In vivo AAV-SB-CRISPR screens of tumor-infiltrating primary NK cells identify genetic checkpoints of CAR-NK therapy.

Natural killer (NK) cells have clinical potential against cancer; however, multiple limitations hinder the success of NK cell therapy. Here, we performed unbiased functional mapping of tumor-infiltrating NK (TINK) cells using in vivo adeno-associated virus (AAV)-SB (Sleeping Beauty)-CRISPR (clustered regularly interspaced short palindromic repeats) screens in four solid tumor mouse models. In parallel, we characterized single-cell transcriptomic landscapes of TINK cells, which identified previously unexplored subpopulations of NK cells and differentially expressed TINK genes. As a convergent hit, CALHM2-knockout (KO) NK cells showed enhanced cytotoxicity and tumor infiltration in mouse primary NK cells and human chimeric antigen receptor (CAR)-NK cells. CALHM2 mRNA reversed the CALHM2-KO phenotype. CALHM2 KO in human primary NK cells enhanced their cytotoxicity, degranulation and cytokine production. Transcriptomics profiling revealed CALHM2-KO-altered genes and pathways in both baseline and stimulated conditions. In a solid tumor model resistant to unmodified CAR-NK cells, CALHM2-KO CAR-NK cells showed potent in vivo antitumor efficacy. These data identify endogenous genetic checkpoints that naturally limit NK cell function and demonstrate the use of CALHM2 KO for engineering enhanced NK cell-based immunotherapies.

AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells.

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.

Immunogenetic Metabolomics Reveals Key Enzymes That Modulate CAR T-cell Metabolism and Function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we investigated whether it is possible to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism whereby cancer cells suppress T-cell function by generating a metabolically unfavorable tumor microenvironment (TME). In an in silico screen, we identified ADA and PDK1 as metabolic regulators. We then showed that overexpression (OE) of these genes enhanced the cytolysis of CD19-specific chimeric antigen receptor (CAR) T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampened this effect. ADA-OE in CAR T cells improved cancer cytolysis under high concentrations of adenosine, the ADA substrate, and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics analysis of these CAR T cells revealed alterations of global gene expression and metabolic signatures in both ADA- and PDK1-engineered CAR T cells. Functional and immunologic analyses demonstrated that ADA-OE increased proliferation and decreased exhaustion in CD19-specific and HER2-specific CAR T cells. ADA-OE improved tumor infiltration and clearance by HER2-specific CAR T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR T cells and reveal potential targets for improving CAR T-cell therapy.

Perturbomics of tumor-infiltrating NK cells.

Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration. To unbiasedly reveal the functional genetic landscape underlying critical NK cell characteristics against cancer, we perform perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing. We establish a strategy with AAV-SleepingBeauty(SB)- CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes, and perform four independent in vivo tumor infiltration screens in mouse models of melanoma, breast cancer, pancreatic cancer, and glioblastoma. In parallel, we characterize single-cell transcriptomic landscapes of tumor-infiltrating NK cells, which identifies previously unexplored sub-populations of NK cells with distinct expression profiles, a shift from immature to mature NK (mNK) cells in the tumor microenvironment (TME), and decreased expression of mature marker genes in mNK cells. CALHM2, a calcium homeostasis modulator that emerges from both screen and single cell analyses, shows both in vitro and in vivo efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells. Differential gene expression analysis reveals that CALHM2 knockout reshapes cytokine production, cell adhesion, and signaling pathways in CAR- NKs. These data directly and systematically map out endogenous factors that naturally limit NK cell function in the TME to offer a broad range of cellular genetic checkpoints as candidates for future engineering to enhance NK cell-based immunotherapies.

Therapeutic immune cell engineering with an mRNA : AAV- Sleeping Beauty composite system.

Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC ( m RNA A AV-Sleeping-Beauty J oint E ngineering of S table T herapeutic I mmune C ells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo . This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

Immunogenetic metabolomics revealed key enzymes that modulate CAR-T metabolism and function.

