Crispr-based compositions and methods of use

Patent metadata

Field	Value
Publication number	US-2016177304-A1
Application number	US-201514975709-A
Country	US
Kind code	A1
Filing date	Dec 18, 2015
Priority date	Dec 18, 2014
Publication date	Jun 23, 2016
Grant date	—

Abstract

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 and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRIPSR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA 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.

First claim

What is claimed is:

1 . An isolated tracrRNA comprising a length-modified form of SEQ ID NO.:18, wherein the isolated tracrRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.

2 . The isolated tracrRNA of claim 1 , wherein the length-modified form of SEQ ID NO.:18 consists of a shortened form of SEQ ID NO.:18.

3 . The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of a member selected from a group consisting of the following:
SEQ ID NO.:18 lacking from 1 to 20 nucleotides at the 5′-end; SEQ ID NO.:18 lacking from 1-10 nucleotides at the 3′-end; and SEQ ID NO.:18 lacking from 1 to 20 nucleotides at the 5′-end and from 1-10 nucleotides at the 3′-end.

4 . The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of a member selected from a group consisting of SEQ ID NOs.: 2, 30-33 and 36-39.

5 . The isolated tracrRNA of claim 2 , wherein the shortened form of SEQ ID NO.:18 consists of SEQ ID NO.: 2 or 38.

6 . The isolated tracrRNA of claim 1 , further comprising at least one chemically-modified nucleotide.

7 . An isolated crRNA comprising a length-modified form of formula (I):
5′-X—Z-3′  (I),
wherein X represents sequences comprising a target-specific protospacer domain comprising about 20 universal nucleotides, and Z represents sequences comprising a tracrRNA-binding domain comprising about 20 nucleotides, wherein the isolated crRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.

8 . The isolated crRNA of claim 7 , wherein the length-modified form of formula (I) consists of a shortened form of formula (I).

9 . The isolated crRNA of claim 8 , wherein the shortened form of formula (I) consists of a member selected from a group consisting of the following:
formula (I) lacking from 1 to 8 nucleotides at the 3′-end of the Z domain; and formula (I) lacking nucleotides at the 5′-end of the X domain to accommodate a target-specific protospacer domain having 17, 18, 19 or 20 nucleotides.

10 . The isolated crRNA of claim 7 , further comprising at least one chemically-modified nucleotide.

11 . The isolated crRNA of claim 10 , wherein the length-modified form of formula (I) consists of SEQ ID NOs.:429-439.

12 . An isolated tracrRNA comprising a chemically-modified form of one of SEQ ID NOs.:2, 18, 30-33 and 36-39, wherein the isolated tracrRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.

13 . The isolated tracrRNA of claim 12 , wherein the chemically-modified form of one of SEQ ID NOs.:2, 18, 30-33 and 36-39 comprises a chemically-modified nucleotide having a modification selected from a group consisting of a ribose modification, an end-modifying group, and an internucleotide modifying linkage.

14 . The isolated tracrRNA of claim 13 , wherein the chemically-modified nucleotide having a modification consists of a ribose modification selected from a group consisting of 2′OMe, 2′F, a bicyclic nucleic acid and a locked nucleic acid (LNA).

15 . The isolated tracrRNA of claim 13 , wherein the chemically-modified nucleotide having a modification consists of an end-modifying group selected from a group consisting of a propanediol (C3) spacer, N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (“ZEN”), and an inverted-dT residue.

16 . The isolated tracrRNA of claim 13 , wherein the chemically-modified nucleotide having a modification consists of an internucleotide modifying linkage consisting of phosphorothioate modification.

17 . The isolated tracrRNA of claim 13 , wherein the isolated tracrRNA is selected from a group consisting of SEQ ID NOs.:100, 129, 130, 131, 132, 134, 136, 449 and 551.

18 . An isolated crRNA comprising a chemically-modified form of formula (I):
5′-X—Z-3′  (I),
wherein X represents sequences comprising a target-specific protospacer domain comprising from about 17 nucleotides to about 20 nucleotides and Z represents sequences comprising a tracrRNA-binding domain comprising from about 12 nucleotides about 19 nucleotides, wherein the isolated crRNA displays activity in a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) endonuclease system.

19 . The isolated crRNA of claim 18 , wherein the chemically-modified form of formula (I) comprises a chemically-modified nucleotide having a modification selected from a group consisting of a ribose modification, an end-modifying group, and an internucleotide modifying linkage.

20 . The isolated crRNA of claim 19 , wherein the chemically-modified nucleotide having a modification consists of a ribose modification selected from a group consisting of 2′OMe, 2′F, a bicyclic nucleic acid and locked nucleic acid (LNA).

21 . The isolated crRNA of claim 19 , wherein the chemically-modified nucleotide having a modification consists of an end-modifying group selected from a group consisting of a propanediol (C3) spacer, N,N-diethyl-4-(4-nitronaphthalen-1-ylazo)-phenylamine (“ZEN”), and an inverted-dT residue.

22 . The isolated tracrRNA of claim 19 , wherein the chemically-modified nucleotide having a modification consists of an internucleotide modifying linkage consisting of phosphorothioate modification.

23 . The isolated tracrRNA of claim 19 , wherein the chemically-modified form of formula (I) is selected from SEQ ID NOs.:429-439.

Assignees

• Integrated Dna Tech Inc

Inventors

• Collingwood Michael Allen
• Jacobi Ashley Mae
• Rettig Garrett Richard
• Schubert Mollie Sue
• Behlke Mark Aaron

Classifications

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2320/53

  reducing unwanted side-effects · CPC title

• C12N2320/51

  modulating the chemical stability, e.g. nuclease-resistance · CPC title

• C12N2310/531

  Stem-loop; Hairpin · CPC title

• C12N2310/346

  having a combination of backbone and sugar modifications · CPC title