Abstract Bacterial life employs diverse immune mechanisms to protect themselves against predatory phage, which are thought to outnumber them by ten to one. CRISPR systems in particular engage their constituent Cas nucleases with programmable guide RNAs to target invading nucleic acids, endowing the host cell with adaptive immunity. They can be divided into six broad types, and the Type VI Cas13 systems contain the only known CRISPR nucleases that exclusively target RNA. CRISPR systems have been broadly adapted as genetic engineering technologies, and over the last few years, platforms based on Type II Cas9 have significantly accelerated basic research and biotechnology. Much like Cas9 for DNA targeting, Cas13 enzymes can be adapted into a modular and efficient platform for RNA targeting in cells, greatly advancing the RNA manipulation toolbox. However, many Cas13 enzymes are limited by variable and unpredictable activity, a challenge that has limited RNA interference technologies. More broadly speaking, a central problem in the genome and transcriptome engineering field is predicting robust and generalizable cleavage efficiency and specificity across different target nucleic acids and cell types within newly developed nuclease effectors. Recently, the Hsu lab reported the discovery of a subtype of Cas13, the Cas13d system, which is significantly smaller, more efficient, and more specific than other Cas13 subtypes or short hairpin RNAs for RNA interference and manipulation of alternative splicing. The Lyumkis lab recently leveraged state-of-the-art cryo- electron microscopy (cryo-EM) advances to solve high-resolution structures of Cas13d bound to guide RNA and target RNA. However, there are gaps in our understanding of Cas13d molecular structure and function and disconnects between the molecular/structural biology defining Cas13d activity and what is observed in mammalian cells in transcriptome engineering efforts. The overarching goal is to elucidate the diverse mechanisms of CRISPR-Cas adaptive immunity to engineer improved CRISPR-associated enzymes for gene regulation and other biotechnological applications. The proposed work will systematically address these challenges using interdisciplinary structural biology, biochemical, protein engineering, bioinformatic, and genetic approaches in collaboration between the Hsu and Lyumkis labs. The combined results from the proposed work will (1) provide mechanistic insight into the complete enzymatic cycle of Cas13d, (2) shed light on the evolutionary pathways involved in Cas13d structure and function, (3) define the mechanism of CRISPR- associated factors that can modulate Cas13 activity, and (4) enable the structure-guided engineering of next- generation RNA-targeting effectors for therapeutic and diagnostic applications. Importantly, the principles and approaches elucidated here will provide a blueprint for the design of diverse forthcoming tools beyond CRISPR-Cas13 for a comprehensive genome engineer...