Construction and Characterization of S. Pyogenes Mutants Influencing CRISPR-Cas9 Memory Formation

Bacteria employ diverse immunological strategies to detect and eliminate foreign DNA, including that of invading viral genomes such as bacteriophages (phages). Unique among these defenses are Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems, which act as the only known form of adaptive immunity in prokaryotes, generating and retaining genetic memories of previously encountered phages in the form of "spacers" stored within the CRISPR array. Despite the pivotal role of CRISPR in defense against phage, the process of acquiring new spacers during infection remains exceptionally rare and poorly understood. To identify genetic factors that influence CRISPR-mediated immunity, a transposon mutagenesis screen (Tn-seq) was performed in Streptococcus pyogenes, a native host of a Type II-A CRISPR system. Notably, insertions in recJ, a well-conserved single-stranded DNA exonuclease involved in DNA repair, experienced strong enrichment in our S. pyogenes survivor pool after exposure to lytic phage A1. Generation of a deletion strain, ΔrecJ, validated Tn-seq results with ΔrecJ cells exhibiting a 2-3-fold enhanced CRISPR immunization rate against an untargeted phage. Surprisingly, the ΔrecJ strain also demonstrates increased survival rates against phage already targeted by CRISPR spacers, suggesting that this mutant influences both naive spacer acquisition as well as utilization of spacers once encoded in the CRISPR array. Furthermore, we have isolated a novel phage from wastewater, Faranak, that is not sensitive to the absence of recJ. We aim to leverage this insensitivity to further elucidate the mechanism of RecJ during phage infection and CRISPR immunization.