IP Library › Granted Patent US 12,227,776
Granted Patent B2
US 12,227,776 · App. 17/120,081 · Granted Feb 18, 2025

Engineered cascade components and cascade complexes

Inventors: Peter Sean Cameron (San Francisco, CA); Scott David Gradia (Albany, CA); Sanne Eveline Klompe (New York, NY); Samuel Henry Sternberg (New York, NY); Matthew Scott Thompson (San Francisco, CA)
Assignee: Caribou Biosciences, Inc.
C12N9/22C07K7/06C07K7/08C07K14/00C12N15/11C12N15/113C12N15/902C12N15/907C07K2319/00C07K2319/09C07K2319/21C07K2319/22C07K2319/50C12N2310/20C12N2800/80
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Quick Facts
Patent No.
US 12,227,776
App. No.
17/120,081
Granted
Feb 18, 2025
Kind
B2
Abstract

The present disclosure provides engineered Class 1 Type I CRISPR-Cas (Cascade) systems that comprise multi-protein effector complexes, nucleoprotein complexes comprising Type I CRISPR-Cas subunit proteins and nucleic acid guides, polynucleotides encoding Type I CRISPR-Cas subunit proteins, and guide polynucleotides. Also, disclosed are methods for making and using the engineered Class 1 Type I CRISPR-Cas systems of the present invention.

Claims (22)

1. An engineered Type I CRISPR-Cas effector composition comprising:

a Type I CRISPR-Cas subunit protein;

a Type I guide polynucleotide; and

an engineered Type I CRISPR mCas3 (mCas3) protein, wherein the engineered mCas3 protein is selected from the group consisting of Pseudomonas sp. S-6-2 mCas3 proteins D448A mCas3, D75A mCas3, and D448A D75A mCas3.

2. The engineered Type I CRISPR-Cas effector composition of claim 1 , wherein the Pseudomonas sp. S-6-2 mCas3 protein comprises SEQ ID NO: 1919.

3. The engineered Type I CRISPR-Cas effector composition of claim 1 further comprising a linker polypeptide covalently connecting the engineered mCas3 protein and the Type I CRISPR-Cas subunit protein.

4. The engineered Type I CRISPR-Cas effector composition of claim 1 , wherein the Type I CRISPR-Cas subunit protein is selected from the group consisting of a Cas8 protein, a Cas5 protein, and a Cas7 protein.

5. The engineered Type I CRISPR-Cas effector composition of claim 4 further comprising a Type I CRISPR Cas6 protein.

6. The engineered Type I CRISPR-Cas effector composition of claim 4 further comprising a Type I CRISPR Cse2 protein.

7. A cell comprising the engineered Type I CRISPR-Cas effector composition of claim 1 .

8. The cell of claim 7 further comprising a second engineered Type I CRISPR-Cas effector composition of claim 1 .

9. The cell of claim 7 further comprising a donor polynucleotide.

10. The cell of claim 7 , wherein the cell is a eukaryotic cell.

11. The eukaryotic cell of claim 10 wherein the cell is induced pluripotent stem cell.

12. A method of nicking double-stranded DNA (dsDNA) comprising:

contacting a first nick site in a first target region in the dsDNA with a first engineered Type I CRISPR-Cas effector composition of claim 1

thereby resulting in the first engineered Type I CRISPR-Cas effector composition binding the first target region and the first engineered mCas3 binding and nicking the dsDNA at the first nick site.

13. The method of claim 12 further comprising:

contacting a second nick site in a second target region in the dsDNA with a second engineered Type I CRISPR-Cas effector composition of claim 1 ;

thereby resulting in the second engineered Type I CRISPR-Cas effector composition binding the second target region and the second engineered mCas3 binding and nicking the dsDNA at the second nick site.

14. The method of claim 13 , wherein the distance between the first nick site and the second nick site on the dsDNA is between 1 and 120 base pairs.

15. The method of claim 12 , wherein the method is performed in vitro.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 18, 2020
From: CAMERON, PETER SEAN; GRADIA, SCOTT DAVID; THOMPSON, MATTHEW SCOTT; KLOMPE, SANNE EVELINE; STERNBERG, SAMUEL HENRY
To: CARIBOU BIOSCIENCES, INC.
Reel/Frame 054692/0203 →
Continuity (4)
Continuation PCTUS2019036864 · Jun 12, 2019
Provisional Application 62684735 · Jun 13, 2018
Provisional Application 62807717 · Feb 19, 2019
Related Publication 20210102183A1 · Apr 8, 2021
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