# A conjugal gene drive-like system efficiently suppresses antibiotic resistance in a bacterial population

## TL;DR

A new gene drive system efficiently reduces antibiotic resistance in bacteria by spreading anti-resistance genes and includes a safeguard to control its spread.

## Contribution

Integration of a CRISPR-based gene drive with conjugative transfer and a homology-based deletion mechanism for controlled microbiome engineering.

## Key findings

- Pro-AG combined with conjugative transfer reduces antibiotic-resistant bacteria by five logs.
- Homology-based deletion (HBD) allows precise removal of gene cassettes flanked by short repeats.
- HBD components can be delivered via plasmids or phages to prevent uncontrolled gene spread.

## Abstract

Antibiotic resistance (AR) is an escalating public health threat, necessitating innovative strategies to control resistant bacterial populations. One promising approach involves engineering genetic elements that can spread within microbial communities to eliminate AR genes. Previously, we developed Pro-Active Genetics (Pro-AG), a CRISPR-based gene-drive-like system capable of reducing AR colony-forming units (CFU) by approximately five logs. Here, we advance this technology by integrating Pro-AG into a conjugative transfer system, enabling efficient dissemination of an anti-AR gene cassette between two bacterial strains. Additionally, we characterize a complementary homology-based deletion (HBD) process, a CRISPR-driven mechanism that precisely removes target DNA sequences flanked by short direct repeats. Our findings reveal that Pro-AG and HBD are differentially influenced by the bacterial RecA pathway and that HBD components can be delivered via plasmids or phages to selectively delete Pro-AG cassettes. This built-in safeguard prevents uncontrolled spread of a gene cassette and mitigates unanticipated side effects. These refinements enhance the efficiency and flexibility of Pro-AG, expanding its potential applications in microbiome engineering, environmental remediation, and clinical interventions aimed at combating antibiotic resistance. More broadly, this work establishes a proof-of-principle for microbiome engineering strategies that could be leveraged to improve health and restore ecological balance.