Compositions and methods for genome engineering with Cas12a proteins
The present disclosure provides novel Cas12a proteins, which cleave target nucleic acids and methods of use thereof.
1. A composition comprising:
at least one of i)-ii):
i) a polypeptide comprising at least 80% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
ii) a polypeptide comprising at least 80% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3;
and
a guide RNA coupled to the at least one of i)-ii), wherein the guide RNA comprises a sequence that is reverse complementary to a eukaryotic nucleic acid sequence comprising from 6 to 60 bases.
2. The composition of claim 1 , wherein the polypeptide is a type V CRISPR-associated protein, optionally wherein the type V CRISPR-associated protein is a Cas12a protein.
3. The composition of claim 1 , wherein the polypeptide further comprises a purification tag.
4. The composition of claim 3 , wherein the polypeptide comprises at least one tag selected from the group consisting of a His tag, a FLAG tag, an AU1 epitope tag, an AU5 epitope tag, a bacteriophage T7 tag, a bacteriophage V5 epitope tag, a Bluetongue virus tag (B-tag), a Glu-Glu tag (EE-tag), an HSV epitope tag, a KT3 epitope tag, a Myc epitope tag, a PDZ ligand tag, a polyarginine tag, a polyaspartate tag, a polycysteine tag, a polyphenylalanine tag, a protein C tag, an S1-tag, an S-tag, a Step-tag, and a VSV-G tag.
5. The composition of claim 1 , wherein the guide RNA comprises an A-rich protospacer adjacent motif (PAM) sequence, a G-rich PAM sequence, a T-rich PAM sequence, or a C-rich PAM sequence.
6. The composition of claim 5 , wherein the PAM sequence comprises 3 T nucleobases, 2 T nucleobases, 1 T nucleobase, or TTTN.
7. The composition of claim 1 , wherein the composition exhibits at least 2-fold increased genome editing efficiency than AsCas12a, FnCas12a, or LbCas12a.
8. The composition of claim 1 , wherein the composition comprises:
the polypeptide comprising at least 85% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
the polypeptide comprising at least 85% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3.
9. The composition of claim 1 , wherein the composition comprises:
a polypeptide comprising at least 90% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
a polypeptide comprising at least 90% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3.
10. The composition of claim 1 , wherein the composition comprises:
a polypeptide comprising at least 95% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
a polypeptide comprising at least 95% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3.
11. A method of gene editing, wherein the method comprises:
providing a composition comprising: at least one of i)-ii):
i) a polypeptide comprising at least 80% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
ii) a polypeptide comprising at least 80% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; and
a guide RNA coupled to the at least one of i)-ii), wherein the guide RNA comprises a sequence that is reverse complementary to a eukaryotic nucleic acid sequence comprising from 6 to 60 bases; and
contacting a cell with the composition, thereby cleaving the eukaryotic nucleic acid sequence.
12. The method of claim 11 , wherein the polypeptide comprises at least one tag selected from the group consisting of: a His tag, a FLAG tag, an AU1 epitope tag, an AU5 epitope tag, a bacteriophage T7 tag, a bacteriophage V5 epitope tag, a Bluetongue virus tag (B-tag), a Glu-Glu tag (EE-tag), an HSV epitope tag, a KT3 epitope tag, a Myc epitope tag, a PDZ ligand tag, a polyarginine tag, a polyaspartate tag, a polycysteine tag, a polyphenylalanine tag, a protein C tag, an S1-tag, an S-tag, a Step-tag, and a VSV-G tag.
13. The method of claim 11 , wherein the guide RNA comprises an A-rich protospacer adjacent motif (PAM) sequence, a G-rich PAM sequence, a T-rich PAM sequence, or a C-rich PAM sequence.
14. The method of claim 11 , wherein the PAM sequence comprises 3 T nucleobases, 2 T nucleobases, 1 T nucleobase, or TTTN.
15. The method of claim 11 , wherein the composition exhibits at least 2-fold increased cleaving efficiency than AsCas12a, FnCas12a, or LbCas12a.
16. The method of claim 11 , wherein the composition comprises:
the polypeptide comprising at least 85% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
the polypeptide comprising at least 85% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3.
17. The method of claim 11 , wherein the composition comprises:
a polypeptide comprising at least 90% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
a polypeptide comprising at least 90% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3.
18. The method of claim 11 , wherein the composition comprises:
a polypeptide comprising at least 95% sequence identity with residue 825 through residue 996 of SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3; or
a polypeptide comprising at least 95% sequence identity with SEQ ID NO: 3, wherein the polypeptide comprises an amino acid sequence comprising a Lysine aligned to a Lysine at position 930 of SEQ ID NO: 3.