Investigating the molecular mechanism of CRISPR-Cas adaptation of Streptococcus thermophilus

Abstract

Abstract Bacteria are constantly challenged by viral infection. In order to combat potentially lethal viruses, bacteria possess adaptive immune defenses called CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated) systems. These systems are RNA-based defense systems that memorize past viral infections to protect prokaryotes like bacteria from future infections. The acquisition of short DNA sequences (spacers) from previously encountered invaders allows bacteria to generate immunity. These systems capture spacers and incorporated them into their CRISPR loci to initialize immunity. Once integrated, spacers are then transcribed and processed into small RNA molecules called crRNAs, which are eventually bound by Cas proteins and form targeting complexes capable of cleaving viral DNA to achieve immunity. Genetic evidence indicates that Cas1, Cas2, Cas9 and Csn2 are each essential for CRISPR adaptation in the Type II-A system of Streptococcus thermophilus. However, the role of each of these proteins in adaptation is not clear. We have systematically constructed single cas gene deletion strains. We aim to detect and analyze low level of adaptation events in these genetic backgrounds, and in turn gain insight into the specific roles of each of these proteins. We also plan to apply biochemical approaches using purified proteins to investigate the molecular mechanistic steps of adaptation.