Endogenous CRISPR-assisted microhomology-mediated end joining enables rapid genome editing in Zymomonas mobilis

Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required.

We developed a novel gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat-Spacer-Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500 bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a σ²⁸ deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem.

Here, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I-F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.