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

Raphael Hofmann (), Calvin Herman, Charlie Y. Mo, Jacob Mathai and Luciano A. Marraffini ()
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Raphael Hofmann: The Rockefeller University
Calvin Herman: The Rockefeller University
Charlie Y. Mo: The Rockefeller University
Jacob Mathai: The Rockefeller University
Luciano A. Marraffini: The Rockefeller University

Nature Communications, 2025, vol. 16, issue 1, 1-13

Abstract: 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.

Date: 2025
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DOI: 10.1038/s41467-025-60925-9

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