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

Area of Science:

• Microbiology
• Synthetic Biology
• Environmental Science

Background:

• *Alcaligenes faecalis* is vital for pollutant biodegradation and denitrification.
• Genetic engineering of *A. faecalis* is limited by inefficient tools and cytotoxicity of heterologous CRISPR-Cas systems.

Purpose of the Study:

• To develop a high-throughput genome editing toolkit for *A. faecalis*.
• To overcome limitations of existing genetic engineering methods in this bacterium.

Main Methods:

• Developed a genome editing toolkit utilizing the endogenous type I-F CRISPR-Cas system of *A. faecalis* J481.
• Engineered a *PheS*-mutant counterselection marker for rapid plasmid curing.
• Demonstrated efficient single-gene knockout and precise deletion of large genomic fragments (~47 kb).

Main Results:

• The toolkit enables efficient single-gene knockout and large genomic deletions, previously unattainable.
• Achieved two rounds of large-fragment removal within 5 days.
• Established the first CRISPR-based platform for complex genome engineering in *A. faecalis*.

Conclusions:

• This work provides a scalable genetic toolbox to enhance *A. faecalis* for bioremediation and eutrophication control.
• Harnessing endogenous CRISPR-Cas systems offers a blueprint for genome editing in other prokaryotes.
• Sets a precedent for developing advanced genetic tools in challenging-to-engineer microbes.