The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3′ region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.

There is a need in molecular biology and biomedical research for open-ended, hypothesis-generating research, in order to discover previously unknown molecular mechanisms. Genetic screening provides a powerful approach for identifying genes, pathways and mechanisms involved in a given phenotype or biological process. This is illustrated by the many successes of forward genetics in cell lines 1 and in model organisms such as flies 2,3 , worms 4 , yeast 5 , plants 6 and fish 7 , and pioneering work in RNA interference (RNAi) screens 8,9 .CRISPR screens exploit the efficiency and flexibility of CRISPR-Cas genome editing 10 . They have become a popular and productive tool for biological discovery in a broad range of applications 11,12 . In a typical pooled CRISPR screen (FIg. 1), a CRISPR guide RNA (gRNA) library is introduced in bulk into cells, such that individual cells receive different gRNAs and are perturbed according to the gRNA received by the cell. These gRNAs are usually delivered by lentiviral transduction and are integrated into the DNA of the target cells, making it possible to efficiently determine the induced perturbations based on the gRNA sequence.

Highlights d In vivo and in vitro genome-scale CD8 T cell CRISPR screen in immunotherapy contexts d Dhx37 knockout in CD8 T cells enhances adoptive transfer efficacy d Dhx37 modulates CD8 T cell activation, cytokine production, and cytotoxicity d DHX37 interacts with PDCD11 and influences NF-kB activity

A causative understanding of genetic factors that regulate glioblastoma (GBM) pathogenesis is of central importance. Here, we developed an adeno-associated virus (AAV)-mediated autochthonous CRISPR screen in GBM. Stereotaxic delivery of an AAV library targeting genes commonly mutated in human cancers into the brains of conditional Cas9 mice resulted in tumors that recapitulate human GBM. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlate with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as Mll2, B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express Homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo.