Abstract Forward genetic screening associates phenotypes with genotypes by randomly inducing mutations and then identifying those that result in phenotypic changes of interest. Here we present spa tially r esolved C RISPR s creening (SPARCS), a platform for microscopy-based genetic screening for spatial cellular phenotypes. SPARCS uses automated high-speed laser microdissection to physically isolate phenotypic variants in situ from virtually unlimited library sizes. We demonstrate the potential of SPARCS in a genome-wide CRISPR-KO screen on autophagosome formation in 40 million cells. Coupled to deep learning image analysis, SPARCS recovered almost all known macroautophagy genes in a single experiment and discovered a role for the ER-resident protein EI24 in autophagosome biogenesis. Harnessing the full power of advanced imaging technologies, SPARCS enables genome-wide forward genetic screening for diverse spatial phenotypes in situ .

SPARCS, a platform for genome-scale CRISPR screening for spatial cellular phenotypes