Methods and Compositions for Sequences Guiding Cas9 Targeting

• US 20170002339A1
• Filed: 01/23/2015
• Published: 01/05/2017
• Est. Priority Date: 01/24/2014
• Status: Active Grant

• Current Assignee: North Carolina State University (University of North Carolina System)
• Original Assignee: North Carolina State University (University of North Carolina System)
• Inventors: BARRANGOU, Rodolphe, SELLE, Kurt M., BRINER, Alexandra E.
• Granted Patent: US 10,787,654 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/00 Hydrolases acting on ester bonds (3.1)

Abstract

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

Claims

1. A synthetic trans-encoded CRISPR(tracr) nucleic acid (e.g., tracrRNA, tracrDNA) construct comprising from 5′

to 3′

,

an anti-zipper sequence comprising at least about three nucleotides;

a bulge sequence comprising at least about three nucleotides;

an anti-stitch sequence comprising a nucleotide sequence of NNANN;

a nexus sequence comprising a nucleotide sequence of TNANNC, T(A/C)A(A/G)(G/A)C, TCAAAC, TAAGGC, GATAAGG, GATAAGGCTT, TCAAG, TCAAGCAA, T(C/A)AA(A/C)(C/A)(A/G)(A/T), GATAAGGCCATGCC, TAAGGCTAGTCC, TCAAGCAAAGC, or TCAAACAAAGCTTCAGC, said nexus forming a hairpin structure comprising at least one double stranded stem; and

a hairpin sequence comprising a nucleotide sequence having at least one hairpin, said hairpin comprising 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.

4. A chimeric nucleic acid construct comprising:

(a) the synthetic tracr nucleic acid construct of claim 1 and

(b) a synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) construct comprising, from 3′

to 5′

, a zipper sequence comprising at least about three nucleotides, a bulge sequence comprising a nucleotide sequence having at least two nucleotides, a stitch sequence comprising a nucleotide sequence of NNTNN, a G_R1 comprising a nucleotide G or GTT, 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 bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the G_R1, and the G_R1 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 bulge sequence of the synthetic CRISPR nucleic acid construct and the bulge sequence of the synthetic CRISPR nucleic acid construct are non-complementary and the zipper sequence of the synthetic CRISPR nucleic acid construct is at least about 70% complementary to and is hybridized to the anti-zipper sequence of said synthetic tracr nucleic acid construct.

5. The chimeric nucleic acid construct of claim 4, further comprising a nucleotide sequence encoding an amino acid sequence having at least 70% identity to an amino acid sequence encoding a Cas9 nuclease.

6. The chimeric nucleic acid construct of claim 5, wherein the Cas9 nuclease is a Cas9 from a Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, the Cas9 nuclease is a Cas9 from Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, the Cas9 nuclease is a Cas9 nuclease from a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, or the Cas9 nuclease is a Cas9 nuclease from a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases.

7. The chimeric nucleic acid construct of claim 5, wherein the amino acid sequence encoding a Cas9 nuclease is selected from the group of amino acid sequences of SEQ ID NO:

1 to SEQ ID NO;

53, or any combination thereof.

8. The chimeric nucleic acid construct of claim 5, wherein the nexus sequence is GATAAGGC or GATAAGGCCATGCC and the Cas9 nuclease is from a Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases;

the nexus sequence is TAAGGC or TAAGGCTAGTCC and the Cas9 nuclease is from a Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases;

the nexus sequence is TCAAGC or TCAAGCAAAGC and the Cas9 nuclease is from a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases;

or the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC and the Cas9 nuclease is from a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases, wherein each nexus forms a hairpin structure comprising at least one double stranded stem.

9. The synthetic tracr nucleic acid construct, the synthetic CRISPR chimeric nucleic acid construct of claim 5, wherein the Cas9 nuclease comprises a mutation in the RuvC active site motif.

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

11. The chimeric nucleic acid construct of claim 5, 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.

12. An expression cassette comprising the chimeric nucleic acid construct of claim 4.

13. A cell comprising the expression cassette of claim 12.

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

contacting the chimeric nucleic acid construct of claim 4 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.

16. The method of claim 15, 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.

21. The method of claim 15, wherein the Cas9 nuclease is a Cas9 from a Streptococcus thermophilus CRISPR 1 (Sth CR1) group of Cas9 nucleases, the Cas9 nuclease is a Cas9 from Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases, the Cas9 nuclease is a Cas9 nuclease from a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases, or the Cas9 nuclease is a Cas9 nuclease from a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases.

22. The method of claim 15, wherein the amino acid sequence encoding a Cas9 nuclease is selected from the group of amino acid sequences of SEQ ID NO:

1 to SEQ ID NO;

53, or any combination thereof.

23. The method of claim 15, wherein the nexus sequence is GATAAGGC or GATAAGGCCATGCC and the Cas9 nuclease is from a Streptococcus thermophilus CRISPR 1 (STh CR1) group of Cas9 nucleases;

the nexus sequence is TAAGGC or TAAGGCTAGTCC and the Cas9 nuclease is from a Streptococcus thermophilus CRISPR 3 (Sth CR3) group of Cas9 nucleases;

the nexus sequence is TCAAGC or TCAAGCAAAGC and the Cas9 nuclease is from a Lactobacillus buchneri CD034 (Lb) group of Cas9 nucleases;

or the nexus sequence is TCAAAC or TCAAACAAAGCTTCAGC and the Cas9 nuclease is from a Lactobacillus rhamnosus GG (Lrh) group of Cas9 nucleases, wherein each nexus forms a hairpin structure comprising at least one double stranded stem.

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

contacting the chimeric nucleic acid construct of claim 11, thereby targeting the polypeptide of interest fused to the Cas9 to a specific site on the target DNA, said site defined by complementary hybridization of the spacer sequence to the target DNA.

26. The method of claim 15, wherein the PAM comprises the nucleotide sequence of GG, GGNG, AGAA, AAAA or GAA.

27. The method of claim 15, wherein the Cas9 nuclease is encoded by a nucleotide sequence that is codon optimized for an organism comprising the target DNA.

28. The method of claim 15, wherein the Cas9 nuclease comprises at least one nuclear localization sequence.

2. A synthetic CRISPR nucleic acid (e.g., crRNA, crDNA) construct comprising, from 3′

to 5′

, a zipper sequence comprising at least about three nucleotides, a bulge sequence comprising a nucleotide sequence having at least two nucleotides, a stitch sequence comprising a nucleotide sequence of NNTNN, a G_R1 comprising a nucleotide G or GTT, 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 bulge sequence, the bulge sequence is located immediately upstream of the stitch sequence, the stitch sequence is located immediately upstream of the G_R1, and the G_R1 is located immediately upstream of the spacer sequence.

3. A synthetic CRISPR nucleic acid array comprising, a nucleotide sequence encoding two or more CRISPR nucleic acid constructs of claim 2, wherein the two or more CRISPR nucleic acid constructs are located immediately adjacent to one another on said nucleotide sequence and the stitch sequences of said two or more CRISPR nucleic acid constructs are identical, and the spacer sequences of said two or more CRISPR nucleic acid constructs are identical or non-identical and the zipper sequences of said two or more CRISPR nucleic acid constructs are identical.

14. (canceled)

17-20. -20. (canceled)

25. (canceled)

29-50. -50. (canceled)