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Prime editing enables diverse genomic alterations to be written into target sites without requiring double-strand breaks or donor templates. The design of prime-editing guide RNAs (pegRNAs), which must be customized for each edit, can however be complex and time consuming. Compared with single guide RNAs (sgRNAs), pegRNAs have an additional 3' extension composed of a primer binding site and a reverse-transcription template. Here we report a web tool, which we named pegFinder ( http://pegfinder.sidichenlab.org ), for the rapid design of pegRNAs from reference and edited DNA sequences. pegFinder can incorporate sgRNA on-target and off-target scoring predictions into its ranking system, and nominates secondary nicking sgRNAs for increasing editing efficiency. CRISPR-associated protein 9 variants with expanded targeting ranges are also supported. To facilitate downstream experimentation, pegFinder produces a comprehensive table of candidate pegRNAs, along with oligonucleotide sequences for cloning.

Immunotherapy has transformed cancer treatment. However, current immunotherapy modalities face various limitations. In the present study, we developed multiplexed activation of endogenous genes as an immunotherapy (MAEGI), a new form of immunotherapy that elicits antitumor immunity through multiplexed activation of endogenous genes in tumors. We leveraged CRISPR activation (CRISPRa) to directly augment the in situ expression of endogenous genes, and thereby the presentation of tumor antigens, leading to dramatic antitumor immune responses. Deploying this as a cell-based vaccination strategy showed efficacy in both prophylactic and therapeutic settings. Intratumoral adeno-associated virus delivery of CRISPRa libraries elicited strong antitumor immunity across multiple cancer types. Precision targeting of mutated gene sets eradicated a large fraction of established tumors at both local and distant sites. This treatment modality led to alterations in the tumor microenvironment, marked by enhanced T cell infiltration and antitumor immune signatures. Multiplexed endogenous gene activation is a versatile and highly scalable strategy to elicit potent immune responses against cancer, distinct from all existing cancer therapies. CRISPR activation (CRISPRa) can target select genes and, rather than being used to delete them, can be used to activate their expression. Chen and colleagues use a CRISPRa-based approach to drive the expression of multiple endogenous genes in tumors and presentation of the antigens encoded, thus enhancing antitumor immune responses.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host–viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air–liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway., Single-cell analysis of human airway epithelial cells infected with SARS-CoV-2 provides novel insights into viral replication, cell tropism, and host-viral interactions. This study reveals novel polyadenylated viral transcripts, preferential tropism for ciliated cells that later extends to other epithelial cell types, and cell-intrinsic expression of type I and type III IFNs and IL6 induced by infection, resulting in expression of interferon-stimulated genes in both infected and bystander cells.

Age is a major risk factor for severe coronavirus disease-2019 (COVID-19). Here, we interrogate the transcriptional features and cellular landscape of the aging human lung. By intersecting these age-associated changes with experimental data on SARS-CoV-2, we identify several factors that may contribute to the heightened severity of COVID-19 in older populations. The aging lung is transcriptionally characterized by increased cell adhesion and stress responses, with reduced mitochondria and cellular replication. Deconvolution analysis reveals that the proportions of alveolar type 2 cells, proliferating basal cells, goblet cells, and proliferating natural killer/T cells decrease with age, whereas alveolar fibroblasts, pericytes, airway smooth muscle cells, endothelial cells and IGSF21+ dendritic cells increase with age. Several age-associated genes directly interact with the SARS-CoV-2 proteome. Age-associated genes are also dysregulated by SARS-CoV-2 infection in vitro and in patients with severe COVID-19. These analyses illuminate avenues for further studies on the relationship between age and COVID-19., Age is one of the strongest risk factors for severe illness from COVID-19. By integrating human lung transcriptomes with experimental data on SARS-CoV-2, the authors pinpoint specific age-associated factors that could contribute to the heightened severity of COVID-19 in older populations.

Immune checkpoint blockade (ICB) has shown remarkable clinical efficacy in several cancer types. However, only a fraction of patients will respond to ICB. Here, we performed pooled mutagenic screening with CRISPR-mediated genetically engineered mouse models (CRISPR-GEMM) in ICB settings, and identified KMT2D as a major modulator of ICB response across multiple cancer types. KMT2D encodes a histone H3K4 methyltransferase and is among the most frequently mutated genes in patients with cancer. Kmt2d loss led to increased DNA damage and mutation burden, chromatin remodeling, intron retention, and activation of transposable elements. In addition, Kmt2d-mutant cells exhibited increased protein turnover and IFNγ-stimulated antigen presentation. In turn, Kmt2d-mutant tumors in both mouse and human were characterized by increased immune infiltration. These data demonstrate that Kmt2d deficiency sensitizes tumors to ICB by augmenting tumor immunogenicity, and also highlight the power of CRISPR-GEMMs for interrogating complex molecular landscapes in immunotherapeutic contexts that preserve the native tumor microenvironment.Significance:ICB is ineffective in the majority of patients. Through direct in vivo CRISPR mutagenesis screening in GEMMs of cancer, we find Kmt2d deficiency sensitizes tumors to ICB. Considering the prevalence of KMT2D mutations, this finding potentially has broad implications for patient stratification and clinical decision-making.This article is highlighted in the In This Issue feature, p. 1775

Clustered regularly interspaced short palindromic repeats (CRISPR) screening is a powerful toolset for investigating diverse biological processes. Most CRISPR screens to date have been performed with in vitro cultures or cellular transplant models. To interrogate cancer in animal models that more closely recapitulate the human disease, autochthonous direct in vivo CRISPR screens have recently been developed that can identify causative drivers in the native tissue microenvironment. By empowering multiplexed mutagenesis in fully immunocompetent animals, direct in vivo CRISPR screens enable the rapid generation of patient-specific avatars that can guide precision medicine. This Opinion article discusses the current status of in vivo CRISPR screens in cancer and offers perspectives on future applications.

