Microbial treasure trove : Unravelling the potential of class 2 CRISPR–Cas systems

Animals are known to possess a large repertoire of immune systems with a high degree of sophistication. On the other hand, the immune systems found in bacteria and archaea appeared to be much more rudimentary. However, the ground-breaking discovery of a novel immune system, CRISPR-Cas, proved otherwise. CRISPR-Cas, is unique in being both adaptive and heritable, and it relies on small RNA molecules that specifically guide the defence system to matching invader DNA sequences. This natural defence system has been successfully repurposed into a valuable molecular technology which is a robust, efficient, easy-to-use method to precisely alter DNA sequences of living organisms. The current CRISPR-based technologies, mostly employ the Cas9 protein, for diverse biotechnological applications. Nevertheless, the natural diversity of CRISPR-Cas systems is remarkably extensive, including systems that target DNA, systems that target RNA, and systems that target both DNA and RNA. The diverse class 2 CRISPR nucleases have unique molecular features that contribute to an expansive toolbox for genome and transcriptome engineering. These nucleases differ greatly in their structure and mechanisms. These differences could be exploited as complementary applications creating numerous CRISPR-based technologies possibly with favourable specificity, efficiency and/or delivery. This thesis explores the diversity of Class 2 CRISPR–Cas systems and provides mechanistic insights into different class 2 nucleases. In addition, it describes potential applications to expand the current repertoire of CRISPR-based technologies. Due to the everlasting arms race between prokaryotes and their viruses, the rapid evolution of CRISPR–Cas systems has resulted in extreme structural and functional diversity. As a result, a plethora of distinct CRISPR–Cas systems are represented in genomes of most archaea and almost half of the bacteria. The key players of this system are the crRNA binding effector complexes, and