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

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.