CRISPR-转座酶 基因簇基因组多位点整合.pdfVIP

Articles /10.1038/s41587-020-00745-y CRISPR RNA-guided integrases for high-efficiency, multiplexed bacterial genome engineering 1 2 3 4 3 Phuc Leo H. Vo    , Carlotta Ronda , Sanne E. Klompe    , Ethan E. Chen    , Christopher Acree    , Harris H. Wang2,5 and Samuel H. Sternberg   3 ✉ 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 dem- onstrated robust function in biomedically and industrially relevant bacteria and achieved target- and species-specific integra- tion in a complex bacterial community. This work establishes INTEGRATE as a versatile tool for multiplexed, kilobase-scale genome engineering. NA technologies to stably integrate genes and pathways into (for example, antibi