Mutations and genetic alterations are often sequentially acquired in various biological and pathological processes, such as development, evolution, and cancer. Certain phenotypes only manifest with precise temporal sequences of genetic events. While multiple approaches have been developed to model the effects of mutations in tumorigenesis, few recapitulate the stepwise nature of cancer evolution. Here we describe a flexible sequential mutagenesis system, Cpf1-Flip, with inducible inversion of a single crRNA array (FlipArray), and demonstrate its application in stepwise mutagenesis in murine and human cells. As a proof-of-concept, we further utilize Cpf1-Flip in a pooled-library approach to model the acquisition of diverse resistance mutations to cancer immunotherapy. Cpf1-Flip offers a simple, versatile, and controlled approach for precise mutagenesis of multiple loci in a sequential manner., Few models recapitulate the stepwise nature of cancer evolution. Here the authors introduce Cpf1-Flip, a system to introduce mutations sequentially through inducible inversion of a crRNA array.

A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as 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.

Small nucleolar RNAs (snoRNAs) constitute a family of non-coding RNAs that are classically known as guide RNAs for processing and modification of ribosomal RNAs. Recently, it was discovered that snoRNAs can be further processed into sno-derived RNAs (sdRNAs), some of which are known to exhibit microRNA-like properties. SdRNAs have been implicated in human cancer; however, a systems-level sdRNA landscape in human cancers is lacking. Through integrative analysis of ~22 nt size-selected smRNA-seq datasets from 10,262 patient samples across 32 cancer types, we mapped a pan-cancer sdRNAome and interrogated its signatures in multiple clinically relevant features, particularly cancer immunity and clinical outcome. Aggregating sdRNA abundances by parental snoRNAs, these expression signatures alone are sufficient to distinguish patients with distinct cancer types. Interestingly, a large panel of sdRNAs are significantly correlated with features of the tumor-immune microenvironment, such as immunosuppressive markers, CD8+ T cell infiltration, cytolytic T cell activity, and tumor vasculature. A set of individual sdRNAs with tumor-immune signatures can also stratify patient survival. These findings implicate snoRNAs and their derivative sdRNAs as a class of prevalent non-coding molecular markers of human cancer immunity.

Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas somemutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct highthroughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adenoassociated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and diedwithin 4 months.We usedmolecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence of multiple variants in driving liver tumorigenesis in immunocompetent mice. AAV-mediated autochthonous CRISPR screens provide a powerful means for mapping a provisional functional cancer genome atlas of tumor suppressors in vivo., Damon Runyon Cancer Research Foundation (DRG-2117-12; DFS-13-15), Melanoma Research Foundation (412806, 16-003524), St. Baldrick’s Foundation (426685), American Cancer Society (IRG 58-012-54), Cancer Research Institute (New York, N.Y.), Breast Cancer Alliance, Cancer Research Institute (New York, N.Y.) (Clinic and Laboratory Integration Program), American Association for Cancer Research (499395), United States. Department of Defense (W81XWH-17-1-0235), National Cancer Institute (U.S.) (1U54CA209992), National Cancer Institute (U.S.) (5P50CA196530-A10805), National Cancer Institute (U.S.) (4P50CA121974-A08306), National Institutes of Health (U.S.) (R01-CA133404), National Institutes of Health (U.S.) (R01-GM034277), National Institutes of Health (U.S.) (CCNE), Skolkovo Foundation, Casimir-Lambert Fund, National Institutes of Health (U.S.) (grant 1R01-HG009761), National Institutes of Health (U.S.) (grant R01-MH110049), National Institutes of Health (U.S.) (grant DP1-HL141201), Howard Hughes Medical Institute, New York Stem Cell Foundation, Simons, Paul G. Allen Family, Vallee Foundations, Poitras Center for Affective Disorders Research at MIT, Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT

Summary Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.

We show that the loss or gain of transcription factor programs that govern embryonic cell-fate specification is associated with a form of tumor plasticity characterized by the acquisition of alternative cell fates normally characteristic of adjacent organs. In human non-small cell lung cancers, downregulation of the lung lineage-specifying TF NKX2-1 is associated with tumors bearing features of various gut tissues. Loss of Nkx2-1 from murine alveolar, but not airway, epithelium results in conversion of lung cells to gastric-like cells. Superimposing oncogenic Kras activation enables further plasticity in both alveolar and airway epithelium, producing tumors that adopt midgut and hindgut fates. Conversely, coupling Nkx2-1 loss with foregut lineage-specifying SOX2 overexpression drives the formation of squamous cancers with features of esophageal differentiation. These findings demonstrate that elements of pathologic tumor plasticity mirror the normal developmental history of organs in that cancer cells acquire cell fates associated with developmentally related neighboring organs.