Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition

Nat Commun. 2025 Jul 1;16(1):5730. doi: 10.1038/s41467-025-60925-9.

Abstract

A remarkable feature of CRISPR-Cas systems is their ability to acquire short sequences from invading viruses to create a molecular record of infection. These sequences, called spacers, are inserted into the CRISPR locus and mediate sequence-specific immunity in prokaryotes. In type II-A CRISPR systems, Cas1, Cas2 and Csn2 form a supercomplex with Cas9 to integrate viral sequences. While the structure of the integrase complex has been described, a detailed functional analysis of the spacer acquisition machinery is lacking. We developed a genetic system that combines deep mutational scanning (DMS) of Streptococcus pyogenes cas genes with a method to select bacteria that acquire new spacers. Here, we show that this procedure reveals key interactions at the Cas1-Cas2 interface critical for spacer integration, identifies Cas variants with enhanced spacer acquisition and immunity against phage infection, and provides insights into the molecular determinants of spacer acquisition, offering a platform to improve CRISPR-Cas-based applications.

MeSH terms

  • Bacterial Proteins* / genetics
  • Bacterial Proteins* / metabolism
  • Bacteriophages / genetics
  • CRISPR-Associated Proteins* / genetics
  • CRISPR-Associated Proteins* / metabolism
  • CRISPR-Cas Systems* / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Endonucleases* / genetics
  • Endonucleases* / metabolism
  • Mutation
  • Streptococcus pyogenes* / genetics
  • Streptococcus pyogenes* / virology

Substances

  • CRISPR-Associated Proteins
  • Bacterial Proteins
  • Endonucleases