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

Nat Biotechnol. 2021 Apr;39(4):480-489. doi: 10.1038/s41587-020-00745-y. Epub 2020 Nov 23.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • CRISPR-Cas Systems
  • DNA Transposable Elements
  • Gene Editing / methods*
  • Genome, Bacterial
  • Plasmids / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Vibrio cholerae / genetics*

Substances

  • DNA Transposable Elements
  • RNA, Guide, CRISPR-Cas Systems