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

Vo, Phuc Leo H ; Ronda, Carlotta ; Klompe, Sanne E ; Chen, Ethan E ; Acree, Christopher ; Wang, Harris H ; Sternberg, Samuel H

Nature biotechnology, 2021-04, Vol.39 (4), p.480-489 [Periódico revisado por pares]

United States: Nature Publishing Group

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Título:
CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering
Autor: Vo, Phuc Leo H ; Ronda, Carlotta ; Klompe, Sanne E ; Chen, Ethan E ; Acree, Christopher ; Wang, Harris H ; Sternberg, Samuel H
Assuntos: Bacteria ; Control ; CRISPR ; CRISPR-Cas Systems ; Deoxyribonucleic acid ; DNA ; DNA Transposable Elements ; Efficiency ; Gene Editing - methods ; Gene sequencing ; Gene therapy ; Genome, Bacterial ; Genomes ; Integrases ; 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
É parte de: Nature biotechnology, 2021-04, Vol.39 (4), p.480-489
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SourceType-Scholarly Journals-1
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P.L.H.V. and S.H.S. conceived of and designed the project, with input from C.R. and H.H.W. P.L.H.V. performed experiments and analyzed data for most E. coli experiments. C.R. performed experiments and analyzed data in K. oxytoca, P. putida and complex bacterial communities, with input from H.H.W. S.E.K. performed target immunity, ShoINT and random fragmentation NGS experiments. E.E.C. helped with cloning and transposition experiments. C.A. assisted with computational analyses of NGS data and the guide RNA design algorithm. P.L.H.V., S.H.S. and all other authors discussed the data and wrote the manuscript.
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Descrição: 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.
Editor: United States: Nature Publishing Group
Idioma: Inglês

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

Vo, Phuc Leo H ; Ronda, Carlotta ; Klompe, Sanne E ; Chen, Ethan E ; Acree, Christopher ; Wang, Harris H ; Sternberg, Samuel H

United States: Nature Publishing Group

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