CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering.
Journal
Nature biotechnology
ISSN: 1546-1696
Titre abrégé: Nat Biotechnol
Pays: United States
ID NLM: 9604648
Informations de publication
Date de publication:
04 2021
04 2021
Historique:
received:
24
03
2020
accepted:
20
10
2020
revised:
19
10
2020
pubmed:
25
11
2020
medline:
23
4
2021
entrez:
24
11
2020
Statut:
ppublish
Résumé
Existing technologies for site-specific integration of kilobase-sized DNA sequences in bacteria are limited by low efficiency, a reliance on recombination, the need for multiple vectors, and challenges in multiplexing. To address these shortcomings, we introduce a substantially improved version of our previously reported Tn7-like transposon from Vibrio cholerae, which uses a Type I-F CRISPR-Cas system for programmable, RNA-guided transposition. The optimized insertion of transposable elements by guide RNA-assisted targeting (INTEGRATE) system achieves highly accurate and marker-free DNA integration of up to 10 kilobases at ~100% efficiency in bacteria. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplexed insertions in three genomic loci and facile, multi-loci deletions by combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integration in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering.
Identifiants
pubmed: 33230293
doi: 10.1038/s41587-020-00745-y
pii: 10.1038/s41587-020-00745-y
pmc: PMC10583764
mid: NIHMS1935542
doi:
Substances chimiques
DNA Transposable Elements
0
RNA, Guide
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
480-489Subventions
Organisme : NIAID NIH HHS
ID : R01 AI132403
Pays : United States
Organisme : NIBIB NIH HHS
ID : R01 EB031935
Pays : United States
Organisme : NIGMS NIH HHS
ID : U01 GM110714
Pays : United States
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