CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering

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 o...

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Bibliographic Details
Published in:Nature biotechnology 2021-04, Vol.39 (4), p.480-489
Main Authors: Vo, Phuc Leo H., Ronda, Carlotta, Klompe, Sanne E., Chen, Ethan E., Acree, Christopher, Wang, Harris H., Sternberg, Samuel H.
Format: Article
Language:English
Subjects:
631/337
631/61
Agriculture
Bacteria
Bioinformatics
Biomedical and Life Sciences
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Control
CRISPR
CRISPR-Cas Systems
Deoxyribonucleic acid
DNA
DNA Transposable Elements
Efficiency
Gene Editing - methods
Gene sequencing
Gene therapy
Genome editing
Genome, Bacterial
Genomes
Integrases
Life Sciences
Loci
Mathematical analysis
Methods
Microbial genetic engineering
Multiplexing
Nucleotide sequence
Plasmids - genetics
Recombination
Ribonucleic acid
RNA
RNA, Guide, CRISPR-Cas Systems - genetics
Transposition
Transposons
Vibrio cholerae - genetics
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Summary: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 Tn 7 -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. Optimized RNA-guided transposons efficiently integrate large DNA sequences in multiple bacterial species.
ISSN:1087-0156
1546-1696
DOI:10.1038/s41587-020-00745-y