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

By P. Vo, Carlotta Ronda, Sanne E. Klompe, Ethan E. Chen, Christopher Acree, Harris H. Wang, S. H. Sternberg. Nature Biotechnology, 2020

311 citations recorded by the source corpus.

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. Optimized RNA-guided transposons efficiently integrate large DNA sequences in multiple bacterial species.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/de0e2dfb6cc79476168b76dd273d9351a921b392