Mapping the genetic landscape of human cells

Seminal yeast studies have established the value of comprehensively mapping genetic interactions (GIs) for inferring gene function. Efforts in human cells using focused gene sets underscore the utility of this approach, but the feasibility of generating large-scale, diverse human GI maps remains unresolved. We developed a CRISPR interference platform for large-scale quantitative mapping of human GIs. We systematically perturbed 222,784 gene pairs in two cancer cell lines. The resultant maps cluster functionally related genes, assigning function to poorly characterized genes, including TMEM261, a new electron transport chain component. Individual GIs pinpoint unexpected relationships between pathways, exemplified by a specific cholesterol biosynthesis intermediate whose accumulation induces deoxynucleotide depletion, causing replicative DNA damage and a synthetic-lethal interaction with the ATR/9-1-1 DNA repair pathway. Our map provides a broad resource, establishes GI maps as a high-resolution tool for dissecting gene function, and serves as a blueprint for mapping the genetic landscape of human cells. A library of constructs expressing two sgRNAs and a joint barcode corresponding to the sgRNA pair were cloned and transduced as a pool in K562 or Jurkat cells. Cells were passaged in standard culture conditions and T0 and endpoint samples were harvested. Genomic DNA was processed and sequenced to obtain a readout of sgRNA pair read counts and phenotypes were quantified by comparing representation at T0 and endpoint. Genetic interaction scores and functional gene clustering were derived from these phenotypes.