The simplicity of programming the CRISPR-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 that inhibits mutant protein kinase BRAF. 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.

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N6-methyladenosine (m6A) is a common modification of mRNA, with potential roles in fine-tuning the RNA life-cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of the methyltransferase complex, and show that three of them, WTAP, METTL14 and KIAA1429, are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps, and their classification into WTAP-dependent and independent sites. WTAP-dependent sites are located at internal positions in transcripts, are topologically static across a variety of systems we surveyed, and are inversely correlated with mRNA stability, consistent with a role in establishing ‘basal’ degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure, and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data sheds new light on proteomic and transcriptional underpinnings of this epitranscriptomic modification.

Genome-wide, targeted loss-of-function pooled screens using the CRISPR (clustered regularly interspaced short palindrome repeats)?associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi). Initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, limiting the range of biological systems amenable to screening. In this work, we present 1- and 2-vector lentiCRISPR systems capable of producing higher viral titers and, in these vectors, new human and mouse libraries for genome-scale CRISPR knock-out (GeCKO) screening.

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified from the screen, we further describe strategies for confirming the screening phenotype as well as genetic perturbation through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9–15 weeks followed by 4–5 weeks of validation.

Forward genetic screens are powerful tools for the discovery and functional annotation of genetic elements. Recently, the RNA-guided CRISPR (clustered regularly interspaced short palindromic repeat)-associated Cas9 nuclease has been combined with genome-scale guide RNA libraries for unbiased, phenotypic screening. In this Review, we describe recent advances using Cas9 for genome-scale screens, including knockout approaches that inactivate genomic loci and strategies that modulate transcriptional activity. We discuss practical aspects of screen design, provide comparisons with RNA interference (RNAi) screening, and outline future applications and challenges.

Summary Enhancers, critical determinants of cellular identity, are commonly identified by correlative chromatin marks and gain-of-function potential, though only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously we identified an erythroid enhancer of BCL11A, subject to common genetic variation associated with fetal hemoglobin (HbF) level, whose mouse ortholog is necessary for erythroid BCL11A expression. Here we develop pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for HbF reinduction. The detailed enhancer map will inform therapeutic genome editing. The screening approach described here is generally applicable to functional interrogation of noncoding genomic elements.

Somatic gene mutations can alter the vulnerability of cancer cells to T cell-based immunotherapies. To mimic loss-of-function mutations involved in resistance to these therapies, we perturbed genes in tumour cells using a genome-scale CRISPR-Cas9 library comprising ~123,000 single guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells (EFT). We correlated these genes with cytolytic activity in ~11,000 patient tumours from The Cancer Genome Atlas. Among the genes validated using different cancer cell lines and antigens, we identified multiple loss-of-function mutations in APLNR, encoding Apelin receptor, in patient tumours refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-gamma responses in tumours, and its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in murine models. Collectively, our study links the loss of essential genes for EFT with the resistance or non-responsiveness of cancer to immunotherapies.