Recombinant type I CRISPR-CAS system

Patent metadata

Field	Value
Publication number	US-11680259-B2
Application number	US-202015930678-A
Country	US
Kind code	B2
Filing date	May 13, 2020
Priority date	Oct 1, 2018
Publication date	Jun 20, 2023
Grant date	Jun 20, 2023

Abstract

This invention relates to recombinant Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays and recombinant nucleic acid constructs encoding Type I-E CASCADE complexes as well as plasmids, retroviruses and bacteriophage comprising the same.

First claim

That which is claimed is:

1. A ribonucleoprotein comprising a
(a) Type I-E CRISPR associated complex for antiviral defense complex (CASCADE complex) comprising a Cse1 polypeptide, a Cse2 polypeptide, a Cas7 polypeptide, a Cas5 polypeptide and a Cas6 polypeptide, and
(b) a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) array comprising two or more repeat sequences and one or more spacer sequence(s), wherein each of the one or more spacer sequences is linked at its 5′ end and at its 3′ end to a repeat sequence, and each of the one or more spacer sequences is complementary to one or more target sequences in a target DNA of a target organism, wherein the target DNA is located immediately adjacent to a protospacer adjacent motif (PAM), wherein the two or more repeat sequences comprise 20 to 28 consecutive nucleotides of a nucleotide sequences having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:1, 10, 19, 28, 37, 42, 51, or 60.

2. The ribonucleoprotein of claim 1 , wherein the Cse1 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:82, the Cse2 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:83, the Cas7 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:84, the Cas5 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:85, and the Cas6 polypeptide is encoded by the nucleotide sequence of SEQ ID NO:86.

3. The ribonucleoprotein of claim 1 , further comprising a Cas3 polypeptide.

4. The ribonucleoprotein of claim 3 , wherein the Cas3 polypeptide comprises the amino acid sequence of SEQ ID NO:119 or is encoded by the nucleotide sequence of SEQ ID NO:87.

5. The ribonucleoprotein of claim 1 , wherein the PAM comprises a nucleotide sequence of 5′-NAA-3′, 5′-AAA-3′ or 5′-AA-3′ that is immediately adjacent to and 5′ of the target sequence.

6. The ribonucleoprotein of claim 1 , wherein the spacer sequence is about 80 to 100% complementary to the target sequence.

7. The ribonucleoprotein of claim 1 , wherein the one or more spacer sequence(s) each have a length of about 25 nucleotides to about 40 nucleotides.

8. The ribonucleoprotein of claim 1 , wherein the one or more spacer sequence(s) each comprise a 5′ region and a 3′ region, wherein the 5′ region comprises a seed sequence and the 3′ region comprises a remaining portion of the one or more spacer sequence(s).

9. The ribonucleoprotein of claim 8 , wherein the seed sequence comprises the first 8 nucleotides of the 5′ end of each of the one or more spacer sequence(s), and is fully complementary to the target sequence, and the remaining portion of the one or more spacer sequence(s) is at least 80% complementary to the target sequence.

10. The ribonucleoprotein of claim 1 , wherein the target sequence is located in a gene, optionally in the sense or coding strand of the gene or in the antisense or non-coding strand of the gene.

11. The ribonucleoprotein of claim 10 , wherein the target sequence is located in an intragenic region of the gene, optionally located in the sense or coding strand of the gene or in the antisense or non-coding strand of the gene.

12. The ribonucleoprotein of claim 1 , wherein the target sequence is located in an intergenic region.

13. The ribonucleoprotein of claim 1 , wherein the target sequence is located on a chromosome, a mobile element, an extrachromosomal nucleic acid or a plasmid.

14. The ribonucleoprotein of claim 10 , wherein the gene encodes a transcription factor or a promoter.

15. The ribonucleoprotein of claim 10 , wherein the gene encodes non-coding RNA.

16. The ribonucleoprotein of claim 1 , wherein the target organism is a prokaryote or a eukaryote.

17. A recombinant cell comprising the ribonucleoprotein of claim 1 , wherein the cell is not a mammalian cell.

18. The recombinant cell of claim 17 , wherein the cell is a fungal cell, a plant cell, an insect cell, a bacterial cell, or an archaeon cell.

19. The recombinant cell of claim 17 , wherein the cell is a Lactobacillus spp. cell.

20. The recombinant cell of claim 19 , wherein the Lactobacillus spp. cell is a Lactobacillus crispatus cell.

21. An isolated recombinant mammalian cell comprising the ribonucleoprotein of claim 1 .

Assignees

• Univ North Carolina State

Inventors

• Barrangou Rodolphe
• Hidalgo-Cantabrana Claudio

Classifications

• C12N2795/00061

  Methods of inactivation or attenuation · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N1/04

  Preserving or maintaining viable microorganisms (immobilised microorganisms C12N11/00) · CPC title

• C12N15/03

  Bacteria · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title