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.

细菌中千碱基大小 DNA 序列的位点特异性整合的现有技术受到效率低、对重组的依赖、需要多个载体以及多重化方面的挑战的限制。为了解决这些缺点，我们引入了先前报道的霍乱弧菌Tn 7样转座子的大幅改进版本，它使用 IF 型 CRISPR-Cas 系统进行可编程、RNA 引导的转座。通过引导 RNA 辅助靶向 (INTEGRATE) 系统优化转座元件的插入，可在细菌中以约 100% 的效率实现高达 10 kilobase 的高精度且无标记的 DNA 整合。使用多间隔区 CRISPR 阵列，我们通过结合正交整合酶和重组酶实现了三个基因组位点的同时多重插入和轻松的多位点删除。最后，我们在生物医学和工业相关细菌中展示了强大的功能，并在复杂的细菌群落中实现了目标和物种特异性整合。这项工作将 INTEGRATE 确立为多重千碱基级基因组工程的多功能工具。