Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus

Abstract

Significance Lactobacillus crispatus primarily inhabits 2 ecological niches: the human vagina and the poultry intestine. The predominance of this commensal species is indicative of a healthy status, and this species has become intriguing as an emerging probiotic. Historically, there has been a paucity of functional studies of L. crispatus due to its recalcitrance to transformation and limited molecular biology tools available. Here, we show how the endogenous type I-E CRISPR-Cas system of L. crispatus can be harnessed for flexible and efficient genetic engineering encompassing insertions, deletions, and single base substitutions. These findings expand the CRISPR toolbox with abundant and diverse type I CRISPR-Cas systems, and illustrate how endogenous systems can be harnessed to develop next-generation probiotics for human and animal health. CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5′-AAA-3′ protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.