Generation of DNA memory by bacterial CRISPR-Cas9 systems

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Project Summary Prokaryotic horizontal gene transfer (HGT) underlines the spread of antibiotic resistance and pathogenic traits. The battle against antibiotic resistance must be fought on multiple fronts, including the understanding of natural barriers that microbes use to restrict HGT. Most bacteria rely on CRISPR-Cas to establish adaptive immunity against invasive elements. DNA from these invaders’ genome can be captured and stored as immunological memories termed spacers, at the CRISPR loci. Small, antisense RNAs produced from CRISPR (crRNAs) will guide Cas enzymes to destroy invaders with a matching target. In the past decade, much progress has been made in understanding the CRISPR interference enzymes and their applications in genetic engineering. However, how microbes acquire their CRISPR memories remains poorly understood. In this proposal, we aim to uncover the molecular basis for CRISPR memorization (i.e. spacer adaptation). We use the gram-negative pathogen Neisseria meningitidis (Nme) as a model organism, due to of its clinical importance and tractable genetics. Current knowledge about spacer adaptation mostly comes from studies of the type I CRISPR native to E. coli; products of its conserved cas1-cas2 integrase genes can create functional memories independently of the interference enzymes. Our recent preliminary findings suggest that the type II CRISPR of N. meningitidis creates memory by a distinct mechanism. The interference enzyme, NmeCas9 and its tracrRNA and crRNA partners, play key but non-conventional roles in the acquisition of functional spacers. We use molecular genetic, genomic and biochemical approaches to address fundamental questions, including: What are the molecular roles of Cas9 and the CRISPR-encoded tracrRNA in spacer acquisition? What are the rules governing memory DNA selection? How does Cas9/tracr cooperate with the Cas1-2 integrase? And finally, how would the anti-CRISPR proteins affect the memorization process? This administrative equipment supplement request will allow access to ddPCR instrument, which enables high throughput and sensitive quantification of nucleic acids. This is essential to decipher CRISPR immunization beyond DNA transformation, in the context of filamentous phage infection. The impact of research supported by the parent grant will therefore be greatly enhanced. Collectively, the proposed research will illuminate the interplay between pathogenic bacteria, their CRISPR systems, and different horizontal gene transfer routes.