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.
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NGS data are available in the NCBI Sequence Read Archive (BioProject accession code PRJNA668381). Published genomes used for analyses were obtained from the NCBI (accessions codes CP001509.3, U00096.3, CP009273.1 and AE015451.2). Datasets generated and analyzed in the current study, as well as custom scripts used for the described data analyses, are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Custom Python scripts used for the described NGS data analyses are available online via GitHub (https://github.com/sternberglab/Vo_etal_2020). The INTEGRATE guide RNA design tool and associated documentation are available online via GitHub (https://github.com/sternberglab/INTEGRATE-guide-RNA-tool).
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We thank N. Jaber for laboratory support, J. Bondy-Denomy for discussions, L.F. Landweber for qPCR instrument access, J. Mohabir for assistance with NGS read alignment, the JP Sulzberger Columbia Genome Center for NGS support and M.L. Smith, I. Oussenko and the Genomics Technology Laboratory at the Icahn School of Medicine at Mount Sinai for SMRT sequencing. H.H.W. acknowledges funding support for this work from the National Science Foundation (MCB-1453219), the National Institutes of Health (1U01GM110714 and 1R01AI132403), the Office of Naval Research (N00014-17-1-2353) and the Burroughs Wellcome Fund (PATH1016691). C.R. is supported by a Junior Fellows Scholarship from the Simons Society of Fellows. S.H.S. acknowledges a generous startup package from the Columbia University Irving Medical Center Dean’s Office and the Vagelos Precision Medicine Fund.
P.L.H.V., S.E.K. and S.H.S. are inventors on patents and patent applications related to CRISPR–Cas systems and uses thereof. H.H.W. is a scientific advisor to SNIPR Biome. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences and an equity holder in Dahlia Biosciences and Caribou Biosciences.
Peer review information Nature Biotechnology thanks Joseph Bondy-Denomy and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Vo, P.L.H., Ronda, C., Klompe, S.E. et al. CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering. Nat Biotechnol 39, 480–489 (2021). https://doi.org/10.1038/s41587-020-00745-y
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