Elucidating cellular mechanisms and therapeutic strategies for neurodegenerative diseases with CRISPRi and CRISPRa
Ruilin Tian1, Connor Ludwig1, Nina Draeger1, Avi Samelson1, Diane Nathaniel1, Michael Ward3, Martin Kampmann1,2,*
1University of California, San Francisco, Institute for Neurodegenerative Diseases and Department for Biochemistry and Biophysics, San Francisco, CA; 2Chan Zuckerberg Biohub, San Francisco, CA; 3National Institute of Neurological Disorders and Stroke, Bethesda, MD.
Human genes associated with brain-related diseases are being discovered at an accelerating pace. A major challenge is the identification of the mechanisms through which these genes act, and of potential therapeutic strategies. To elucidate such mechanisms in human cells, we are leveraging a CRISPR-based platform for genetic screening that we recently co-developed. Our approach relies on CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), in which a catalytically dead version of the bacterial Cas9 protein recruits transcriptional repressors or activators, respectively, to endogenous genes to control their expression, as directed by a small guide RNA (sgRNA). Complex libraries of sgRNAs enable us to conduct genome-wide loss-of-function and gain-of-function screens in mammalian cells. We have adapted this strategy for use in human iPSC-derived neurons and other cell types, in order to elucidate disease mechanisms and therapeutic strategies in patient-derived cells and isogenic control cells. Our pilot screens systematically reveal genes and cellular pathways relevant for neuronal survival and response to oxidative stress.