Graduate Research Assistant Iowa State University Ames, Iowa, United States
Yukti Dhingra (Iowa State University)| Hayun Lee (Iowa State University)| Dipali Sashital (Iowa State University)
CRISPR-Cas systems (Clustered regularly interspaced short palindromic repeats and associated proteins) are adaptive immune systems that protect bacteria and archaea against invading phages. Spacer sequences derived from invader genome are arranged between repeat sequences in the CRISPR array. Upgrading the CRISPR array with new spacers is a crucial step to ensure bacterial systems are at pace with rapidly evolving phages. This is carried out by Cas1 and Cas2 proteins, universally present in most CRISPR systems. Many systems require proteins in addition to Cas1 and Cas2 to maintain the efficiency and fidelity of adaptation. Cas4 is one such protein and is part of the core family of Cas proteins in over half of known CRISPR systems. Cas4 carries a RecB like nuclease domain and initial studies on Cas4 characterized its function as a 5’ to 3’ exonuclease. Recent in-vivo and in-vitro studies have shown that Cas4 plays a role in selection, processing, and integration of functional spacers. Our lab has shown that Cas4 performs endonucleolytic processing of 3’ overhangs of prespacers in the presence of Cas1 and Cas2. Using negative stain EM, we have shown that Cas4 forms a complex with Cas1 and Cas2 in the presence of a potential spacer substrate. However, the exact mechanistic steps in Cas4-Cas1-Cas2 mediated integration remain unknown. Moreover, some systems encode Cas4-1 fusion proteins that provide an increased efficiency of spacer acquisition observed in-vivo. These fusions remain to be characterized biochemically. With this work, we aim to determine the sequence of events in integration in systems that have Cas4 and understand the structure function relationship of Cas4 in fusion with Cas1 and complex with Cas1-Cas2.