# CRISPR-Cas is beneficial in plasmid competition, but limited by competitor toxin–antitoxin activity when horizontally transferred

## TL;DR

Plasmids use CRISPR-Cas to compete with other plasmids, but this defense is limited when the plasmid is newly transferred and the competitor has toxin-antitoxin systems.

## Contribution

The study reveals how toxin-antitoxin systems can limit the effectiveness of plasmid-borne CRISPR-Cas after horizontal transfer.

## Key findings

- Plasmid-borne CRISPR-Cas is beneficial when the plasmid is resident in a host.
- CRISPR-Cas is selected against after horizontal transfer if the competitor plasmid has toxin-antitoxin systems.
- Naturally occurring CRISPR-Cas plasmids avoid targeting plasmids with toxin-antitoxin systems.

## Abstract

Bacteria can encode dozens of different immune systems that protect them from infection by mobile genetic elements (MGEs). MGEs themselves may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs. It is unclear when this is favored by natural selection, and whether toxin–antitoxin (TA) systems—common competitive mechanisms carried by plasmids—can alter their efficacy. Here, we develop and test novel theory to analyze the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our mathematical model and experiments using Escherichia coli and competing IncP plasmids reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid has not recently transferred to a new host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if a resident competitor plasmid encodes a TA system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems across bacterial genera. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.

Plasmids use immune systems like CRISPR-Cas to compete with other plasmids, but it is unclear when these systems provide a selective advantage. This study shows that CRISPR-Cas benefits resident plasmids but is constrained by toxin-antitoxin systems after horizontal transfer.