IP Library › Granted Patent US 12,698,490
Granted Patent B2
US 12,698,490 · App. 17/002,133 · Granted Aug 4, 2026

Methods and compositions for sequences guiding Cas9 targeting

Inventors: Rodolphe Barrangou (Raleigh, NC); Kurt M. Selle (Raleigh, NC); Alexandra Briner Crawley (Cary, NC)
Assignee: NORTH CAROLINA STATE UNIVERSITY
C12N9/22C12N15/85C12Y301/00
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Quick Facts
Patent No.
US 12,698,490
App. No.
17/002,133
Filed
Aug 25, 2020
Granted
Aug 4, 2026
Kind
B2
Art Unit
1612
USPC
506/16
Abstract

The present invention is directed to methods and compositions for genome editing and DNA targeting of proteins.

Claims (33)

1 . A chimeric nucleic acid construct comprising (a) a synthetic trans-encoded CRISPR (tracr) nucleic acid construct and (b) a synthetic CRISPR nucleic acid construct,

the synthetic tracr nucleic acid construct comprising, from 5′ to 3′, (i) nucleotides 4 to 93 of SEQ ID NO:63, wherein the synthetic tracr nucleic acid construct comprises an anti-zipper sequence comprising at least four nucleotides; wherein the at least four nucleotides comprises nucleotides 22 -25 of SEQ ID NO:63; a bulge sequence comprising at least three nucleotides, wherein the at least three nucleotides comprises nucleotides 30-32 of SEQ ID NO:63; an anti-stitch sequence comprising nucleotides 33-39 of SEQ ID NO:63; a nexus sequence comprising the nucleotides 40-65 of SEQ ID NO: 63, wherein said nexus sequence forms a hairpin structure comprising a double stranded double stem; and a hairpin sequence comprising a stem having at least three matched base pairs,

wherein the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; and

the synthetic CRISPR nucleic acid construct comprising, from 3′ to 5′, a zipper sequence comprising at least four nucleotides, wherein the at least four nuleotides comprises the nucleotide sequence of GAUG, a stitch sequence comprising a nucleotide sequence of GACUCUG, and a spacer sequence having a 5′ end and a 3′ end and comprising at least seven nucleotides at its 3′ end having 100% complementarity to a target DNA, and the zipper sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the spacer sequence,

wherein the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and is hybridized to the anti-stitch sequence of said synthetic tracr nucleic acid construct and the zipper sequence of the synthetic CRISPR nucleic acid construct is at least about 90% complementary to and is hybridized to the anti-zipper sequence of said synthetic tracr nucleic acid construct,

wherein the chimeric nucleic acid construct is functional with a Cas9 nuclease having at least 90% identity to the amino acid sequence of SEQ ID NO:42.

2 . The chimeric nucleic acid construct of claim 1 , wherein the chimeric nucleic acid construct is capable of forming a nucleic acid-protein complex with a Cas9 nuclease having at least 90% identity to the amino acid sequence of SEQ ID NO:42.

3 . The chimeric nucleic acid construct of claim 2 , wherein the Cas9 nuclease is a Cas9 nuclease from a Lactobacillus rhamnosus (Lrh) group of Cas9 nucleases.

4 . The chimeric nucleic acid construct of claim 2 , wherein the Cas9 nuclease comprises a mutation in the RuvC active site motif.

5 . The chimeric nucleic acid construct of claim 2 , wherein the Cas9 nuclease comprises a mutation in the HNH active site motif.

6 . The chimeric nucleic acid construct of claim 2 , wherein the Cas9 nuclease comprises a mutation in the HNH active site motif and in the RuvC active site motif and is fused to a polypeptide of interest.

7 . An expression cassette encoding the chimeric nucleic acid construct of claim 1 .

8 . A cell comprising the expression cassette of claim 7 .

9 . A target nucleic acid modification system, comprising a chimeric nucleic acid construct comprising (a) a synthetic trans-encoded CRISPR (tracr) nucleic acid construct and (b) a synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) construct,

the synthetic tracr nucleic acid construct comprising, from 5′ to 3′, nucleotides 4 to 110 of SEQ ID NO:63, wherein the synthetic tracr nucleic acid construct comprises an anti-zipper sequence comprising at least four nucleotides, wherein the at least four nucleotides comprises nucleotides 22-25 of SEQ ID NO:63; a bulge sequence comprising at least three nucleotides, wherein the at least three nucleotides comprises nucleotides 30-32 of SEQ ID NO:63; an anti-stitch sequence comprising nucleotides 33-39 of SEQ ID NO:63; a nexus sequence comprising nucleotides 40-65 of SEQ ID NO: 63, wherein said nexus sequence forms a hairpin structure comprising a double stranded double stem; and a hairpin sequence comprising a stem having at least three matched base pairs, wherein the anti-zipper sequence is located immediately upstream of the bulge sequence, the bulge sequence is located immediately upstream of the anti-stitch sequence, the anti-stitch sequence is located immediately upstream of the nexus sequence, and the nexus sequence is located immediately upstream of the hairpin sequence; and

