Identification of Required Host Factors for SARS-CoV-2 Infection in Human Cells.

To better understand host-virus genetic dependencies and find potential therapeutic targets for COVID-19, we performed a genome-scale CRISPR loss-of-function screen to identify host factors required for SARS-CoV-2 viral infection of human alveolar epithelial cells. Top-ranked genes cluster into distinct pathways, including the vacuolar ATPase proton pump, Retromer, and Commander complexes. We validate these gene targets using several orthogonal methods such as CRISPR knockout, RNA interference knockdown, and small-molecule inhibitors. Using single-cell RNA-sequencing, we identify shared transcriptional changes in cholesterol biosynthesis upon loss of top-ranked genes. In addition, given the key role of the ACE2 receptor in the early stages of viral entry, we show that loss of RAB7A reduces viral entry by sequestering the ACE2 receptor inside cells. Overall, this work provides a genome-scale, quantitative resource of the impact of the loss of each host gene on fitness/response to viral infection.

Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.

A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks.

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.

Voices on technology: The molecular biologists' ever-expanding toy box.

With the focus on technology for this issue of Molecular Cell, a group of scientists working in different areas of molecular biology provide their perspective on the most recent important technological advance in their field, where the field is lacking, and their wish list for future technology development.

A genome-scale screen for synthetic drivers of T cell proliferation.

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However, further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-γ. In addition, we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-ß receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells, LTBR induced profound transcriptional and epigenomic remodelling, leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-κB pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and γδ T cells, highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes.

Autism genes converge on asynchronous development of shared neuron classes.

Genetic risk for autism spectrum disorder (ASD) is associated with hundreds of genes spanning a wide range of biological functions1-6. The alterations in the human brain resulting from mutations in these genes remain unclear. Furthermore, their phenotypic manifestation varies across individuals7,8. Here we used organoid models of the human cerebral cortex to identify cell-type-specific developmental abnormalities that result from haploinsufficiency in three ASD risk genes-SUV420H1 (also known as KMT5B), ARID1B and CHD8-in multiple cell lines from different donors, using single-cell RNA-sequencing (scRNA-seq) analysis of more than 745,000 cells and proteomic analysis of individual organoids, to identify phenotypic convergence. Each of the three mutations confers asynchronous development of two main cortical neuronal lineages-γ-aminobutyric-acid-releasing (GABAergic) neurons and deep-layer excitatory projection neurons-but acts through largely distinct molecular pathways. Although these phenotypes are consistent across cell lines, their expressivity is influenced by the individual genomic context, in a manner that is dependent on both the risk gene and the developmental defect. Calcium imaging in intact organoids shows that these early-stage developmental changes are followed by abnormal circuit activity. This research uncovers cell-type-specific neurodevelopmental abnormalities that are shared across ASD risk genes and are finely modulated by human genomic context, finding convergence in the neurobiological basis of how different risk genes contribute to ASD pathology.

Next-generation forward genetic screens: uniting high-throughput perturbations with single-cell analysis.

Programmable genome-engineering technologies, such as CRISPR (clustered regularly interspaced short palindromic repeats) nucleases and massively parallel CRISPR screens that capitalize on this programmability, have transformed biomedical science. These screens connect genes and noncoding genome elements to disease-relevant phenotypes, but until recently have been limited to individual phenotypes such as growth or fluorescent reporters of gene expression. By pairing massively parallel screens with high-dimensional profiling of single-cell types/states, we can now measure how individual genetic perturbations or combinations of perturbations impact the cellular transcriptome, proteome, and epigenome. We review technologies that pair CRISPR screens with single-cell multiomics and the unique opportunities afforded by extending pooled screens using deep multimodal phenotyping.

Genome-wide CRISPR screens in spheroid culture reveal that the tumor suppressor LKB1 inhibits growth via the PIKFYVE lipid kinase.

The tumor suppressor LKB1 is a serine/threonine protein kinase that is frequently mutated in human lung adenocarcinoma (LUAD). LKB1 regulates a complex signaling network that is known to control cell polarity and metabolism; however, the pathways that mediate the tumor-suppressive activity of LKB1 are incompletely defined. To identify mechanisms of LKB1-mediated growth suppression, we developed a spheroid-based cell culture assay to study LKB1-dependent growth. We then performed genome-wide CRISPR screens in spheroidal culture and found that LKB1 suppresses growth, in part, by activating the PIKFYVE lipid kinase. Finally, we used chemical inhibitors and a pH-sensitive reporter to determine that LKB1 impairs growth by promoting the internalization of wild-type EGFR in a PIKFYVE-dependent manner.

Heterozygous deletion of the autism-associated gene CHD8 impairs synaptic function through widespread changes in gene expression and chromatin compaction.

Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.

Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq.

