Target-specific CRISPR mutant
The present invention relates to an artificially engineered CRISPR/Cas9 system. More particularly, the present invention relates to an artificially engineered CRISPR enzyme having enhanced target specificity and a use of an artificially engineered CRISPR/Cas9 system including the same enzyme in genome and/or epigenome manipulation or modification, genome targeting, genome editing, and in vitro diagnosis, etc.
1 . A SpCas9 ( Streptococcus pyogenes Cas9) variant comprising:
all of SEQ ID NO:1 except for:
an aspartic acid at the position corresponding to position 203 of the polypeptide of SEQ ID NO: 1;
a histidine at the position corresponding to position 277 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 366 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1;
an asparagine at the position corresponding to position 601 of the polypeptide of SEQ ID NO: 1;
an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1;
a tyrosine at the position corresponding to position 965 of the polypeptide of SEQ ID NO: 1;
a tyrosine at the position corresponding to position 1038 of the polypeptide of SEQ ID NO: 1;
a glutamic acid at the position corresponding to position 1127 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1, and an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1, and an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 1038 of the polypeptide of SEQ ID NO: 1;
an asparagine at the position corresponding to position 601 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 965 of the polypeptide of SEQ ID NO: 1;
an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1, and an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1;
an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 1038 of the polypeptide of SEQ ID NO: 1;
a tyrosine at the position corresponding to position 965 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 1038 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1, an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 1038 of the polypeptide of SEQ ID NO: 1;
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1, an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1, an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 965 of the polypeptide of SEQ ID NO: 1; or
a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1, an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1, an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1, and a tyrosine at the position corresponding to position 1038 of the polypeptide of SEQ ID NO: 1,
wherein the SpCas9 variant has an endonuclease activity.
2 . The SpCas9 variant of claim 1 , wherein the SpCas9 variant further comprises one or more functional domains selected from the group consisting of
a tag for isolation and purification;
a deaminase; and
an activity domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity.
3 . The SpCas9 variant of claim 1 , wherein the SpCas9 variant further comprises a reporter protein, a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
4 . A composition for gene modification, comprising:
the Streptococcus pyogenes Cas9 (SpCas9) variant of claim 1 , and
a guide RNA,
wherein the guide RNA is capable of targeting the interest gene and complexing with the SpCas9 variant.
5 . The composition of claim 4 , wherein the guide RNA and the SpCas9 variant form a complex which is a ribonucleoprotein (RNP).
6 . The composition of claim 4 , wherein the composition further comprises a donor nucleic acid that comprises a gene of interest.
7 . A composition for gene modification, comprising:
the Streptococcus pyogenes Cas9 (SpCas9) variant of claim 1 , and
a nucleic acid encoding a guide RNA.
8 . The composition of claim 7 , wherein the composition comprises a vector comprising the nucleic acid encoding the guide RNA.