Recently, RNA based adaptive immune systems (CRISPR-Cas systems) have been discovered. They protect bacteria and archaea from invasions by foreign nucleic acid elements such as phages and plasmids. The young and fast-moving field of CRIPSR provides exciting opportunities in biomedical research to fight pathogens, in biotechnology to develop CRIPSR based molecular tools, and in industry to reduce loss of bacteria due to phage infection during fermentation. There are three major steps in CRIPSR-Cas actions: the integration of foreign genetic elements into the CRIPSR array (adaptation), the biogenesis of CRISPR RNA (crRNA), and the processing of invading nucleic acid (interference). Here I propose to uncover the detailed mechanism of adaptation, the equally important but under-studied process in CRISPR-Cas systems, focusing on the CRISPR system1 of Streptococcus thermophilus. With a novel reporter system, I aim to identify both trans-acting factors such as specific Cas proteins, and cis-acting factors such as elements of CRISPR leader and the repeat sequences, which are critical for CRISPR adaptation. I plan to use a powerful combination of in vivo and in vitro approaches to identify and examine Cas protein complexes that function in adaptation. I will determine their protein compositions and nucleic acid binding and cleavage activities to gain mechanistic insights on adaptation. My study will provide fundamental understanding of the mechanism of adaptation at the molecular level, and help develop industrially and medically related CRISPR-based technologies.
Existing or engineered CRISPR-Cas systems are being exploited in both biomedical research and biotechnology. CRISPR-Cas systems are also being exploited to reduce horizontal gene transfer of antibiotic resistance genes and virulence factors, which could result in a novel way to treat serious human pathogens such as methicillin-resistant Staphylococcus aureus. Phage infections cause drastic loss in fermentation, i.e., in dairy industries. Bacteria strains with engineered CRISPR systems could provide resistance to common phages and therefore benefit related industries. Knowledge provided in my studies will help tremendously to manipulate CRISPR-Cas immune systems to fight pathogens (inhibit spreading of antibiotics resistance genes), to protect beneficial bacteria from phage attack, and to develop new CRISPR-based molecular technologies