CRISPR-BASED COMPOSITIONS AND METHODS OF USE
This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRISPR systems. The resultant length-modified and chemically-modified forms of crRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRIPSR Cas9 endonuclease system.
1 . An isolated crRNA comprising a length-modified and chemically modified form of formula (I):
5′-X—Z-3′ (I);
wherein X is a target-specific protospacer domain and Z is a tracrRNA-binding domain;
wherein the tracrRNA binding domain further comprises at least one chemically modified nucleotide;
and wherein the isolated crRNA is active in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein endonuclease system.
2 . The isolated crRNA of claim 1 , wherein the protospacer domain consists of 17,18, 19 or 20 nucleotides.
3 . The isolated crRNA of claim 1 , wherein the at least one chemically modified nucleotide is at or near the 3′ end.
4 . The isolated crRNA of claim 3 , wherein the at least one chemically modified nucleotide consists of a 2-O-Methyl modification, a phosphorothioate internucleotide linkage, a locked nucleic acid, or a combination thereof.
5 . The isolated crRNA of claim 1 , wherein the tracrRNA-binding domain is selected from the group consisting of SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43 and SEQ ID No. 44.
6 . A method of performing gene editing, comprising: contacting a candidate editing target site locus with an active CRISPR/Cas endonuclease system having a suitable crRNA;
wherein the crRNA has a tracrRNA binding domain; and
wherein the tracrRNA binding domain further comprises at least one chemically modified nucleotide.
7 . The method of claim 6 , wherein the tracrRNA binding domain is selected form the group consisting of SEQ ID No. 41, SEQ ID No. 42, SEQ ID No. 43 and SEQ ID No. 44.
8 . A method of performing gene editing, comprising: contacting a candidate editing target site locus in bacteria with an active CRISPR/Cas endonuclease system having a suitable crRNA;
wherein the crRNA has a tracrRNA binding domain; and
wherein the tracrRNA binding domain further comprises at least one chemically modified nucleotide.
9 . The method of claim 8 , wherein the tracrRNA binding domain is SEQ ID No. 46.