The phenotypic landscape of essential human genes

Abstract

Understanding the basis for cellular growth, proliferation, and function requires determining the contributions of essential genes to diverse cellular processes. Here, we combined pooled CRISPR/Cas9-based functional screening of 5,072 fitness-conferring genes in human cells with microscopy-based visualization of DNA, DNA damage, actin, and microtubules. Analysis of >31 million individual cells revealed measurable phenotypes for >90% of genes. Using multi-dimensional clustering based on hundreds of quantitative phenotypic parameters, we identified co-functional genes across diverse cellular activities, revealing novel gene functions and associations. Pooled live-cell screening of ∼450,000 cell division events for 239 genes further identified functional contributions to chromosome segregation. Our work creates a resource for the phenotypic analysis of core cellular processes and defines the functional landscape of essential human genes.

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

“…Thus, INTS10-13-14-C7orf26 represents a functionally and biochemically distinct module of Integrator, consistent with concurrent studies ( Figure 3H ; Funk et al, 2021 ; Pan et al, 2022 ; Wainberg et al, 2021 ). Although Integrator has been subjected to extensive structural analyses, it has been difficult to resolve the INTS10-13-14 components in relation to the rest of the complex, and inclusion of C7orf26 may facilitate future structural efforts.…”

“…In every case, the affected ribosomal subunit corresponded to the Perturb-seq clustering across two independent sgRNAs. Although this study was in progress, another group identified C1orf131 as a structural component of the pre-A1 small subunit processome by cryo-EM, complementing our functional work (Singh et al, 2021). This validation suggests that many poorly characterized genes can be assigned functional roles through Perturb-seq, although a subset of these relationships may be explained by off-target effects (Figure S4G…”

The PRP19 Ubiquitin Ligase, Standing at the Cross-Roads of mRNA Processing and Genome Stability

“…A direct association between CDC5L and securin was also found, pointing towards a direct role for the NTC in meiosis promotion. Finally, in a recent high-throughput CRISPR-based optical pooled screen, the NTC and NTC-associated components (PRP19, CDC5L, PLRG, SNW1 and BUD31) co-clustered with other mRNA splicing factors as key regulators of mitosis, confirming prior work [ 146 ]. In addition to its splicing roles, the NTC also participates directly in mitosis progression.…”

Exploring the genetic space of the DNA damage response for cancer therapy through CRISPR‐based screens

“…This is typically done by sorting cells based on their expression for a particular marker. More recently, microscopy has also been utilized as a readout, for example, by measuring γH2AX foci (a marker of DNA damage) coupled with in situ sequencing [ 13 ]. Arrayed screens, which have a physical separation between each perturbation, are typically used for validation and follow‐up studies.…”