RNA-guided DNA integration using Tn7-like transposons

Patent metadata

Field	Value
Publication number	US-10947534-B2
Application number	US-202016812138-A
Country	US
Kind code	B2
Filing date	Mar 6, 2020
Priority date	Mar 7, 2019
Publication date	Mar 16, 2021
Grant date	Mar 16, 2021

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).

First claim

What is claimed is:

1. A system for RNA-guided DNA integration, the system comprising one or more vectors heterologous to Vibrio cholerae encoding:
a) an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system, the engineered CRISPR-Cas system comprising: Cas5, Cas6, Cas7 and Cas8; and
b) an engineered transposon 7-like (Tn7-like) transposon system, the engineered Tn7-like transposon system comprising: i) Transposon 7 protein A (TnsA) TnsA, ii) Transposon 7 protein B (Tns B), iii) Transposon 7 protein C (Tns C), and iv) transposition of integron protein Q (TniQ),
wherein the engineered Tn7-like transposon system is derived from Vibrio cholerae Tn6677.

2. The system of claim 1 , wherein the CRISPR-cas system is a Type I-F CRISPR-cas system.

3. The system of claim 1 , wherein said CRISPR-cas system is a Type I-F variant where the Cas8 and Cas5 form a Cas8-Cas5 fusion.

4. The system of claim 1 , further comprising a guide RNA (gRNA), wherein the gRNA is specific for a target site.

5. The system of claim 1 , further comprising a donor DNA to be integrated, wherein the donor DNA comprises a cargo nucleic acid sequence and first and second transposon end sequences, wherein said cargo nucleic acid sequence is flanked by said first and second transposon end sequences, and wherein each of said first and second transposon end sequences comprises at least one TnsB binding site.

6. The system of claim 5 , wherein said first and second transposon end sequences are Tn7-like transposon end sequences.

7. The system of claim 1 , wherein the CRISPR-Cas system and the Tn7-like transposon system are on the same vector.

8. The system of claim 1 , wherein the engineered CRISPR-Cas system is nuclease-deficient.

9. The system of claim 1 , wherein said one or more vectors are plasmids.

Assignees

• Univ Columbia

Inventors

• Sternberg Samuel Henry
• Klompe Sanne Eveline

Classifications

• C12N2800/90

  Vectors containing a transposable element · CPC title

• C12N2310/20

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

• C12N15/90

  Stable introduction of foreign DNA into chromosome · CPC title

• C12N15/85

  for animal cells · CPC title

• C12N15/82

  for plant cells {, e.g. plant artificial chromosomes (PACs)} · CPC title