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

Abstract

Tn7-like transposons are pervasive mobile genetic elements in bacteria that mobilize using heteromeric transposase complexes comprising distinct targeting modules. We recently described a Tn7-like transposon from Vibrio cholerae that employs a Type I-F CRISPR-Cas system for RNA-guided transposition, in which Cascade directly recruits transposition proteins to integrate donor DNA downstream of genomic target sites complementary to CRISPR RNA. However, the requirement for multiple expression vectors and low overall integration efficiencies, particularly for large genetic payloads, hindered the practical utility of the transposon. Here, we present a significantly improved INTEGRATE (insertion of transposable elements by guide RNA-assisted targeting) system for targeted, multiplexed, and marker-free DNA integration of up to 10 kilobases at ~100% efficiency. Using multi-spacer CRISPR arrays, we achieved simultaneous multiplex insertions in three genomic loci, and facile multi-loci deletions when combining orthogonal integrases and recombinases. Finally, we demonstrated robust function in other biomedically- and industrially-relevant bacteria, and developed an accessible computational algorithm for guide RNA design. This work establishes INTEGRATE as a versatile and portable tool that enables multiplex and kilobase-scale genome engineering.

Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation

“…Lastly, transposaseassociated Type I-F systems have been shown to specifically insert synthetic "cargo" DNA up to 10 kb (kilobases) in length in E. coli with high fidelity, and thus holds promise as a technique to knock-in genes without requiring homologous recombination (Fig 4A)(66). Indeed, recent preprints have reported the use of transposaseassociated Type I-F systems for targeted insertion of antibiotic resistance genes in members of a microbial community, and multiplexed gene insertion in several medically and industrially important bacterial species (133,134).…”

The pIT5 Plasmid Series, an Improved Toolkit for Repeated Genome Integration in E. coli

“…Common methods for chromosomal integration in bacteria include lambda RED based homologous recombineering 5 , Tn7 based transposition 6 , and phage integrase mediated recombination [7][8][9] . In addition, variations of these methods have been reported that combine CRISPR-Cas based targeting for improved efficiency 10 .…”

Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase

“…AAVs, on the other hand, have fewer adverse effects, but their payload capacity is limited to <5 kb (22). DNA transposases, such as Tn7 combined with catalytically inactive Cas9 for RNA-guided site-specificity, have been developed recently to perform targeted transgenesis of up to 10 kb of DNA, it is limited to prokaryotes (23)(24)(25)(26). Further, transposases exhibit high off-target integration (27) requiring extensive engineering to achieve precise transgenesis in human cells, mice, and other animals.…”