the synthetic CRISPR nucleic acid construct comprising, from 3′ to 5′, a zipper sequence comprising at least four nucleotides, wherein the at least four nucleotides comprises the nucleotide sequence of GAUG, a stitch sequence comprising a nucleotide sequence of GACUCUG, and a spacer sequence having a 5′ end and a 3′ end and comprising at least seven nucleotides at its 3′ end having 100% complementarity to a target DNA, and the zipper sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the spacer sequence,

wherein the stitch sequence of the synthetic CRISPR nucleic acid construct is 100% complementary to and is hybridized to the anti-stitch sequence of said synthetic tracr nucleic acid construct and the zipper sequence of the synthetic CRISPR nucleic acid construct is at least about 90% complementary to and is hybridized to the anti-zipper sequence of said synthetic tracr nucleic acid construct; and

(c) a Cas9 nuclease having at least 90% identity to the amino acid sequence of SEQ ID NO:42,

wherein the Cas9 nuclease binds to and is capable of forming a complex with the chimeric nucleic acid, and the spacer sequence of the synthetic CRISPR nucleic acid construct hybridizes to a portion of the target nucleic acid and adjacent to a protospacer adjacent motif (PAM) on the target nucleic acid, thereby the Cas9 nuclease is guided to the target nucleic acid and modifies the target nucleic acid.

10 . The system of claim 9 , wherein the Cas9 nuclease having at least 90% identity to the amino acid sequence of SEQ ID NO:42 is a Cas9 nuclease from a Lactobacillus gasseri (Lga) group of Cas9 nucleases.

11 . The system of claim 9 , wherein the Cas9 nuclease comprises a mutation in the RuvC active site motif and/or a mutation in the HNH active site motif.

12 . The system of claim 9 , wherein the nucleic acid modification is a site-specific cleavage of a double stranded target DNA.

13 . The system of claim 9 , wherein the Cas9 nuclease comprises a mutation in the HNH active site motif and in the RuvC active site motif and is fused to a polypeptide of interest.

14 . The system of claim 9 , wherein the PAM comprises the nucleotide sequence of GAAA, CCCC, CAAA, GAAC, GACC, CAAC, or GCCC.

15 . A target nucleic acid modification system, comprising a chimeric nucleic acid construct comprising (a) a synthetic trans-encoded CRISPR (tracr) nucleic acid construct comprising nucleotides 4 to 110 of SEQ ID NO:63; (b) a synthetic CRISPR nucleic acid (crRNA or crDNA) construct; and (c) a Cas9 nuclease having at least 90% identity to the amino acid sequence of SEQ ID NO:42.

16 . A method for site-specific cleavage of a double stranded target DNA, comprising:

contacting the chimeric nucleic acid construct of claim 1 with the target DNA in the presence of a Cas9 nuclease, and the spacer sequence of the synthetic CRISPR nucleic acid construct hybridizes to a portion of the target DNA and adjacent to a protospacer adjacent motif (PAM) on the target DNA, thereby producing a site-specific cleavage of the target DNA in a region defined by complementary hybridization of the spacer sequence to the target DNA.

17 . The method of claim 16 , wherein the site-specific cleavage is a site-specific nicking of a (+) strand of the double stranded target DNA and said Cas9 nuclease comprises a mutation in a RuvC active site motif, thereby cleaving the (+) strand of the double stranded target and producing a site-specific nick in said (+) strand the double stranded target DNA, or the site-specific cleavage is a site-specific nicking of the (−) strand of the double stranded target DNA and said Cas9 nuclease comprises a point mutation in a HNH active site motif, thereby cleaving the (−) strand of the double stranded target DNA and producing a site-specific nick in said (−) strand the double stranded target DNA.

18 . A method of site-specific targeting of a polypeptide of interest to a double stranded (ds) target DNA, comprising

contacting the target DNA with the chimeric nucleic acid construct of claim 6 , thereby targeting the polypeptide of interest fused to the Cas9 nuclease to a specific site on the target DNA, said site defined by complementary hybridization of the spacer sequence to the target DNA.

19 . The method of claim 18 , wherein the target DNA comprises a protospacer adjacent motif (PAM), said PAM comprising the nucleotide sequence of GAAA, CCCC, CAAA, GAAC, GACC, CAAC, or GCCC.

20 . The method of claim 18 , wherein the Cas9 nuclease is encoded by a nucleotide sequence that is codon optimized for an organism comprising the target DNA.

21 . The method of claim 18 , wherein the Cas9 nuclease comprises at least one nuclear localization sequence.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 26, 2020
From: BARRANGOU, RODOLPHE; SELLE, KURT M.; CRAWLEY, ALEXANDRA BRINER
To: NORTH CAROLINA STATE UNIVERSITY
Reel/Frame 053599/0875 →
Continuity (4)
Division 15113656 · Jan 23, 2015
Provisional Application 61986427 · Apr 30, 2014
Provisional Application 61931515 · Jan 24, 2014
Related Publication 20210017507A1 · Jan 21, 2021
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