Genome-Wide CRISPR Activation: A Novel Strategy for Identifying Anti-Aging Targets - The instrumental role of systematic gene knockdown studies, using RNA interference (RNAi), in identifying lifespan-extending gene alterations in model organisms like C. elegans is well established. In contrast, systematic gene overexpression remains mostly unexplored, representing a major knowledge gap in our understanding of the genetic basis of longevity and health. This gap exists primarily due to previous technological limitations which restricted studies to a limited selection of candidate genes. The recent advancements in CRISPR technology for C. elegans overcome those hurdles. These innovations allow rapid, conditional overexpression of specific genes simply by expressing dead Cas9 (dCas9) fused to a transcriptional activation domain in chosen tissues and feeding the organism bacteria carrying a particular guide RNA at the desired time (feeding CRISPRa). This technological leap allows for a systematic identification of genes that modulate longevity and health. This novel approach could identify a new class of genes pivotal for longevity and health, transforming our understanding of aging and disease. Our objective in this proposal is to identify new determinants of lifespan and health by extending these gene-specific overexpression tools to a genome scale. Results enabled by our labs’ collaborations and joint expertise make us uniquely well prepared to undertake the proposed research. Specifically, (i) the Apfeld lab pioneered genetic approaches to aging in C. elegans, (ii) the Levine lab pioneered systems biology approaches in C. elegans, (iii) both labs developed innovative, high- throughput approaches to study aging and resilience, including the automated Lifespan Machine scanner cluster, (iv) both labs have robust molecular biology expertise, and (v) we confirmed that feeding CRISPRa works in our hands for several genes. The rationale of the proposed research is twofold: identifying genes that prolong lifespan can pave the way for the development of new therapies to promote healthy aging and treat age-related diseases, while understanding genes that shorten lifespan can provide insights into disorders of accelerated aging and guide strategies to manage or mitigate their impact. We will accomplish these goals by pursuing two specific aims: in Aim 1 we will build a well-characterized gene-activation toolkit that enables time-dependent and tissue-specific systematic gene activation in C. elegans; in Aim 2 we will use this toolkit to identify the set of genes that modulate C. elegans lifespan and health when activated. The approach is innovative, in our opinion, because it pioneers the use of a rapid and simple CRISPR-activation technology to systematically identify new determinants of lifespan and health. This contribution will be significant because it is anticipated to provide a foundation for understanding how deliberate genetic activation can influence longevity and health, with potential applications to aging-related diseases and general health maintenance. Moreover, by equipping the C. elegans research community with a novel genome-scale toolkit for precise gene overexpression control, we are removing a major obstacle hindering the study of gene functions associated with aging.