Pooled CRISPR screens coupled with single-cell RNA-sequencing have enabled systematic interrogation of gene function and regulatory networks. Here, we introduce Cas13 RNA Perturb-seq (CaRPool-seq), which leverages the RNA-targeting CRISPR-Cas13d system and enables efficient combinatorial perturbations alongside multimodal single-cell profiling. CaRPool-seq encodes multiple perturbations on a cleavable CRISPR array that is associated with a detectable barcode sequence, allowing for the simultaneous targeting of multiple genes. We compared CaRPool-seq to existing Cas9-based methods, highlighting its unique strength to efficiently profile combinatorially perturbed cells. Finally, we apply CaRPool-seq to perform multiplexed combinatorial perturbations of myeloid differentiation regulators in an acute myeloid leukemia (AML) model system and identify extensive interactions between different chromatin regulators that can enhance or suppress AML differentiation phenotypes.

High-Throughput Approaches to Pinpoint Function within the Noncoding Genome.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease system is a powerful tool for genome editing, and its simple programmability has enabled high-throughput genetic and epigenetic studies. These high-throughput approaches offer investigators a toolkit for functional interrogation of not only protein-coding genes but also noncoding DNA. Historically, noncoding DNA has lacked the detailed characterization that has been applied to protein-coding genes in large part because there has not been a robust set of methodologies for perturbing these regions. Although the majority of high-throughput CRISPR screens have focused on the coding genome to date, an increasing number of CRISPR screens targeting noncoding genomic regions continue to emerge. Here, we review high-throughput CRISPR-based approaches to uncover and understand functional elements within the noncoding genome and discuss practical aspects of noncoding library design and screen analysis.

Transcription control by the ENL YEATS domain in acute leukaemia.

Recurrent chromosomal translocations producing a chimaeric MLL oncogene give rise to a highly aggressive acute leukaemia associated with poor clinical outcome. The preferential involvement of chromatin-associated factors as MLL fusion partners belies a dependency on transcription control. Despite recent progress made in targeting chromatin regulators in cancer, available therapies for this well-characterized disease remain inadequate, prompting the need to identify new targets for therapeutic intervention. Here, using unbiased CRISPR-Cas9 technology to perform a genome-scale loss-of-function screen in an MLL-AF4-positive acute leukaemia cell line, we identify ENL as an unrecognized gene that is specifically required for proliferation in vitro and in vivo. To explain the mechanistic role of ENL in leukaemia pathogenesis and dynamic transcription control, a chemical genetic strategy was developed to achieve targeted protein degradation. Acute loss of ENL suppressed the initiation and elongation of RNA polymerase II at active genes genome-wide, with pronounced effects at genes featuring a disproportionate ENL load. Notably, an intact YEATS chromatin-reader domain was essential for ENL-dependent leukaemic growth. Overall, these findings identify a dependency factor in acute leukaemia and suggest a mechanistic rationale for disrupting the YEATS domain in disease.

Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood.

Mammalian genomes contain thousands of loci that transcribe long noncoding RNAs (lncRNAs), some of which are known to carry out critical roles in diverse cellular processes through a variety of mechanisms. Although some lncRNA loci encode RNAs that act non-locally (in trans), there is emerging evidence that many lncRNA loci act locally (in cis) to regulate the expression of nearby genes-for example, through functions of the lncRNA promoter, transcription, or transcript itself. Despite their potentially important roles, it remains challenging to identify functional lncRNA loci and distinguish among these and other mechanisms. Here, to address these challenges, we developed a genome-scale CRISPR-Cas9 activation screen that targets more than 10,000 lncRNA transcriptional start sites to identify noncoding loci that influence a phenotype of interest. We found 11 lncRNA loci that, upon recruitment of an activator, mediate resistance to BRAF inhibitors in human melanoma cells. Most candidate loci appear to regulate nearby genes. Detailed analysis of one candidate, termed EMICERI, revealed that its transcriptional activation resulted in dosage-dependent activation of four neighbouring protein-coding genes, one of which confers the resistance phenotype. Our screening and characterization approach provides a CRISPR toolkit with which to systematically discover the functions of noncoding loci and elucidate their diverse roles in gene regulation and cellular function.

Erratum: Genome-scale activation screen identifies a lncRNA locus regulating a gene neighbourhood.

This corrects the article DOI: 10.1038/nature23451.

Identification of essential genes for cancer immunotherapy.

Somatic gene mutations can alter the vulnerability of cancer cells to T-cell-based immunotherapies. Here we perturbed genes in human melanoma cells to mimic loss-of-function mutations involved in resistance to these therapies, by using a genome-scale CRISPR-Cas9 library that consisted of around 123,000 single-guide RNAs, and profiled genes whose loss in tumour cells impaired the effector function of CD8+ T cells. The genes that were most enriched in the screen have key roles in antigen presentation and interferon-γ signalling, and correlate with cytolytic activity in 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 the apelin receptor, in patient tumours that were refractory to immunotherapy. We show that APLNR interacts with JAK1, modulating interferon-γ responses in tumours, and that its functional loss reduces the efficacy of adoptive cell transfer and checkpoint blockade immunotherapies in mouse models. Our results link the loss of essential genes for the effector function of CD8+ T cells with the resistance or non-responsiveness of cancer to immunotherapies.

