PROJECT SUMMARY The CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by employing guide RNAs and endonuclease effectors to specifically recognize and cleave invasive nucleic acids. The specific DNA targeting and cleavage activities of CRISPR-Cas systems have been adopted and developed for genome editing and various other applications, which are revolutionizing biomedical research and beyond. However, safety concerns are raised because of off-target genome editing and the dependence on endogenous DNA repair pathways, hindering clinical applications of CRISPR-Cas systems. Exploration of alternative CRISPR-Cas systems in nature not only offers an opportunity to overcome those challenges, but may also inspires new applications. Structural and biochemical characterizations of CRISPR-Cas systems are critical for understanding of their mechanisms and repurposing them for precise genome editing. Our long-term goals are to unravel the mechanisms underlying target nucleic acid recognition and cleavage mediated by diverse CRISPR-Cas systems, which provide essential knowledge for safe and reliable use of this technology in treating human diseases. In this proposal, we will work on the molecular mechanisms for four newly discovered CRISRP-Cas systems, covering DNA targeting (Cas12i), RNA targeting (Cas12g) and CRISPR RNA guided DNA transposition (VcCascade and Cas12k). As revealed in our structure, Cas12i accommodates longer crRNA-DNA heteroduplex than currently used Cas effectors, thus potentially improve the specificity for genome editing. The RNA-guided RNase Cas12g is compact and thermostable, and thus potentially expand the toolkits for RNA editing and RNA-targeting applications. VcCascade and Cas12k direct transposition machinery for RNA-guided DNA transposition, opening a new paradigm for genome editing independent of DNA repair pathways. 1