Mutations that Enhance the DNA Cleavage Activity of Acidaminococcus Sp. CPF1
The present disclosure concerns polynucleotides and amino acids of Acidaminococcus sp. Cas12a (Cpf1) and methods for their use for genome editing in eukaryotic cells.
1 - 41 . (canceled)
42 . A method of performing therapeutic gene editing in a eukaryotic cell, comprising, introducing a CRISPR/Cas endonuclease system into the eukaryotic cell, wherein the CRISPR/Cas endonuclease system comprises:
i. a nucleic acid encoding a Cas12a variant, wherein the Cas12a variant comprises one or more substitutions selected from the group consisting of M537R, F870L, and M537R/F870L substitutions relative to the wild-type Cas12a sequence of SEQ ID NO: 462; and
ii. at least one guide RNA that hybridizes to a target sequence of a DNA molecule in a eukaryotic cell,
wherein the eukaryotic cell is selected from the group consisting of human cells, and non-human animal cells, further wherein the target sequence of the DNA molecule in the eukaryotic cell is edited.
43 . The method of claim 42 wherein the Cas12a variant further comprises at least one nuclear localization signal.
44 . The method of claim 42 wherein the Cas12a variant is a mutant Acidaminococcus sp. Cas12a (AsCas12a) variant, and/or wherein the CRISPR/Cas endonuclease system displays reduced off-target editing activity and maintains on-target editing activity relative to a wild-type CRISPR/Cas endonuclease system having the wild-type Cas 12a sequence of SEQ ID NO: 462.
45 . The method of claim 42 wherein the Cas12a variant comprises an amino acid sequence of SEQ ID NO.: 472,
46 . The method of claim 42 wherein the Cas12a variant comprises an amino acid sequence of SEQ ID NO.: 473, and
47 . The method of claim 42 wherein the Cas12a variant comprises an amino acid sequence of SEQ ID NO.: 465.
48 . The method of claim 42 wherein the nucleic acid is an mRNA encoding the Cas12a variant comprising one or more substitutions selected from the group consisting of M537R, F870L, and M537R/F870L substitutions relative to the wild-type Cas12a sequence of SEQ ID NO: 462.
49 . The method of claim 42 wherein the nucleic acid encoding the Cas12a variant comprises the nucleic acid sequence of SEQ ID NO: 466.
50 . The method of claim 42 wherein the nucleic acid encoding the Cas12a variant comprises the nucleic acid sequence of SEQ ID NO: 467.
51 . The method of claim 42 wherein the nucleic acid encoding the Cas12a variant comprises the nucleic acid sequence of SEQ ID NO: 489.
52 . The method of claim 42 wherein the nucleic acid encoding an isolated CRISPR-associated protein comprises the nucleic acid sequence of SEQ ID NO: 491.
53 . An mRNA encoding a Cas12a variant, wherein the Cas12a variant comprises one or more substitutions selected from the group consisting of M537R, F870L, and M537R/F870L substitutions relative to the wild-type Cas12a sequence of SEQ ID NO: 462.
54 . The mRNA of claim 53 wherein the Cas12a variant further comprises at least one nuclear localization signal.
55 . The mRNA of claim 53 wherein the Cas12a variant comprises an amino acid sequence of SEQ ID NO.: 472,
56 . The mRNA of claim 53 wherein the Cas12a variant comprises an amino acid sequence of SEQ ID NO.: 473, and
57 . The mRNA of claim 53 wherein the Cas12a variant comprises an amino acid sequence of SEQ ID NO.: 465.
58 . The mRNA of claim 53 wherein the Cas12a variant comprises the amino acid sequence of SEQ ID NO: 493.
59 . The mRNA of claim 53 wherein the Cas12a variant comprises the nucleic acid sequence of SEQ ID NO: 466.
60 . The mRNA of claim 53 wherein the Cas12a variant comprises the nucleic acid sequence of SEQ ID NO: 467.
61 . The mRNA of claim 53 wherein the Cas12a variant comprises the nucleic acid sequence of SEQ ID NO: 489.