Multiplexed CRISPR-Cpf1-Mediated Genome Editing in Clostridium difficile toward the Understanding of Pathogenesis of C. difficile Infection

Clostridium difficile is often the primary cause of nosocomial diarrhea, leading to thousands of deaths annually worldwide. The availability of an efficient genome editing tool for C. difficile is essential to understanding its pathogenic mechanism and physiological behavior. Although CRISPR-Cas9 has been extensively employed for genome engineering in various organisms, large gene deletion and multiplex genome editing is still challenging in microorganisms with underdeveloped genetic engineering tools. Here, we describe a streamlined CRISPR-Cpf1-based toolkit to achieve precise deletions of fur, tetM, and ermB1/2 in C. difficile with high efficiencies. All of these genes are relevant to important phenotypes (including iron uptake, antibiotics resistance, and toxin production) as related to the pathogenesis of C. difficile infection (CDI). Furthermore, we were able to delete an extremely large locus of 49.2-kb comprising a phage genome ( phiCD630-2) and realized multiplex genome editing in a single conjugation with high efficiencies (simultaneous deletion of cwp66 and tcdA). Our work highlighted the first application of CRISPR-Cpf1 for multiplexed genome editing and extremely large gene deletion in C. difficile, which are both crucial for understanding the pathogenic mechanism of C. difficile and developing strategies to fight against CDI. In addition, for the DNA cloning, we developed a one-step-assembly protocol along with a Python-based algorithm for automatic primer design, shortening the time for plasmid construction to half that of conventional procedures. The approaches we developed herein are easily and broadly applicable to other microorganisms. Our results provide valuable guidance for establishing CRISPR-Cpf1 as a versatile genome engineering tool in prokaryotic cells.