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 (i) a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1; (ii) an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1; and (iii) an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1, and 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 2 , wherein the SpCas9 variant further comprises a reporter protein, a NLS (nuclear localization sequence or signal), or a NES (nuclear export sequence or signal).
4. A composition for gene modification comprising a Cas9 and a guide RNA, wherein the Cas9 is a Streptococcus pyogenes Cas9 (SpCas9) variant comprising all of SEQ ID NO: 1 except for (i) a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1; (ii) an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1; and (iii) an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1, and wherein the guide RNA is complementary to a target gene.
5. The composition of claim 4 , wherein the guide RNA and the Cas9 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.