Generation of DNA memory by bacterial CRISPR-Cas9 systems

Project Summary Prokaryotes have evolved a myriad of defense systems to protect themselves from viruses (i.e., bacteriophages) and other parasitic elements. Uniquely among these, CRISPR-Cas enables adaptive immunity through RNA- programmed destruction of the invader genome. During infection, bacteria can capture viral DNA snippets into their CRISPR loci as immune memories (termed spacers), in a process known as spacer acquisition or CRISPR adaptation. CRISPR RNAs transcribed from this memory will then guide Cas enzymes to locate and degrade complementary targets in the invader to achieve immunity. Much progress has been made in understanding the Cas enzymes and their applications in genetic engineering. However, how microbes acquire CRISPR memories from viruses remains poorly understood. Our research programs aim to decipher the molecular basis of viral memory acquisition in CRISPR-Cas9 and unveil the contributions of bacterial host gene networks to this mysterious phenomenon. We also thrive to uncover novel mechanisms by which bacteriophages evade CRISPR-Cas9 immunity. Gram-negative pathogen Neisseria meningitidis (Nme) and its cryptic inoviruses (i.e., filamentous phages) serve as our model systems. We recently found a novel role of guide-less apoCas9 in regulating spacer acquisition and safeguarding immunity depth in meningococci. In the next five years, we will use bacteria genetic, deep sequencing, bioinformatic, and biochemical approaches to tackle outstanding questions, including: How does Cas9 coordinate with the Cas1- Cas2 integrase mechanistically to regulate acquisition? What host factors play key roles during viral spacer acquisition, and how? What diverse anti-defense approaches have Neisseria phages evolved to counteract CRISPR-Cas9 and CRISPR-Cas3 immunity? This proposed research will generate new insights into CRISPR-Cas adaptability. This work will also illuminate the co-evolutionary arms between pathogenic bacteria, their CRISPR-Cas, and viral predators. Moreover, findings from our research promises to guide technology advances, including phage-encoded CRISPR inhibitors for controlling genome editing applications to improve safety, and Cas9-Cas1-Cas2 based genome-tagging and molecular recording devices.