Immune evasion is a critical step of cancer progression that remains a major obstacle for current T cell-based immunotherapies. Hence, we seek to genetically reprogram T cells to exploit a common tumor-intrinsic evasion mechanism, whereby cancer cells suppress T cell function by generating a metabolically unfavorable tumor microenvironment (TME). Specifically, we use an in silico screen to identify ADA and PDK1 as metabolic regulators, in which gene overexpression (OE) enhances the cytolysis of CD19-specific CD8 CAR-T cells against cognate leukemia cells, and conversely, ADA or PDK1 deficiency dampens such effect. ADA -OE in CAR-T cells improves cancer cytolysis under high concentrations of adenosine, the ADA substrate and an immunosuppressive metabolite in the TME. High-throughput transcriptomics and metabolomics in these CAR-Ts reveal alterations of global gene expression and metabolic signatures in both ADA- and PDK1- engineered CAR-T cells. Functional and immunological analyses demonstrate that ADA -OE increases proliferation and decreases exhaustion in α-CD19 and α-HER2 CAR-T cells. ADA-OE improves tumor infiltration and clearance by α-HER2 CAR-T cells in an in vivo colorectal cancer model. Collectively, these data unveil systematic knowledge of metabolic reprogramming directly in CAR-T cells, and reveal potential targets for improving CAR-T based cell therapy. Synopsis: The authors identify the adenosine deaminase gene (ADA) as a regulatory gene that reprograms T cell metabolism. ADA-overexpression (OE) in α-CD19 and α-HER2 CAR-T cells increases proliferation, cytotoxicity, memory, and decreases exhaustion, and ADA-OE α-HER2 CAR-T cells have enhanced clearance of HT29 human colorectal cancer tumors in vivo .

Double knockout CRISPR screen for cancer resistance to T cell cytotoxicity.

Immunotherapy has transformed cancer treatments; however, a large fraction of patients encounter resistance. Such resistance is mediated by complex factors, often involving interactions between multiple genes. Thus, it is crucially important to identify genetic interactions between genes that are significantly mutated in cancer patients and those involved in immune responses, ideally the ones that currently have chemical compounds for direct targeting. To systematically interrogate such genetic interactions that mediate cancer cells' response to T cell killing, we designed an asymmetric dual perturbation library targeting the matched combinations between significantly mutated tumor suppressors and immune resistance genes. We performed a combinatorial double knockout screen on 1159 gene pairs and identified those where joint loss-of-function renders altered cellular response to T cell cytotoxicity. We also performed comparative transcriptomics-based analyses on tumor and normal samples from TCGA and GTEx cohorts, mutational profiling analyses, and survival analyses to further characterize the significance of identified hits in clinical patients. Interactions between significantly mutated tumor suppressors and potentially druggable immune resistance genes may offer insights on potential new concepts of how to target clinically relevant cancer mutations with currently available agents. This study also provides a technology platform and an asymmetric double knockout library for interrogating genetic interactions between cancer mutations and immune resistance pathways under various settings.

A genome-scale gain-of-function CRISPR screen in CD8 T cells identifies proline metabolism as a means to enhance CAR-T therapy.

Chimeric antigen receptor (CAR)-T cell-based immunotherapy for cancer and immunological diseases has made great strides, but it still faces multiple hurdles. Finding the right molecular targets to engineer T cells toward a desired function has broad implications for the armamentarium of T cell-centered therapies. Here, we developed a dead-guide RNA (dgRNA)-based CRISPR activation screen in primary CD8+ T cells and identified gain-of-function (GOF) targets for CAR-T engineering. Targeted knockin or overexpression of a lead target, PRODH2, enhanced CAR-T-based killing and in vivo efficacy in multiple cancer models. Transcriptomics and metabolomics in CAR-T cells revealed that augmenting PRODH2 expression reshaped broad and distinct gene expression and metabolic programs. Mitochondrial, metabolic, and immunological analyses showed that PRODH2 engineering enhances the metabolic and immune functions of CAR-T cells against cancer. Together, these findings provide a system for identification of GOF immune boosters and demonstrate PRODH2 as a target to enhance CAR-T efficacy.

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617.

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identify two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generate a bispecific antibody. Lead antibodies show strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solve several cryo-EM structures at ~3 Å resolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and reveal distinct epitopes, binding patterns, and conformations. The lead clones also show potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generate and characterize a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

Prdm6 controls heart development by regulating neural crest cell differentiation and migration.

The molecular mechanisms that drive the acquisition of distinct neural crest cell (NCC) fates is still poorly understood. Here, we identified Prdm6 as an epigenetic modifier that temporally and spatially regulates the expression of NCC specifiers and determines the fate of a subset of migrating cardiac NCCs (CNCCs). Using transcriptomic analysis and genetic and fate mapping approaches in transgenic mice, we showed that disruption of Prdm6 was associated with impaired CNCC differentiation, delamination, and migration and led to patent ductus arteriosus (DA) and ventricular noncompaction. Bulk and single-cell RNA-Seq analyses of the DA and CNCCs identified Prdm6 as a regulator of a network of CNCC specification genes, including Wnt1, Tfap2b, and Sox9. Loss of Prdm6 in CNCCs diminished its expression in the pre-epithelial-mesenchymal transition (pre-EMT) cluster, resulting in the retention of NCCs in the dorsal neural tube. This defect was associated with diminished H4K20 monomethylation and G1-S progression and augmented Wnt1 transcript levels in pre-EMT and neural tube clusters, which we showed was the major driver of the impaired CNCC migration. Altogether, these findings revealed Prdm6 as a key regulator of CNCC differentiation and migration and identified Prdm6 and its regulated network as potential targets for the treatment of congenital heart diseases.

Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617.

COVID-19 pathogen SARS-CoV-2 has infected hundreds of millions and caused over 5 million deaths to date. Although multiple vaccines are available, breakthrough infections occur especially by emerging variants. Effective therapeutic options such as monoclonal antibodies (mAbs) are still critical. Here, we report the development, cryo-EM structures, and functional analyses of mAbs that potently neutralize SARS-CoV-2 variants of concern. By high-throughput single cell sequencing of B cells from spike receptor binding domain (RBD) immunized animals, we identified two highly potent SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity, and generated a bispecific antibody. Lead antibodies showed strong inhibitory activity against historical SARS-CoV-2 and several emerging variants of concern. We solved several cryo-EM structures at ∼3 Šresolution of these neutralizing antibodies in complex with prefusion spike trimer ectodomain, and revealed distinct epitopes, binding patterns, and conformations. The lead clones also showed potent efficacy in vivo against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We also generated and characterized a humanized antibody to facilitate translation and drug development. The humanized clone also has strong potency against both the original virus and the B.1.617.2 Delta variant. These mAbs expand the repertoire of therapeutics against SARS-CoV-2 and emerging variants.

CRISPR-Sunspot: Imaging of endogenous low-abundance RNA at the single-molecule level in live cells.

CRISPR/Cas-based mRNA imaging has been developed to labeling of high-abundance mRNAs. A lack of non-genetically encoded mRNA-tagged imaging tools has limited our ability to explore the functional distributions of endogenous low-abundance mRNAs in cells. Here, we developed a CRISPR-Sunspot method based on the SunTag signal amplification system that allows efficient imaging of low-abundance mRNAs with CRISPR/Cas9. Methods: We created a stable TRE3G-dCas9-EGFP cell line and generated an Inducible dCas9-EGFP imaging system for assessment of two factors, sgRNA and dCas9, which influence imaging quality. Based on SunTag system, we established a CRISPR-Sunspot imaging system for amplifying signals from single-molecule mRNA in live cells. CRISPR-Sunspot was used to track co-localization of Camk2a mRNA with regulatory protein Xlr3b in neurons. CRISPR-Sunspot combined with CRISPRa was used to determine elevated mRNA molecules. Results: Our results showed that manipulating the expression of fluorescent proteins and sgRNA increased the efficiency of RNA imaging in cells. CRISPR-Sunspot could target endogenous mRNAs in the cytoplasm and amplified signals from single-molecule mRNA. Furthermore, CRISPR-Sunspot was also applied to visualize mRNA distributions with its regulating proteins in neurons. CRISPR-Sunspot detected the co-localization of Camk2a mRNA with overexpressed Xlr3b proteins in the neuronal dendrites. Moreover, we also manipulated CRISPR-Sunspot to detect transcriptional activation of target gene such as HBG1 in live cells. Conclusion: Our findings suggest that CRISPR-Sunspot is a novel applicable imaging tool for visualizing the distributions of low-abundance mRNAs in cells. This study provides a novel strategy to unravel the molecular mechanisms of diseases caused by aberrant mRNA molecules.

CRISPR-GEMM Pooled Mutagenic Screening Identifies KMT2D as a Major Modulator of Immune Checkpoint Blockade.

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.

Comparative mRNA and LncRNA Analysis of the Molecular Mechanisms Associated With Low Silk Production in Bombyx mori.

Naked pupa sericin and Naked pupa are two mutant strains of Bombyx mori with extremely low or no fibroin production compared to the Qiufeng and Baiyu strains, both of which exhibit very high silk fibroin production. However, the molecular mechanisms by which long non-coding RNAs regulate fibroin synthesis need further study. In this study, we performed high-throughput RNA-seq to investigate lncRNA and mRNA expression profiles in the posterior silk gland of Qiufeng, Baiyu, Nd-sD, and Nd silkworms at the third day of the 5th instar. Our efforts yielded 26,767 novel lncRNAs and 6,009 novel mRNAs, the expression levels of silk protein genes and silk gland transcription factors were decreased in Qiufeng vs. Nd-sD and Qiufeng vs. Nd, while those of many genes related to autophagy, apoptosis, RNA degradation, ubiquitin-mediated proteolysis and heat shock proteins were increased. Moreover, the expression of a large number of genes responsible for protein synthesis and secretion was significantly decreased in Nd. GO and KEGG analysis results showed that nucleotide excision repair, mRNA surveillance pathways, amino acid degradation, protein digestion and absorption, ER-associated degradation and proteasome pathways were significantly enriched for the Qiufeng vs. Nd-sD and Qiufeng vs. Nd comparisons. In conclusion, our findings contribute to the lncRNA and mRNA database of Bombyx mori, and the identified differentially expressed mRNAs and lncRNAs help to reveal the molecular mechanisms of low silk production in Nd-sD and Nd, providing new insights for improvement of silk yield and elucidation of silk mechanical properties.

