Components of the prokaryotic clustered, regularly interspaced, short palindromic repeats (CRISPR) loci have recently been repurposed for use in mammalian cells. The CRISPR-associated (Cas)9 can be ...programmed with a single guide RNA (sgRNA) to generate site-specific DNA breaks, but there are few known rules governing on-target efficacy of this system. We created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. We discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens. We provide an online tool for the design of highly active sgRNAs for any gene of interest.
The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that ...our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.
CRISPR loss of function screens are powerful tools to interrogate biology but exhibit a number of biases and artifacts that can confound the results. Here, we introduce Chronos, an algorithm for ...inferring gene knockout fitness effects based on an explicit model of cell proliferation dynamics after CRISPR gene knockout. We test Chronos on two pan-cancer CRISPR datasets and one longitudinal CRISPR screen. Chronos generally outperforms competitors in separation of controls and strength of biomarker associations, particularly when longitudinal data is available. Additionally, Chronos exhibits the lowest copy number and screen quality bias of evaluated methods. Chronos is available at https://github.com/broadinstitute/chronos .
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene ...function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
Cancer cells that express oncogenic alleles of RAS typically require sustained expression of the mutant allele for survival, but the molecular basis of this oncogene dependency remains incompletely ...understood. To identify genes that can functionally substitute for oncogenic RAS, we systematically expressed 15,294 open reading frames in a human KRAS-dependent colon cancer cell line engineered to express an inducible KRAS-specific shRNA. We found 147 genes that promoted survival upon KRAS suppression. In particular, the transcriptional coactivator YAP1 rescued cell viability in KRAS-dependent cells upon suppression of KRAS and was required for KRAS-induced cell transformation. Acquired resistance to Kras suppression in a Kras-driven murine lung cancer model also involved increased YAP1 signaling. KRAS and YAP1 converge on the transcription factor FOS and activate a transcriptional program involved in regulating the epithelial-mesenchymal transition (EMT). Together, these findings implicate transcriptional regulation of EMT by YAP1 as a significant component of oncogenic RAS signaling.
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•YAP1 substitutes for loss of oncogenic KRAS in human and murine cancers•YAP1 expression is required for KRAS-induced cell transformation•KRAS and YAP1 converge on FOS to regulate the epithelial-mesenchymal transition•YAP1 and FOS colocalize at promoters of genes involved in EMT
The transcriptional regulation of the epithelial-mesenchymal transition by the transcriptional coactivator YAP1 is a critical feature of cancer cell dependence on oncogenic KRAS.
Oxidative phosphorylation (OXPHOS) is the major pathway for ATP production in humans. Deficiencies in OXPHOS can arise from mutations in either mitochondrial or nuclear genomes and comprise the ...largest collection of inborn errors of metabolism. At present we lack a complete catalog of human genes and pathways essential for OXPHOS. Here we introduce a genome-wide CRISPR “death screen” that actively selects dying cells to reveal human genes required for OXPHOS, inspired by the classic observation that human cells deficient in OXPHOS survive in glucose but die in galactose. We report 191 high-confidence hits essential for OXPHOS, including 72 underlying known OXPHOS diseases. Our screen reveals a functional module consisting of NGRN, WBSCR16, RPUSD3, RPUSD4, TRUB2, and FASTKD2 that regulates the mitochondrial 16S rRNA and intra-mitochondrial translation. Our work yields a rich catalog of genes required for OXPHOS and, more generally, demonstrates the power of death screening for functional genomic analysis.
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•Death screening is a new method that actively selects and sequences dead cells•A high-quality catalog of genes essential for human OXPHOS is reported•NGRN, WBSCR16, RPUSD3, RPUSD4, TRUB2, and FASTKD2 form a 16S rRNA regulatory module
Arroyo et al. introduce CRISPR “death screening” and apply it to systematically identify human genes essential for oxidative phosphorylation. Among the hits, they reveal how five new genes encode a functional module that regulates mitochondrial 16S rRNA and intra-mitochondrial translation.
The availability of multiple datasets comprising genome-scale RNAi viability screens in hundreds of diverse cancer cell lines presents new opportunities for understanding cancer vulnerabilities. ...Integrated analyses of these data to assess differential dependency across genes and cell lines are challenging due to confounding factors such as batch effects and variable screen quality, as well as difficulty assessing gene dependency on an absolute scale. To address these issues, we incorporated cell line screen-quality parameters and hierarchical Bayesian inference into DEMETER2, an analytical framework for analyzing RNAi screens ( https://depmap.org/R2-D2 ). This model substantially improves estimates of gene dependency across a range of performance measures, including identification of gold-standard essential genes and agreement with CRISPR/Cas9-based viability screens. It also allows us to integrate information across three large RNAi screening datasets, providing a unified resource representing the most extensive compilation of cancer cell line genetic dependencies to date.
Wnt/β-catenin signaling plays a key role in the pathogenesis of colon and other cancers; emerging evidence indicates that oncogenic β-catenin regulates several biological processes essential for ...cancer initiation and progression. To decipher the role of β-catenin in transformation, we classified β-catenin activity in 85 cancer cell lines in which we performed genome-scale loss-of-function screens and found that β-catenin active cancers are dependent on a signaling pathway involving the transcriptional regulator YAP1. Specifically, we found that YAP1 and the transcription factor TBX5 form a complex with β-catenin. Phosphorylation of YAP1 by the tyrosine kinase YES1 leads to localization of this complex to the promoters of antiapoptotic genes, including BCL2L1 and BIRC5. A small-molecule inhibitor of YES1 impeded the proliferation of β-catenin-dependent cancers in both cell lines and animal models. These observations define a β-catenin-YAP1-TBX5 complex essential to the transformation and survival of β-catenin-driven cancers.
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► β-catenin-dependent cancers require YAP1 expression for survival ► β-catenin, YAP1, and TBX5 form a complex that drives expression of BIRC5 and BCL2L1 ► YES1 regulates the activity of the β-catenin-YAP1-TBX5 complex ► The YES1 inhibitor dasatinib inhibits the proliferation of β-catenin-active cells
Loss-of-function screens and β-catenin activity profiling in 85 cancer cell lines identified a transcriptional complex composed of YAP1, a known mediator of Hippo signaling, the transcription factor TBX5, and β-catenin. This complex is essential for the proliferation and tumorigenicity of β-catenin-active cell lines.
Mutations in IDH1 and IDH2, the genes coding for isocitrate dehydrogenases 1 and 2, are common in several human cancers, including leukemias, and result in overproduction of the (R)-enantiomer of ...2-hydroxyglutarate (R)-2HG. Elucidation of the role of IDH mutations and (R)-2HG in leukemogenesis has been hampered by a lack of appropriate cell-based models. Here, we show that a canonical IDH1 mutant, IDH1 R132H, promotes cytokine independence and blocks differentiation in hematopoietic cells. These effects can be recapitulated by (R)-2HG, but not (S)-2HG, despite the fact that (S)-2HG more potently inhibits enzymes, such as the 5'-methylcytosine hydroxylase TET2, that have previously been linked to the pathogenesis of IDH mutant tumors. We provide evidence that this paradox relates to the ability of (S)-2HG, but not (R)-2HG, to inhibit the EglN prolyl hydroxylases. Additionally, we show that transformation by (R)-2HG is reversible.
The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized ...loss-of-function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that microRNA (miRNA)-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a consensus gene signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 single guide RNAs (sgRNAs) in 6 cells lines and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.
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