A conjugative Clustered Regularly Interspaced Short Palindromic Repeats–based gene drive–like system reduced ampicillin-resistant colony-forming units by three to five logs in bacterial populations, with enhanced effects in recipient strains lacking RecA, according to a proof-of-principle study published in npj Antimicrobials and Resistance.
In the study, researchers engineered an approximately 65-kilobase plasmid, pPro-MobV, that integrates Prokaryotic-Active Genetics (Pro-AG) components with the IncP RK2 conjugative system to enable horizontal transfer of an anti–antibiotic resistance cassette. In liquid conjugation assays using Escherichia coli donor and recipient strains, transfer occurred at a frequency of approximately 40% after 72 hours.
Following conjugation, transconjugants harboring both the pPro-MobV plasmid and the pETas target plasmid were selected overnight on low-salt Luria-Bertani (LSLB) agar plates containing ampicillin, spectinomycin, and chloramphenicol. Individual colonies were regrown in LSLB either without arabinose or with arabinose to induce the lambda Red–Cas9 fusion operon, followed by plating on either ampicillin- or spectinomycin-containing agar plates for colony-forming units (CFU) enumeration.
Induction was associated with an approximately 1,000-fold decrease in CFU on ampicillin plates and an approximately 100-fold recovery on spectinomycin plates. This effect was approximately tenfold greater than that observed with the smaller consolidated intermediate pPro-AG plasmid carrying the same pBBR1 origin of replication, the researchers reported.
DNA sequencing of spectinomycin-resistant, ampicillin-sensitive colonies showed that approximately 20% contained the anticipated precise single-guide RNA insertion disrupting the bla gene. The remaining approximately 80% instead carried an approximately 1.2-kilobase deletion of pETas plasmid sequences that removed the entire coding region of the bla gene.
The researchers designated this deletion mechanism homology-based deletion. Homology-based deletion required Cas9-mediated cleavage and direct repeats of approximately 25 to 100 base pairs and was markedly enhanced by lambda Red. Sequencing of individual spectinomycin-resistant, ampicillin-sensitive colonies recovered following arabinose induction showed that 25% contained precise single-guide RNA insertional events, whereas 75% exhibited homology-based deletion events.
In recipient strains lacking RecA function, induction was associated with an approximately 100,000-fold reduction in ampicillin-resistant CFU and an approximately 1,000-fold increase in spectinomycin-resistant CFU. The researchers concluded that pPro-MobV efficiently targets a high-copy-number antibiotic resistance locus via conjugation and that both Pro-AG and homology-based deletion mechanisms are significantly augmented in RecA-deficient strains.
Deletion of Cas9 abrogated both insertional inactivation and homology-based deletion, confirming that double-strand cleavage was required. Removal of lambda Red reduced recovery of edited plasmids, with most outcomes reflecting plasmid destruction rather than repair, although low-frequency editing events persisted.
To evaluate mitigation strategies, the researchers developed a restoration assay in which a green fluorescent protein cassette disrupted the bla locus and was flanked by 100-base pair direct repeats. Delivery of a green fluorescent protein–targeting single-guide RNA with inducible lambda Red–Cas9 restored ampicillin resistance in 100% of colonies analyzed, all showing precise deletion of the intervening cassette. Shortening repeat length reduced baseline deletion frequency.
Phage delivery of a green fluorescent protein–targeting single-guide RNA similarly restored ampicillin resistance. All 24 colonies analyzed showed precise deletion of the green fluorescent protein target cassette, consistent with Cas9–single-guide RNA–mediated cleavage at the green fluorescent protein locus.
Compared with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) approaches that destroy resistance plasmids, the researchers reported that insertional inactivation was two to three logs more efficient than plasmid elimination and avoided potential effects of toxin–antitoxin systems.
The investigators concluded that Pro-AG and homology-based deletion are differentially affected by the bacterial RecA pathway and that homology-based deletion components can be delivered by plasmids or phages to selectively delete Pro-AG cassettes. They described this safeguard as a mechanism to prevent uncontrolled spread of a gene cassette and mitigate unanticipated side effects. Together, these refinements increase the efficiency and flexibility of the Pro-AG platform and broaden its potential utility in microbiome engineering, environmental remediation, and clinical strategies targeting antibiotic resistance.
“This work establishes a proof-of-principle for microbiome engineering strategies that could be leveraged to improve health and restore ecological balance,” the researchers concluded.
Disclosures: The researchers reported affiliations with the University of California, San Diego, and the Tata Institutes for Genetics and Society. Additional disclosures are detailed in the published article.
