Generation of Memory of Infection During the CRISPR-Cas9 Immune Response

Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral attack in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5′-NGG-3′ protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. Before my graduate work, it was not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. My work revealed that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminates or changes PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. This was a newly identified function of Cas9 in the genesis of prokaryotic immunological memory. To further explore the link between Cas9 and spacer acquisition, I performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. I identified a mutation, I473F, which increases the rate of spacer acquisition by more than two orders of magnitude. This patented variant of Cas9 highlights the enzyme’s role during CRISPR immunization, provides a useful tool to study this otherwise rare process, and holds promise to be developed into a biotechnological application. Researching Cas9 and spacer acquisition involved many rounds of high-throughput sequencing of millions of spacers acquired by bacteria during phage infection. These experiments revealed that the abundance of each spacer in the surviving population was highly uneven. Since the molecular mechanisms underlying this bias were not known, I decided to look into the factors that affect the distribution of individual spacer sequences during phage infection of cells harboring the CRISPR system from Streptococcus pyogenes. My work has shown that spacer patterns are established early during infection and correlate with spacer acquisition rates, but not with spacer targeting efficiency. The data suggests that the rate of spacer acquisition depends on unique sequence elements within the spacers and therefore determines the abundance of different spacers within the adapted population. These results elucidate a fundamental mechanism behind the generation of immunological diversity during the type II CRISPR-Cas response

Spacer Acquisition Rates Determine the Immunological Diversity of the Type II CRISPR-Cas Immune Response.

CRISPR-Cas systems provide acquired immunity in prokaryotes. Upon infection, short sequences from the phage genome, known as spacers, are inserted between the CRISPR repeats. Spacers are transcribed into small RNA molecules that guide nucleases to their targets. The forces that shape the distribution of newly acquired spacers, which is observed to be uneven, are poorly understood. We studied the spacer patterns that arise after phage infection of Staphylococcus aureus harboring the Streptococcus pyogenes type II-A CRISPR-Cas system. We observed that spacer patterns are established early during the CRISPR-Cas immune response and correlate with spacer acquisition rates, but not with spacer targeting efficiency. The rate of spacer acquisition depended on sequence elements within the spacer, which in turn determined the abundance of different spacers within the adapted population. Our results reveal how the two main forces of the CRISPR-Cas immune response, acquisition and targeting, affect the generation of immunological diversity

Cas9 specifies functional viral targets during CRISPR–Cas adaptation

International audienceClustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory

Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.

CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application