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 of SEQ ID NO: 17,
wherein the variant consists of the following mutations:
substitution of aspartic acid at 10th position (D10) or histidine at 840th position (H840) of SEQ ID NO: 17 with alanine;
substitution of phenylalanine at 539th position (F539) of SEQ ID NO: 17 with serine;
substitution of methionine 763rd position (M763) of SEQ ID NO: 17 with isoleucine;
substitution of lysine 890th position (K890) of SEQ ID NO: 17 with asparagine; and
substitution of glutamic acid at 1007th position (E1007) of SEQ ID NO: 17 with one amino acid selected from the group consisting of alanine, asparagine, aspartic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
2 . The SpCas9 variant of claim 1 , wherein the SpCas9 variant comprises a NLS (nuclear localization sequence or signal).
3 . The SpCas9 variant of claim 1 , wherein the variant comprises the substitution of glutamic acid at 1007th position (E1007) of SEQ ID NO: 17 with one amino acid selected from leucine or proline.
4 . 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
a domain selected from the group consisting of domains having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.
5 . The SpCas9 variant of claim 4 , wherein the deaminase is a cytidine deaminase or adenine deaminase.
6 . A nucleic acid encoding the SpCas9 variant of claim 1 .
7 . A vector comprising the nucleic acid of claim 6 .
8 . A composition for gene modification in a cell, comprising:
the SpCas9 variant of claim 1 , or a nucleic acid encoding the SpCas9 variant; and
a guide RNA or a nucleic acid encoding the guide RNA;
wherein the guide RNA is capable of binding a target nucleic acid within DNA and complexing with the SpCas9 variant.
9 . The composition of claim 8 , wherein the SpCas9 variant comprises a NLS (nuclear localization sequence or signal).
10 . The composition of claim 8 , 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
a domain selected from the group consisting of domains having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity.
11 . The composition of claim 10 , wherein the deaminase is a cytidine deaminase or adenine deaminase.
12 . The composition of claim 8 , wherein the guide RNA and the SpCas9 variant form a complex of ribonucleoprotein (RNP).
13 . The composition of claim 8 , wherein the nucleic acid encoding the SpCas9 variant and the nucleic acid encoding the guide RNA are included in one or more vectors.