Abstract
•We develop a SHOT methodology for genome engineering of E. coli.•SHOT overcomes the shortcomings of CRISPR-Cas9 and ShCAST.•SHOT enhances on-target integration without off-target effects and co-integration.•SHOT enables ≈97% integration of 14.5 kb DNA and multiplexed integration.•We couple SHOT and CRISPRi to inhibit acid byproducts during protein production.
CRISPR-Cas9 induces double-strand break (DSB) for gene editing, but integration efficiency is low in some E. coli strains such as BL21(DE3). CRISPR-associated transposon from cyanobacterium Scytonema hofmanni (ShCAST) is a novel system comprising transposase and Cas12k for RNA-guided, DSB-free DNA transposition. However, ShCAST was developed in the specialized E. coli strain and its application is limited by off-target effect and plasmid DNA co-integration.
To overcome the drawbacks of ShCAST, we developed a ShCAST-based Optimized Transposition (SHOT) methodology by elevating Cas12k expression with an independent strong promoter, lowering the editing temperature and colony re-streaking.
SHOT enhanced on-target integration into sites difficult to edit by ShCAST without detectable off-target effects, and eliminated plasmid co-integration in BL21(DE3). SHOT enabled ≈97% integration of large DNA (≥14.5 kb) and multiplexed integration into difficult-to-edit loci, hence expediting generation of stable strains. We further leveraged SHOT to integrate a 10.3 kb CRISPR interference module into BL21(DE3) for multiplexed suppression of acid-producing genes, hence attenuating acid byproduct formation and increasing recombinant protein production. SHOT also facilitated DSB-free engineering of other difficult-to-edit E. coli and bacterial strains. Collectively, SHOT overcomes the shortcomings of CRISPR-Cas9 and ShCAST and facilitates genome engineering of difficult-to-edit E. coli strains.
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