Unlocking genome engineering in Alcaligenes faecalis by exploiting its native type I-F CRISPR-Cas

ABSTRACT Alcaligenes faecalis is an environmentally significant bacterium for pollutant biodegradation and aerobic denitrification, yet its genetic engineering has been hindered by a lack of high-throughput tools. Conventional methods like homologous recombination are time-consuming and cannot achieve large genomic deletions, while technologies based on heterologous CRISPR-Cas systems failed due to cytotoxicity. This study resolves these limitations by developing a genome editing toolkit based on the endogenous type I-F CRISPR-Cas of A. faecalis J481. The toolkit enables efficient single-gene knockout and accomplishes the previously unattainable precise deletion of large genomic fragments. By engineering a PheS -mutant counterselection marker, we achieved rapid plasmid curing, allowing two rounds of large-fragment removal (~47 kb total) within 5 days. This breakthrough provides the first CRISPR-based platform for complex genome engineering in A. faecalis , overcoming intrinsic constraints of heterologous systems. The work establishes a scalable genetic toolbox to enhance A. faecalis ’ capabilities in bioremediation and eutrophication control. Moreover, the strategy of harnessing endogenous CRISPR-Cas systems offers a blueprint for developing advanced genome editing tools in other prokaryotes. IMPORTANCE This study breaks through the longstanding genetic engineering bottleneck in an environmentally crucial bacterium, Alcaligenes faecalis , by creating a fast, efficient, and versatile toolkit using its native CRISPR-Cas system. This enables complex edits, such as large genomic deletions previously impossible, unlocking new potential for bioremediation and eutrophication control, providing a blueprint for other prokaryotes, and setting a precedent for genetic tool development in other hard-to-engineer microbes.