A High-Resolution Genome-Wide CRISPR/Cas9 Viability Screen Reveals Structural Features and Contextual Diversity of the Human Cell-Essential Proteome

Abstract

To interrogate genes essential for cell growth, proliferation and survival in human cells, we carried out a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 screen in a B-cell lymphoma line using a custom extended-knockout (EKO) library of 278,754 single-guide RNAs (sgRNAs) that targeted 19,084 RefSeq genes, 20,852 alternatively spliced exons, and 3,872 hypothetical genes. A new statistical analysis tool called robust analytics and normalization for knockout screens (RANKS) identified 2,280 essential genes, 234 of which were unique. Individual essential genes were validated experimentally and linked to ribosome biogenesis and stress responses. Essential genes exhibited a bimodal distribution across 10 different cell lines, consistent with a continuous variation in essentiality as a function of cell type. Genes essential in more lines had more severe fitness defects and encoded the evolutionarily conserved structural cores of protein complexes, whereas genes essential in fewer lines formed context-specific modules and encoded subunits at the periphery of essential complexes. The essentiality of individual protein residues across the proteome correlated with evolutionary conservation, structural burial, modular domains, and protein interaction interfaces. Many alternatively spliced exons in essential genes were dispensable and were enriched for disordered regions. Fitness defects were observed for 44 newly evolved hypothetical reading frames. These results illuminate the contextual nature and evolution of essential gene functions in human cells.

Inducible degron-dependent depletion of the RNA polymerase I associated factor PAF53 demonstrates it is essential for cell growth and allows for the analysis of functional domains

“…When mammalian cells were screened for genes "required for proliferation and survival", Wang et al reported that the PolR1E (PAF53) gene was essential. This result was confirmed by Bertomeu et al (16) and others (50). Thus, we sought to determine the specific effect of knocking down PAF53 using the AID as both physiological and biochemical results should be apparent very rapidly.…”

“…They also reported they were essential for promoter-specific transcription (32,33 (34,35,49). Most recently, several CRISPR/Cas9 based screenings of the mammalian genome identified PAF53 and PAF49 as being "essential" genes (16,17), which was confirmed when we found that we could not isolate cell lines that failed to express PAF53 (50).…”

“…rDNA transcription requires a unique set of transcription factors not used by the other nuclear polymerases, and we are just beginning to understand the biochemistry and functional interactions of these mammalian factors. Many of the genes whose protein products are involved in ribosome biogenesis were identified in CRISPR screenings of essential genes (16)(17)(18)(19). High-throughput screening of mammalian cells for proteins that affect nucleolar number has recently revealed proteins involved in regulating ribosome biogenesis (20,21).…”

Genetic Landscape of Electron Transport Chain Complex I Dependency in Acute Myeloid Leukemia

“…In order to identify Mubritinib’s target pathway, we then carried out a whole-genome CRISPR/Cas9 screen in the presence or absence of Mubritinib treatment (Fig. S4a and Read files 1-3), as previously described 44 . For this purpose, we used the B cell precursor leukemia cell line NALM-6, which, similar to poor outcome AML patient cells, is sensitive to Mubritinib (Fig.…”

Interrogation of Mammalian Protein Complex Structure, Function, and Membership Using Genome-Scale Fitness Screens

“…We and others have observed that cancer cell lines exhibit highly variable genetic dependencies for cellular fitness (Bertomeu et al, 2018; Blomen et al, 2015; Hart et al, 2015; Hart et al, 2017a; McDonald et al, 2017; Meyers et al, 2017; Tsherniak et al, 2017; Wang et al, 2015; Wang et al, 2017b) in a manner that reflects the diverse genomic and transcriptomic alterations a cell may accumulate during tumorigenesis. Project Achilles (Broad Institute of MIT and Harvard) seeks to systematically map genetic vulnerabilities across large collections of cancer cell lines, including the Cancer Cell Line Encyclopedia (CCLE) (Barretina et al, 2012), and has recently performed genome-scale perturbation screens in 501 cancer cell lines using RNA interference (RNAi) (Tsherniak et al, 2017) and in 342 cancer cell lines using CRISPR-Cas9 (Meyers et al, 2017).…”