNLIIKNAVN
(d)
VKVDNKNKNNL
(e)
RKNEIAKLNGEQE
(f)
GYLLQKPSPNKSIYCY
(g)
PGYVPKWQY
(h)
CVFDMRGLLRT
(i)
TVDVGQNNP
(j)
GEKFNCLKCG,
and
(k)
VAITAQSMPKP.
21. The eukaryotic cell of claim 17 , wherein the nucleic acid molecule is an expression vector.
22. The eukaryotic cell of claim 21 , wherein the expression vector is an adeno-associated viral vector.
23. The eukaryotic cell of claim 17 , wherein the eukaryotic cell comprises a DNA donor template.
24. The eukaryotic cell of claim 17 , wherein the nucleic acid molecule is associated with a lipid or liposome.
25. The eukaryotic cell of claim 17 , wherein the nuclease is fused to a heterologous polypeptide.
26. The eukaryotic cell of claim 17 , wherein the eukaryotic cell is selected from the group consisting of a mammalian cell, a human cell, a T cell, and a hematopoietic stem cell.
27. A population of cells comprising the eukaryotic cell of claim 17 .
28. The eukaryotic cell of claim 1 , wherein the nuclease comprises a RuvC nuclease domain.
29. The eukaryotic cell of claim 1 , wherein the recombinant guide nucleic acid comprises at least one modified sugar moiety.
30. The eukaryotic cell of claim 29 , wherein the at least one modified sugar moiety is 2′-O-Methyl.
31. The eukaryotic cell of claim 1 , wherein the recombinant guide nucleic acid comprises a phosphorothioate linkage.
32. The eukaryotic cell of claim 17 , wherein the nuclease comprises a RuvC nuclease domain.