Simultaneous Multiplex Genome Loci Editing of Halomonas bluephagenesis Using an Engineered CRISPR-guided Base Editor

Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various products. However, the simultaneous editing of multiple loci in H. bluephagenesis TD remains a significant challenge. Herein, we report the development of a multiple loci genome editing system, named CRISPR-deaminase-assisted base editor (CRISPR-BE) in H. bluephagenesis TD. This system comprises two components: a cytidine (CRISPR-cBE) and an adenosine (CRISPR-aBE) deaminase-based base editor. CRISPR-cBE can introduce a cytidine to thymidine mutation with an efficiency of up to 100% within a 7-nt editing window in H. bluephagenesis TD. Similarly, CRISPR-aBE demonstrates an efficiency of up to 100% in converting adenosine to guanosine mutation within a 7-nt editing window. CRISPR-cBE has been further validated and successfully employed for simultaneous multiplexed editing in H. bluephagenesis TD. Our findings reveal that CRISPR-cBE efficiently inactivated all six copies of the IS1086 gene simultaneously by introducing stop codon. This system achieved an editing efficiency of 100% and 41.67% in inactivating two genes and three genes, respectively. By substituting the P_cas promoter with the inducible promoter P_Mmp1, we optimized CRISPR-cBE system and ultimately achieved 100% editing efficiency in inactivating three genes. In conclusion, our research offers a robust and efficient method for concurrently modifying multiple loci in H. bluephagenesis TD, opening up vast possibilities for industrial applications in the future.