Multiplexed detection of proteins, transcriptomes, clonotypes and CRISPR perturbations in single cells.

Multimodal single-cell assays provide high-resolution snapshots of complex cell populations, but are mostly limited to transcriptome plus an additional modality. Here, we describe expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-seq) for the high-throughput characterization of at least five modalities of information from each single cell. We demonstrate application of ECCITE-seq to multimodal CRISPR screens with robust direct single-guide RNA capture and to clonotype-aware multimodal phenotyping of cancer samples.

Effects of calcareous marine algae on milk production, feed intake, energy balance, mineral status, and inflammatory markers in transition dairy cows.

The objective of this experiment was to compare the effects of calcareous marine algae (CMA; Acid Buf, Celtic Sea Minerals) with a limestone-based control on feed intake, milk production, energy balance, serum mineral metabolites, and inflammatory markers in transition dairy cows. Twenty-two multiparous and 10 primiparous cows were assigned to 2 treatments from 25 d before expected parturition until 42 d postpartum. Cows were assigned to treatment according to a randomized complete block design based on parity, pre-experimental body condition score, previous 305-d milk yield, and either fat + protein yield (for multiparous cows) or predicted transmitting ability for milk yield and fat + protein yield (for primiparous cows). Cows were fed a negative dietary cation-anion difference [-50 mEq/kg] total mixed ration (TMR) based on corn silage, grass silage, and straw during the prepartum period and a 50:50 forage:concentrate TMR based on grass silage, corn silage, and concentrate during the postpartum period. The 2 dietary treatments consisted of a control (CON), which contained limestone as the primary calcium source, and CMA, in which limestone was replaced by CMA at 0.42% and 0.47% of dry matter for the pre- and postpartum periods, respectively. The dietary treatments were fed as 2 different concentrate pellets added to the TMR. Cows fed the CMA diet had higher dry matter intake in both the prepartum (+1.08 kg) and postpartum (+0.94 kg) periods compared with cows fed the CON diet. Fat yield (+0.11 kg), fat concentration (+0.43%), and 4% fat-corrected milk (+1.56 kg) were higher in cows fed CMA than in cows fed CON. The concentration of plasma serum amyloid A was reduced and that of serum P was increased on the CMA treatment compared with the CON treatment. These findings demonstrate the benefits of supplementing CMA to dairy cows during the transition period compared with a CON treatment containing limestone as the primary Ca source.

Invited review: Effect of subacute ruminal acidosis on gut health of dairy cows.

Subacute ruminal acidosis (SARA) is assumed to be a common disease in high-yielding dairy cows. Despite this, the epidemiological evidence is limited by the lack of survey data. The prevalence of SARA has mainly been determined by measuring the pH of ruminal fluid collected using rumenocentesis. This may not be sufficiently accurate, because the symptoms of SARA are not solely due to ruminal pH depression, and ruminal pH varies among sites in the rumen, throughout a 24-h period, and among days. The impact of SARA has mainly been studied by conducting SARA challenges in cows, sheep, and goats based on a combination of feed restriction and high-grain feeding. The methodologies of these challenges vary considerably among studies. Variations include differences in the duration and amount of grain feeding, type of grain, amount and duration of feed restriction, number of experimental cows, and sensitivity of cows to SARA challenges. Grain-based SARA challenges affect gut health. These effects include depressing the pH in, and increasing the toxin content of, digesta. They also include altering the taxonomic composition of microbiota, reducing the functionality of the epithelia throughout the gastrointestinal tract (GIT), and a moderate inflammatory response. The effects on the epithelia include a reduction in its barrier function. Effects on microbiota include reductions in their richness and diversity, which may reduce their functionality and reflect dysbiosis. Changes in the taxonomic composition of gut microbiota throughout the GIT are evident at the phylum level, but less evident and more variable at the genus level. Effects at the phylum level include an increase in the Firmicutes to Bacteroidetes ratio. More studies on the effects of a SARA challenge on the functionality of gut microbiota are needed. The inflammatory response resulting from grain-based SARA challenges is innate and moderate and mainly consists of an acute phase response. This response is likely a combination of systemic inflammation and inflammation of the epithelia of the GIT. The systemic inflammation is assumed to be caused by translocation of immunogenic compounds, including bacterial endotoxins and bioamines, through the epithelia into the interior circulation. This translocation is increased by the increase in concentrations of toxins in digesta and a reduction of the barrier function of epithelia. Severe SARA can cause rumenitis, but moderate SARA may activate an immune response in the epithelia of the GIT. Cows grazing highly fermentable pastures with high sugar contents can also have a low ruminal pH indicative of SARA. This is not accompanied by an inflammatory response but may affect milk production and gut microbiota. Grain-based SARA affects several aspects of gut health, but SARA resulting from grazing high-digestible pastures and insufficient coarse fiber less so. We need to determine which method for inducing SARA is the most representative of on-farm conditions.

High-throughput functional genomics using CRISPR-Cas9.

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.