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

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 spatially resolved CRISPR screening (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.

Cell Type-Agnostic Optical Perturbation Screening Using Nuclear In-Situ Sequencing (NIS-Seq)

“…Subsequently, cells are dissociated, marked cells are enriched by FACS sorting, and perturbagens are analyzed by deep sequencing 11 . To a similar end, image-based cell sorting 12 or laser microdissection 13 can be used to isolate cells displaying a microscopic phenotype of interest for subsequent perturbagen identification, albeit at limited optical resolution. Instead of enriching for a population of cells for lysis and sequencing, Feldman et al 10 developed a method for microscopic perturbagen identification based on in-situ sequencing of barcodes contained in cellular mRNAs, using signal amplification by rolling circle amplification (RCA).…”