Enhancing multiple genome editing efficiency by employing CRISPR/Cas9 nickases in Erwinia billingiae QL-Z3

• Comparison of suicide plasmid vector system, CRISPR/Cas9 and CRISPR/Cas9n. • CRISPR-Cas9n in Erwinia billingiae QL-Z3. • 100 % efficiency in large genome deletion and multiple editing in many bacteria. • Take only 3.5 days to complete a genome editing cycle. Microbial genome editing is crucial for studying and optimizing enzyme functions, yet multiplex chromosomal editing remains challenging. In this study, we employed two Cas9 nickase (Cas9n) mutants, carrying D10A or H840A mutations, and systematically compared their editing efficiencies with wild-type (WT) Cas9 in Erwinia billingiae QL-Z3. While suicide plasmid-mediated knockout showed 1–4 % efficiency and WT Cas9 achieved 35–50 %, both Cas9n systems reached 100 % efficiency for single-gene deletions (0.6–7.4 kb) and insertions (0.7 kb). The dual-gene mutation approach maintained 100 % efficiency, and in triple-gene edits, pCas9n-H840A reached 75 % editing efficiency, whereas pCas9n-D10A consistently achieved 100 % efficiency and stability across knockouts (0.6–25 kb) in diverse bacteria. We further applied this system to delete three ligninolytic genes ( EDYP_48, ELAC_205, ESOD_1236 ), revealing that their disruption significantly reduced enzyme activity involved in the bioconversion of alkaline lignin. Further studies revealed that multiple gene deletions of EDYP_48, ELAC_205 inhibited ferulic acid consumption, while vanillic acid and protocatechuic acid accumulation suggested synergistic interactions among these enzymes and pathway components. Overall, the pCas9n-D10A mediated multiple gene editing emerged as an efficient and streamlined genome engineering strategy to reveal metabolic pathways, poised to accelerate the metabolic engineering for strain modification and cell factory construction in many bacteria.