CRISPR/Cas9 Recombineering with Bacteriophage Recombinases Enables Efficient and Scarless Genome Editing in...

Cupriavidus necator H16 is a promising chassis for one-carbon (C1) biomanufacturing due to its ability to assimilate CO₂ with H₂-derived reducing power, yet genome engineering in this bacterium remains constrained by inefficient DNA delivery and low homologous recombination efficiency. Here, we establish a modular two-plasmid CRISPR/Cas9 recombineering system that decouples Cas9 expression from sgRNA turnover, incorporates a self-splicing intron riboswitch to tightly control Cas9 counterselection, and introduces a pair of bacteriophage recombinases, Che9c60 and Che9c61, to markedly enhance homologous recombination efficiency. With Che9c60 and Che9c61, the gene knockout efficiency increased from undetectable levels to 58.3% and reached 100% at certain loci. Importantly, introducing these two recombinases enabled scarless chromosomal knock-in by boosting insertion efficiency from undetectable levels to nearly 40%, and supported targeted integration of DNA payloads up to 4.0 kb. To facilitate rapid multiround engineering, we integrated a tdk-based counterselection marker into the sgRNA plasmid, reducing plasmid-retaining cells to 1.1% within 24 h under 5-fluoro-2'-deoxyuridine (FUDR) selection. Overall, this streamlined toolkit enables efficient, iterative, and scarless genome editing in C. necator, accelerating C. necator strain development for C1 metabolic engineering and industrial applications.