# Insight into crRNA Processing in Streptococcus mutans P42S and Application of SmutCas9 in Genome Editing

## TL;DR

This paper explores the use of a unique Cas9 enzyme from Streptococcus mutans for genome editing, particularly in genomes rich in AT nucleotides.

## Contribution

The study introduces SmutCas9, a novel Cas9 variant that recognizes rare PAM motifs, expanding genome editing capabilities.

## Key findings

- SmutCas9 successfully edited genes in a lactococcal phage, demonstrating its potential for genome editing.
- SmutCas9 recognizes the PAM motifs 5′-NAA-3′ and 5′-NGAA-3′, which are rarely used by other Cas9 variants.
- RNA sequencing provided insights into the RNA components of the CRISPR-Cas system in Streptococcus mutans.

## Abstract

CRISPR-Cas is an adaptive immune system found in bacteria and archaea that provides resistance against invading nucleic acids. Elements of this natural system have been harnessed to develop several genome editing tools, including CRISPR-Cas9. This technology relies on the ability of the nuclease Cas9 to cut DNA at specific locations directed by a guide RNA. In addition, the nuclease activity of Cas9 requires the presence of a short nucleotide motif (5′-NGG-3′ for Cas9 from Streptococcus pyogenes) called PAM, flanking the targeted region. As the reliance on this PAM is typically strict, diverse Cas9 variants recognising different PAM motifs have been studied to target a broader range of genomic sites. In this study, we assessed the potential of Cas9 from Streptococcus mutans strain P42S (SmutCas9) in gene editing. SmutCas9 recognises the rarely targeted 5′-NAA-3′ and 5′-NGAA-3′ PAMs. To test its efficacy, two genes of the virulent lactococcal phage p2 were edited, thereby demonstrating the potential of SmutCas9 for gene editing purposes, particularly in AT-rich genomes. Sequencing of total RNA also revealed the RNA components of this system, allowing further molecular characterisation of the type II-A CRISPR-Cas system of S. mutans.