IP Library › Granted Patent US 12,644,138
Granted Patent B2
US 12,644,138 · App. 17/888,027 · Granted Jun 2, 2026

Dual guide CRISPR hybrid DNA/RNA polynucleotides and methods of use

Inventors: Andrew Paul May (San Francisco, CA); Paul Daniel Donohoue (Berkeley, CA)
Assignee: Caribou Biosciences, Inc.
C12N15/907C12N9/16C12N9/22C12N15/111C12N15/63C12N15/8213C12N15/902C12Q1/6827C12Y301/00C12N2310/20C12N2320/51C12N2320/53
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Quick Facts
Patent No.
US 12,644,138
App. No.
17/888,027
Granted
Jun 2, 2026
Kind
B2
Abstract

The present disclosure provides DNA-guided CRISPR systems; polynucleotides comprising DNA, RNA and mixtures thereof for use with CRISPR systems; and methods of use involving such polynucleotides and DNA-guided CRISPR systems.

Claims (16)

1 . A set of two Class 2 Type II CRISPR polynucleotides forming a dual guide Class 2 Type II polynucleotide, comprising:

(i) a first Class 2 Type II CRISPR polynucleotide comprising a targeting region comprising ribonucleic acid (RNA) and an activating region adjacent to said targeting region; and,

(ii) a second Class 2 Type II CRISPR polynucleotide comprising an activating region comprising a sequence that is complementary to a sequence in the activating region of the first Class 2 Type II CRISPR polynucleotide,

wherein the activating region of the first Class 2 Type II CRISPR polynucleotide and the activating region of the second Class 2 Type II CRISPR polynucleotide form a dual guide activating region comprising a mixture of RNA and deoxyribonucleic acid (DNA), wherein the two activating regions are hybridized to each other to form an activating duplex region,

wherein the activating duplex region comprises a bulge, and the second Class 2 Type II CRISPR polynucleotide further comprises a nexus 3′ to the activating region, and at least one hairpin 3′ to the nexus, and

wherein the activating duplex region is capable of binding with a Cas9 protein.

2 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the targeting region comprises a mixture of RNA and DNA.

3 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the at least one hairpin comprises a mixture of DNA and RNA.

4 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the at least one hairpin comprises a first hairpin 3′ to the nexus, and a second hairpin 3′ to the first hairpin, wherein at least one of said hairpins comprises a mixture of RNA and DNA.

5 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the targeting region comprises a mixture of RNA and DNA.

6 . The dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , wherein the targeting region of the first Class 2 Type II CRISPR polynucleotide comprises a compound selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphorotriesters, aminoalkylphosphosphorotriesters, alkylphosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3′-amino phosphoramidate, amino alkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates.

7 . A Class 2 Type II CRISPR complex comprising the dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , and a Cas9 protein.

8 . The Class 2 Type II CRISPR complex of claim 7 , wherein the Cas9 protein comprises a nuclear localization signal (NLS).

9 . A cell comprising the Class 2 Type II CRISPR complex of claim 7 .

10 . The cell of claim 9 , further comprising a donor polynucleotide.

11 . A Class 2 Type II CRISPR system comprising the dual guide Class 2 Type II CRISPR polynucleotide of claim 1 , and a donor polynucleotide.

Assignments (5)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 11, 2025
From: CARIBOU BIOSCIENCES, INC.
To: PIONEER HI-BRED INTERNATIONAL, INC.
Reel/Frame 072222/0136 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 11, 2025
From: PIONEER HI-BRED INTERNATIONAL, INC.
To: CARIBOU BIOSCIENCES, INC.
Reel/Frame 072222/0596 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 11, 2025
From: MAY, ANDREW PAUL; DONOHOUE, PAUL DANIEL
To: CARIBOU BIOSCIENCES, INC.
Reel/Frame 072857/0508 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 11, 2025
From: CARIBOU BIOSCIENCES, INC.
To: PIONEER HI-BRED INTERNATIONAL, INC.
Reel/Frame 072857/0537 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 11, 2025
From: MAY, ANDREW PAUL; DONOHOUE, PAUL DANIEL
To: CARIBOU BIOSCIENCES, INC.
Reel/Frame 072857/0543 →
Continuity (10)
Continuation 17208146 · Mar 22, 2021
Division 16941130 · Jul 28, 2020
Division 16670832 · Oct 31, 2019
Continuation 15845524 · Dec 18, 2017
Continuation 15679555 · Aug 17, 2017
Continuation 15493744 · Apr 21, 2017
Continuation 15008054 · Jan 27, 2016
Provisional Application 62251548 · Nov 5, 2015
Provisional Application 62108931 · Jan 28, 2015
Related Publication 20230193324A1 · Jun 22, 2023
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