IP Library › Granted Patent US 12,331,292
Granted Patent B2
US 12,331,292 · App. 16/913,299 · Granted Jun 17, 2025

RNA-guided DNA integration using Tn7-like transposons

Inventors: Samuel Henry Sternberg (New York, NY); Sanne Eveline Klompe (New York, NY)
Assignee: The Trustees of Columbia University in the City of New York
C12N15/113C12N9/22C12N15/102C12N15/63C12N15/902C12N2310/20
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Quick Facts
Patent No.
US 12,331,292
App. No.
16/913,299
Granted
Jun 17, 2025
Kind
B2
Abstract

In certain embodiments, the present systems and methods use Tn7-like transposons that encode CRISPR-Cas systems for programmable, RNA-guided DNA integration. For example, the CRISPR-Cas machinery directs the Tn7 transposon-associated proteins to integrate DNA downstream of a target site (e.g., a genomic target site) recognized by a guide RNA (gRNA).

Claims (18)

1. A method for RNA-guided DNA integration comprising:

introducing into a cell: i) an engineered CRISPR-Cas system, and/or one or more vectors encoding the engineered CRISPR-Cas system, ii) an engineered transposon system, and/or one or more vectors encoding the engineered transposon system, and iii) a donor sequence comprising a cargo nucleic acid sequence and first and second transposon end sequences,

wherein, when one or more vectors are employed, the CRISPR-Cas system and the transposon system are on the same or different vector(s),

wherein the cell comprises a nucleic acid sequence with a target site,

wherein the CRISPR-Cas system comprises: (a) at least one Cas protein, and (b) a guide RNA (gRNA),

wherein the engineered CRISPR-Cas system is derived from a Type I CRISPR-Cas system,

wherein the CRISPR-Cas system binds to the target site,

wherein the donor sequence is integrated on the PAM-distal side of the target site, and

wherein the engineered transposon system and the engineered CRISPR-Cas system are derived from the same species.

2. The method of claim 1 , wherein the at least one Cas protein comprises Cas5, Cas6, Cas7, and Cas8.

3. The method of claim 1 , wherein the Type I CRISPR-Cas system is Type I-B or Type I-F.

4. The method of claim 3 , wherein the Type I CRISPR-Cas system is a Type I-F variant where Cas8 and Cas5 form a Cas8-Cas5 fusion protein.

5. The method of claim 1 , wherein the transposon system comprises TnsA, TnsB, and TnsC.

6. The method of claim 1 , wherein the transposon system is derived from a Tn7-like transposon system.

7. The method of claim 6 , wherein the Tn7-like transposon system is derived from Vibrio cholerae.

8. The method of claim 1 , wherein the transposon system comprises: i) TnsA, TnsB, and TnsC, and ii) TnsD and/or TniQ.

9. The method of claim 1 , wherein the method further comprises introducing into a cell a second engineered CRISPR-Cas system and a second engineered transposon system,

wherein the second engineered CRISPR-Cas system is from a Type V CRISPR-Cas system.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 15, 2020
From: STERNBERG, SAMUEL HENRY; KLOMPE, SANNE EVELINE
To: THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK
Reel/Frame 053777/0947 →
Continuity (11)
Continuation 16812138 · Mar 6, 2020
Provisional Application 62902171 · Sep 18, 2019
Provisional Application 62884600 · Aug 8, 2019
Provisional Application 62875772 · Jul 18, 2019
Provisional Application 62873455 · Jul 12, 2019
Provisional Application 62866270 · Jun 25, 2019
Provisional Application 62855814 · May 31, 2019
Provisional Application 62845218 · May 8, 2019
Provisional Application 62822544 · Mar 22, 2019
Provisional Application 62815187 · Mar 7, 2019
Related Publication 20200325474A1 · Oct 15, 2020
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