Broad-host-range mutagenesis with CRISPR-associated transposase

Abstract

Transposons have been instrumental tools in microbiology enabling random mutagenesis, with transposons like Tn5 and Mariner, and site-specific DNA integrations with Tn7. However, programmable targeting of transposons was impossible until CRISPR-associated transposase (CasTn) systems were described. Like other CRISPR-derived systems, CasTn can be programmed with a short DNA encoded sequence that is transcribed into a guide-RNA. Here we describe a broad-host-range CasTn system and demonstrate its function in bacteria from three classes of the Proteobacteria. The CasTn genes are expressed from a broad-host-range replicative plasmid, while the guide-RNA and transposon are provided on a high-copy pUC plasmid that is suicidal in most bacteria outside of E. coli. Using our CasTn system, single-gene disruptions were performed with on-target efficiencies approaching 100% in the Beta- and Gammaproteobacteria, Burkholderia thailandensis, and Pseudomonas putida, respectively. The results were more modest in the Alphaproteobacterium Agrobacterium fabrum, with a peak efficiency of 45%, though for routine single-gene disruptions, this efficiency is adequate. In B. thailandensis, the system allowed simultaneous co-integration of transposons at two different target sites. The CasTn system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in P. putida. Given the iterative capabilities and large payload size, this system will be helpful for genome engineering experiments across several fields of research.

RNA-Guided DNA Transposition in Corynebacterium glutamicum and Bacillus subtilis

“…Recently, the successful deployment of VchCAST in Lactococcus lactis also consolidated our hypothesis. However, the transposition efficiency in these bacteria was less than 5%, as in Burkholderia thailandensis and Pseudomonas putida , 100% efficiency can only be achieved when enforcing positive selection. Hence, multiple or multiplexed genome editing using VchCAST is limited with such low efficiency or relying on selection markers.…”

A Targeted Genome-scale Overexpression Platform for Proteobacteria

“…Although several CAST systems have been described [24][25][26] , the Tn6677 system from Vibrio cholerae (VcCAST) is among the best characterized and most precise [27][28][29][30] . In VcCAST, a nuclease-deficient Type I-F CRISPR system is paired with a Tn7-like transposase to achieve guide RNA-directed transposition.…”