Discovery of multiple anti-CRISPRs highlights anti-defense gene clustering in mobile genetic elements

Rafael Pinilla-Redondo,Peter C Fineran,Chris M Brown,Joseph Bondy-Denomy,Saadlee Shehreen,Søren J Sørensen,Nicole D Marino,Robert D Fagerlund

doi:10.1038/s41467-020-19415-3

Abstract

Many prokaryotes employ CRISPR–Cas systems to combat invading mobile genetic elements (MGEs). In response, some MGEs have developed strategies to bypass immunity, including anti-CRISPR (Acr) proteins; yet the diversity, distribution and spectrum of activity of this immune evasion strategy remain largely unknown. Here, we report the discovery of new Acrs by assaying candidate genes adjacent to a conserved Acr-associated (Aca) gene, aca5, against a panel of six type I systems: I–F (Pseudomonas, Pectobacterium, and Serratia), I–E (Pseudomonas and Serratia), and I–C (Pseudomonas). We uncover 11 type I–F and/or I–E anti-CRISPR genes encoded on chromosomal and extrachromosomal MGEs within Enterobacteriaceae and Pseudomonas, and an additional Aca (aca9). The acr genes not only associate with other acr genes, but also with genes encoding inhibitors of distinct bacterial defense systems. Thus, our findings highlight the potential exploitation of acr loci neighborhoods for the identification of previously undescribed anti-defense systems.

Highlights

Many prokaryotes employ clustered regularly interspaced short palindromic repeats (CRISPR)–Cas systems to combat invading mobile genetic elements (MGEs)
To uncover how MGEs within the Enterobacteriales order cope with the pressure of CRISPR–Cas immunity, we performed bioinformatic searches using the Enterobacteriales-enriched aca[5] gene[12] that is encoded downstream of several acrIF11 orthologs
These organisms are enriched with class 1 CRISPR–Cas systems[3], suggesting that their MGEs may rely on unknown type I inhibitors to bypass immunity

Summary

Introduction

Many prokaryotes employ CRISPR–Cas systems to combat invading mobile genetic elements (MGEs). Prokaryotes are outnumbered by a wide spectrum of mobile genetic elements (MGEs) that infect them, including viruses and plasmids. This selective pressure has driven the evolution of diverse defense mechanisms, including restriction-modification systems, abortive infection, and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes[1,2]. CRISPR–Cas loci have been identified in sequenced genomes of around 40% of bacteria and 85% of archaea[3] and are occasionally carried by a wide range of MGEs4–6, bearing testament to their evolutionary and ecological importance This mode of defense allows cells to remember, recognize and thwart recurrently infecting agents. Acr proteins benefit phage-based therapeutics and plasmid-based delivery platforms and provide a means to control CRISPR–Cas-derived biotechnologies[22]

Methods

Results

Conclusion