# Disparate mechanisms counteract extraneous CRISPR RNA production in type II-C CRISPR-Cas systems

## TL;DR

This paper explores how type II-C CRISPR-Cas systems prevent harmful 'extraneous' CRISPR RNAs from interfering with immune defense.

## Contribution

The study identifies three novel strategies used by type II-C systems to counteract extraneous CRISPR RNA production.

## Key findings

- Type II-C systems use Rho-independent terminators to block extraneous CRISPR RNA production.
- Hairpin structures sequester extraneous CRISPR RNA to prevent interference.
- Mutations in CRISPR repeats disrupt the formation of extraneous CRISPR RNA.

## Abstract

CRISPR-Cas adaptive immune systems in bacteria and archaea enable precise targeting and elimination of invading genetic elements. An inherent feature of these systems is the ‘extraneous’ CRISPR RNA (ecrRNA), which is produced via the extra repeat in a CRISPR array lacking a corresponding spacer. As ecrRNAs would interact with the Cas machinery yet not direct acquired immunity, they pose a potential barrier to defence. Type II-A CRISPR-Cas systems resolve this barrier through the leader sequence upstream of a CRISPR array, which forms a hairpin structure with the extra repeat that inhibits ecrRNA production. However, the fate of ecrRNAs in other CRISPR types and subtypes remains to be explored. Here, we report that II-C systems likely employ disparate strategies to resolve the ecrRNA due to their distinct configuration in comparison to II-A. Applying bioinformatics analyses to over 650 II-C systems followed by experimental validation, we identified three strategies applicable to these systems: formation of an upstream Rho-independent terminator, formation of a hairpin that sequesters the ecrRNA guide, and mutations in the repeat expected to disrupt ecrRNA formation. These findings expand the list of mechanisms in CRISPR-Cas systems that could resolve the ecrRNA to optimize immune response.

The majority of the analyzed type II-C CRISPR-Cas systems have at least one mechanism that functions to inhibit the ‘extraneous’ crRNA and its negative side effects.