Application of CRISPR-based technologies in non-model microorganisms is currently very limited. Here, we reported efficient genome engineering of an important industrial microorganism, Zymomonas mobilis, by repurposing the endogenous Type I-F CRISPR–Cas system upon its functional characterization. This toolkit included a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Through this toolkit, various genome engineering purposes were efficiently achieved, including knockout of ZMO0038 (100% efficiency), cas2/3 (100%), and a genomic fragment of >10 kb (50%), replacement of cas2/3 with mCherry gene (100%), in situ nucleotide substitution (100%) and His-tagging of ZMO0038 (100%), and multiplex gene deletion (18.75%) upon optimal donor size determination. Additionally, the Type I-F system was further applied for CRISPRi upon Cas2/3 depletion, which has been demonstrated to successfully silence the chromosomally integrated mCherry gene with its fluorescence intensity reduced by up to 88%. Moreover, we demonstrated that genome engineering efficiency could be improved under a restriction–modification (R–M) deficient background, suggesting the perturbance of genome editing by other co-existing DNA targeting modules such as the R–M system. This study might shed light on exploiting and improving CRISPR–Cas systems in other microorganisms for genome editing and metabolic engineering practices.

Establishment of production platforms through prokaryotic engineering in microbial organisms would be one of the most efficient means for chemicals, protein, and biofuels production. Despite the fact that CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies have readily emerged as powerful and versatile tools for genetic manipulations, their applications are generally limited in prokaryotes, possibly owing to the large size and severe cytotoxicity of the heterogeneous Cas (CRISPR-associated) effector. Nevertheless, the rich natural occurrence of CRISPR-Cas systems in many bacteria and most archaea holds great potential for endogenous CRISPR-based prokaryotic engineering. The endogenous CRISPR-Cas systems, with type I systems that constitute the most abundant and diverse group, would be repurposed as genetic manipulation tools once they are identified and characterized as functional in their native hosts. This article reviews the major progress made in understanding the mechanisms of invading DNA immunity by type I CRISPR-Cas and summarizes the practical applications of endogenous type I CRISPR-based toolkits for prokaryotic engineering.

Establishment of production platform organisms through prokaryotic engineering represents an efficient means to generate alternatives for yielding renewable biochemicals and biofuels from sustainable resources. Zymomonas mobilis, a natural facultative anaerobic ethanologen, possesses many attractive physiological attributes, making it an important industrial microorganism. To facilitate the broad applications of this strain for biorefinery, an efficient genome engineering toolkit for Z.mobilis was established in this study by repurposing the endogenous Type I-F CRISPR-Cas system upon its functional characterization, and further updated. This toolkit includes a series of genome engineering plasmids, each carrying an artificial self-targeting CRISPR and a donor DNA for the recovery of recombinants. Using the updated toolkit, genome engineering purposes were achieved with efficiencies of up to 100%, including knockout of cas3 gene, replacement of cas3 with the mCherry-encoding rfp gene, nucleotide substitutions in cas3, and deletion of two large genomic fragments up to 10 kb. This study established thus far the most efficient, straightforward and convenient genome engineering toolkit for Z. mobilis, and laid a foundation for further native CRISPRi studies in Z. mobilis, which extended the application scope of CRISPR-based technologies, and could also be applied to other industrial microorganisms with unexploited endogenous CRISPR-Cas systems.