Multiplexed activation of endogenous genes by CRISPRa elicits potent antitumor immunity.

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.

In vivo CRISPR screening in CD8 T cells with AAV-Sleeping Beauty hybrid vectors identifies membrane targets for improving immunotherapy for glioblastoma.

Targeting membrane proteins could improve the efficacy of T cell-based immunotherapies. To facilitate the identification of T cell targets, we developed a hybrid genetic screening system where the Sleeping Beauty (SB) transposon and single guide RNA cassette are nested in an adeno-associated virus (AAV). SB-mediated genomic integration of the single guide RNA cassette enables efficient gene editing in primary murine T cells as well as a screen readout. We performed in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma (GBM). We validated screen hits by demonstrating that adoptive transfer of CD8+ T cells with Pdia3, Mgat5, Emp1 or Lag3 gene editing enhances the survival of GBM-bearing mice in both syngeneic and T-cell receptor transgenic models. Transcriptome profiling, single cell sequencing, cytokine assays and T cell signaling analysis showed that Pdia3 editing in T cells enhances effector functions. Engineered PDIA3 mutant EGFRvIII chimeric antigen T cells are more potent in antigen-specific killing of human GBM cells.

Systematic Immunotherapy Target Discovery Using Genome-Scale In Vivo CRISPR Screens in CD8 T Cells.

CD8 T cells play essential roles in anti-tumor immune responses. Here, we performed genome-scale CRISPR screens in CD8 T cells directly under cancer immunotherapy settings and identified regulators of tumor infiltration and degranulation. The in vivo screen robustly re-identified canonical immunotherapy targets such as PD-1 and Tim-3, along with genes that have not been characterized in T cells. The infiltration and degranulation screens converged on an RNA helicase Dhx37. Dhx37 knockout enhanced the efficacy of antigen-specific CD8 T cells against triple-negative breast cancer in vivo. Immunological characterization in mouse and human CD8 T cells revealed that DHX37 suppresses effector functions, cytokine production, and T cell activation. Transcriptomic profiling and biochemical interrogation revealed a role for DHX37 in modulating NF-κB. These data demonstrate high-throughput in vivo genetic screens for immunotherapy target discovery and establishes DHX37 as a functional regulator of CD8 T cells.

Improving Silkworm Genome Annotation Using a Proteogenomics Approach.

The silkworm genome has been deeply sequenced and assembled, but accurate genome annotation, which is important for modern biological research, remains far from complete. To improve silkworm genome annotation, we carried out a proteogenomics analysis using 9.8 million mass spectra collected from different tissues and developmental stages of the silkworm. The results confirmed the translational products of 4307 existing gene models and identified 1701 novel genome search-specific peptides (GSSPs). Using these GSSPs, 74 novel gene-coding sequences were identified, and 121 existing gene models were corrected. We also identified 1182 novel junction peptides based on an exon-skipping database that resulted in the identification of 973 alternative splicing sites. Furthermore, we performed RNA-seq analysis to improve silkworm genome annotation at the transcriptional level. A total of 1704 new transcripts and 1136 new exons were identified, 2581 untranslated regions (UTRs) were revised, and 1301 alternative splicing (AS) genes were identified. The transcriptomics results were integrated with the proteomics data to further complement and verify the new annotations. In addition, 14 incorrect genes and 10 skipped exons were verified using the two analysis methods. Altogether, we identified 1838 new transcripts and 1593 AS genes and revised 5074 existing genes using proteogenomics and transcriptome analyses. Data are available via ProteomeXchange with identifier PXD009672. The large-scale proteogenomics and transcriptome analyses in this study will greatly improve silkworm genome annotation and contribute to future studies.

In vivo profiling of metastatic double knockouts through CRISPR-Cpf1 screens.

Systematic investigation of the genetic interactions that influence metastatic potential has been challenging. Here we developed massively parallel CRISPR-Cpf1/Cas12a crRNA array profiling (MCAP), an approach for combinatorial interrogation of double knockouts in vivo. We designed an MCAP library of 11,934 arrays targeting 325 pairwise combinations of genes implicated in metastasis. By assessing the metastatic potential of the double knockouts in mice, we unveiled a quantitative landscape of genetic interactions that drive metastasis.