Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems

Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems in bacteria and archaea employ CRISPR RNAs to specifically recognize the complementary DNA of foreign invaders, leading to sequence-specific cleavage or degradation of the target DNA. Recent work has shown that the accidental or intentional targeting of the bacterial genome is cytotoxic and can lead to cell death. Here, we have demonstrated that genome targeting with CRISPR-Cas systems can be employed for the sequence-specific and titratable removal of individual bacterial strains and species. Using the type I-E CRISPR-Cas system in Escherichia coli as a model, we found that this effect could be elicited using native or imported systems and was similarly potent regardless of the genomic location, strand, or transcriptional activity of the target sequence. Furthermore, the specificity of targeting with CRISPR RNAs could readily distinguish between even highly similar strains in pure or mixed cultures. Finally, varying the collection of delivered CRISPR RNAs could quantitatively control the relative number of individual strains within a mixed culture. Critically, the observed selectivity and programmability of bacterial removal would be virtually impossible with traditional antibiotics, bacteriophages, selectable markers, or tailored growth conditions. Once delivery challenges are addressed, we envision that this approach could offer a novel means to quantitatively control the composition of environmental and industrial microbial consortia and may open new avenues for the development of “smart” antibiotics that circumvent multidrug resistance and differentiate between pathogenic and beneficial microorganisms.

CRISPR-based screening of genomic island excision events in bacteria

“…S4 A and B) (25). Transformation with plasmids eliciting chromosomal self-targeting by CRISPR-Cas systems appeared cytotoxic as measured by the relative reduction in surviving transformants compared with non-self-targeting plasmids (15,29). Targeting the lacZ gene in S. thermophilus resulted in an ∼2.5-log reduction in recovered transformants (Fig.…”

“…Similarly, Vercoe et al (30) demonstrated that chromosomal targeting by a type-IF CRISPR-Cas system caused elimination of a horizontally acquired pathogenicity island in Pectobacterium atrosepticum (30). The concept of sequence-based removal of specific genotypes was developed further as a tool for manipulation of microbial consortia via CRISPR-Cas targeting, resulting in directed genome evolution at the population level (15). In accordance with previous work, our results demonstrate that wild-type clones were removed from the population, but mutants without CRISPR-Cas-targeted features survived.…”

“…Interference results from a corresponding transcript that is processed selectively at each repeat sequence, forming CRISPR RNAs (crRNAs) that guide Cas proteins for sequence-specific recognition and cleavage of target DNA complementary to the spacer (7). CRISPR-Cas technology has applications in strain typing and detection (8)(9)(10), exploitation of natural/engineered immunity against mobile genetic elements (11), programmable genome editing in diverse backgrounds (12), transcriptional control (13,14), and manipulation of microbial populations in defined consortia (15).…”

Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria

“…These studies, and another study (13), also showed that the transferred CRISPR-Cas system is capable of eliminating specific bacterial populations. Furthermore, they demonstrated that the system might be used against pathogens to effectively treat infected animals.…”

“…CRISPR-Cas systems have also recently been used to phenotypically correct genetic diseases in live animals (11), and their utility is being explored for various therapeutic approaches in mammals. Nevertheless, only limited studies have shown the use of CRISPR-Cas systems to target antibiotic resistance genes or a specific population of virulent bacterial strains (12)(13)(14)(15)(16)(17).…”