Modified CRISPR RNA and modified single CRISPR RNA and uses thereof
The present disclosure provides compounds comprising modified oligonucleotides for use in CRISPR. In certain embodiments, such modified oligonucleotides provide improved properties of crRNA. In certain embodiments, such modified oligonucleotides provide improved properties of scrRNA.
1. A modified crRNA consisting essentially of 29 to 32 nucleosides, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage, wherein:
a. a first portion of the crRNA is a DNA recognition portion, and
b. a second portion of the crRNA is a tracrRNA recognition portion having at least one nucleoside that comprises a bicyclic sugar moiety,
wherein
the DNA recognition portion is located 5′ of the tracrRNA recognition portion,
the seven 5′-terminal nucleosides of the DNA recognition portion comprise modified sugar moieties, at least one of which is a non-bicyclic sugar moiety,
the DNA recognition portion comprises at least 10 consecutive nucleosides comprising unmodified 2′-hydroxy sugar moieties at the 3′ end of the DNA recognition portion, and
the 5′ end of the tracrRNA recognition portion comprises the motif kr 3 wherein k is a cEt sugar moiety and r is an unmodified 2′-hydroxy sugar moiety.
2. The modified crRNA of claim 1 , wherein the non-bicyclic sugar moiety is selected from 2′-O-methyl and 2′-F.
3. The modified crRNA of claim 1 , wherein the modified sugar moieties of the seven 5′-terminal nucleosides are each independently selected from among 2′-O-methyl and 2′-F.
4. The modified crRNA of claim 1 , wherein the modified sugar moieties of the seven 5′-terminal nucleosides are each 2′-F.
5. The modified crRNA of claim 1 , wherein the tracrRNA recognition portion of the modified crRNA comprises at least 4 modified nucleosides.
6. The modified crRNA of claim 5 , wherein the at least 4 modified nucleosides each comprise a modified sugar moiety comprising a bicyclic sugar moiety.
7. The modified crRNA of claim 1 , wherein the 5′ end of the tracrRNA recognition portion comprising the kr 3 motif comprises the motif kr 3 kr 3 , wherein each k is a cEt sugar moiety and each r is an unmodified 2′-hydroxy sugar moiety.
8. The modified crRNA of claim 1 , wherein the seven 5′-terminal nucleosides of the DNA recognition portion comprise modified sugar moieties selected from among 2′-O-methyl and 2′-F and the tracrRNA recognition portion of the modified crRNA comprises at least 4 modified nucleosides selected from cEt and LNA.
9. The modified cRNA of claim 1 , wherein the modified crRNA consists essentially of 29 nucleosides.
10. A compound consisting essentially of the modified crRNA of claim 1 , and a conjugate group.
11. The compound of claim 10 , wherein the conjugate group is selected from a GalNAc moiety and a GalNAc cluster.
12. A pharmaceutical composition comprising the modified crRNA of claim 1 , a plasmid or a viral vector encoding a Cas9 protein, and a pharmaceutically acceptable carrier or diluent.