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

Author

Listed:
  • Raphael Hofmann

    (The Rockefeller University)

  • Calvin Herman

    (The Rockefeller University)

  • Charlie Y. Mo

    (The Rockefeller University
    University of Wisconsin)

  • Jacob Mathai

    (The Rockefeller University)

  • Luciano A. Marraffini

    (The Rockefeller University
    The Rockefeller University)

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.

Suggested Citation

  • Raphael Hofmann & Calvin Herman & Charlie Y. Mo & Jacob Mathai & Luciano A. Marraffini, 2025. "Deep mutational scanning identifies Cas1 and Cas2 variants that enhance type II-A CRISPR-Cas spacer acquisition," Nature Communications, Nature, vol. 16(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-60925-9
    DOI: 10.1038/s41467-025-60925-9
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