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

Abstract

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.

Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to Prokaryotic Engineering

“…Strikingly, the native type I-B system of C. pasteurianum facilitated more efficient genome editing of around 4-fold higher than that of the heterologously expressed Cas9 system (Pyne et al, 2016). Very recently, the type I-E system of Lactobacillus crispatus was also exploited to perform in situ genomic modifications in the host cells (Hidalgo-Cantabrana et al, 2019b).…”

A minimal CRISPR-Cas3 system for genome engineering

“…Type I systems are the most prevalent CRISPR-Cas systems in nature 1 , which has enabled the use of endogenous CRISPR-Cas3 systems for genetic manipulation via self-targeting. This has been accomplished in Pectobacterium atrosepticum (Type I-F) 16 , Sulfolobus islandicus (Type I-A) 17 , in various Clostridium species (Type I-B) 18–20 , and in Lactobacillus crispatus (Type I-E) 21 , leading to deletions as large as 97 kb amongst the self-targeted survivor cells 16 . Additionally, two recent studies have repurposed Type I systems for use in human cells, including the ribonucleoprotein (RNP) based delivery of a Type I-E system 22 , and the use of I-E and I-B systems for transcriptional modulation 23 .…”

Lactobacilli and other gastrointestinal microbiota of Peromyscus leucopus, reservoir host for agents of Lyme disease and other zoonoses in North America

“…The presence or absence of other genes or pathways that differentiated between the four species were an L-rhamnose biosynthesis pathway in one species, a luxS gene associated with a quorum sensing system in L. reuteri and L. johnsonii (41), a type 1 CRISPR-Cas3 array in “L. peromysci” (42), pathways for thiamine biosynthesis (43) and for reduction of nitrate (44) in three species, an arginine deiminase and its repressor in L. reuteri (45), and a type VII secretion system in L. animalis (46).…”