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CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.

CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Simple Summary: Cancer is a disease caused by mutations in DNA differentiating cancer cells from normal cells. Precision medicine could exploit these genetic differences to identify and create therapeutic opportunities that selectively kill cancer cells while sparing normal cells. In some cancers, the same mutations recur, making them desirable biomarkers for genetics-based therapeutic strategies. This is the case for the gene ARID1A which is lost in about 50% of ovarian clear cell carcinomas. We used genetic screening to identify targets for killing an ovarian clear cell carcinoma cell line lacking the ARID1A gene. We identified a cancer stress response regulator called KEAP1 as a potential target for ARID1A-deficient ovarian clear cell carcinomas. Using different approaches and cell lines, we observed a relationship where ARID1A-deficient cells required KEAP1 for fitness. Together these data suggest that KEAP1 could be pursued as a means to selectively kill certain types of ARID1A-deficient cancer. ARID1A is the core DNA-binding subunit of the BAF chromatin remodeling complex and is mutated in about 8% of all cancers. The frequency of ARID1A loss varies between cancer subtypes, with clear cell ovarian carcinoma (CCOC) presenting the highest incidence at > 50% of cases. Despite a growing understanding of the consequences of ARID1A loss in cancer, there remains limited targeted therapeutic options for ARID1A-deficient cancers. Using a genome-wide CRISPR screening approach, we identify KEAP1 as a genetic dependency of ARID1A in CCOC. Depletion or chemical perturbation of KEAP1 results in selective growth inhibition of ARID1A-KO cell lines and edited primary endometrial epithelial cells. While we confirm that KEAP1-NRF2 signalling is dysregulated in ARID1A-KO cells, we suggest that this synthetic lethality is not due to aberrant NRF2 signalling. Rather, we find that KEAP1 perturbation exacerbates genome instability phenotypes associated with ARID1A deficiency. Together, our findings identify a potentially novel synthetic lethal interaction of ARID1A-deficient cells. [ABSTRACT FROM AUTHOR]

Rising cancer-related mortality underscores the importance of biomarkers for treatment and prognosis, with Chromosome Segregation 1 Like (CSE1L) linked to various cancers yet its roles remain partially understood. This study investigates CSE1L's expression and oncogenic mechanisms in solid tumors. We analyzed multi-omics data from 31 solid tumors, measured CSE1L in 41 head and neck carcinoma patients post-chemotherapy via qRT-PCR, and evaluated the impact of CSE1L knockdown on cell proliferation in A549 and HepG2 cells. In this study, we observed significantly elevated levels of CSE1L RNA in 13 tumor tissues and protein levels in 8 tumor tissues compared to their corresponding adjacent normal tissues. Additionally, our investigation unveiled a correlation between heightened CSE1L expression in tumor tissues and worsened patient prognosis, poor response to immunotherapy, and diminished effectiveness of neoadjuvant chemotherapy. Through an analysis of CSE1L mechanisms, we discovered its potential involvement in promoting tumor cell proliferation, enhancing drug resistance, and influencing immune infiltration, thereby impacting patient prognosis and treatment outcomes. Finally, we delved into the potential mechanisms underlying upregulation of CSE1L in tumor tissues. Our findings demonstrate that CSE1L promotes tumor development in various malignancies, highlighting its potential as both a therapeutic target and prognostic indicator. [ABSTRACT FROM AUTHOR]

Background: Immune checkpoint therapy (ICT) provides durable responses in select cancer patients, yet resistance remains a significant challenge, prompting the exploration of underlying molecular mechanisms. Tyrosylprotein sulfotransferase-2 (TPST2), known for its role in protein tyrosine O-sulfation, has been suggested to modulate the extracellular protein-protein interactions, but its specific role in cancer immunity remains largely unexplored. Methods: To explore tumor cell-intrinsic factors influencing anti-PD1 responsiveness, we conducted a pooled loss-of-function genetic screen in humanized mice engrafted with human immune cells. The responsiveness of cancer cells to interferon-γ (IFNγ) was estimated by evaluating IFNγ-mediated induction of target genes, STAT1 phosphorylation, HLA expression, and cell growth suppression. The sulfotyrosine-modified target gene of TPST2 was identified by co-immunoprecipitation and mass spectrometry. The in vivo effects of TPST2 inhibition were evaluated using mouse syngeneic tumor models and corroborated by bulk and single-cell RNA sequencing analyses. Results: Through in vivo genome-wide CRISPR screening, TPST2 loss-of-function emerged as a potential enhancer of anti-PD1 treatment efficacy. TPST2 suppressed IFNγ signaling by sulfating IFNγ receptor 1 at Y397 residue, while its downregulation boosted IFNγ-mediated signaling and antigen presentation. Depletion of TPST2 in cancer cells augmented anti-PD1 antibody efficacy in syngeneic mouse tumor models by enhancing tumor-infiltrating lymphocytes. RNA sequencing data revealed TPST2's inverse correlation with antigen presentation, and increased TPST2 expression is associated with poor prognosis and altered cancer immunity across cancer types. Conclusions: We propose TPST2's novel role as a suppressor of cancer immunity and advocate for its consideration as a therapeutic target in ICT-based treatments. [ABSTRACT FROM AUTHOR]

Advancements in the CRISPR technology, a game-changer in experimental research, have revolutionized various fields of life sciences and more profoundly, cancer research. Cell death pathways are among the most deregulated in cancer cells and are considered as critical aspects in cancer development. Through decades, our knowledge of the mechanisms orchestrating programmed cellular death has increased substantially, attributed to the revolution of cutting-edge technologies. The heroic appearance of CRISPR systems have expanded the available screening platform and genome engineering toolbox to detect mutations and create precise genome edits. In that context, the precise ability of this system for identification and targeting of mutations in cell death signaling pathways that result in cancer development and therapy resistance is an auspicious choice to transform and accelerate the individualized cancer therapy. The concept of personalized cancer therapy stands on the identification of molecular characterization of the individual tumor and its microenvironment in order to provide a precise treatment with the highest possible outcome and minimum toxicity. This study explored the potential of CRISPR technology in precision cancer treatment by identifying and targeting specific cell death pathways. It showed the promise of CRISPR in finding key components and mutations involved in programmed cell death, making it a potential tool for targeted cancer therapy. However, this study also highlighted the challenges and limitations that need to be addressed in future research to fully realize the potential of CRISPR in cancer treatment. Current application of CRISPR system in cancer therapy through a glance. A choosing the appropriate biological model for screening in vitro (using established cell lines, animal derived tumor cells, human derived tumor cells, stem cells or T cells), in vivo (using animal models which can harbor human derived tumor), or ex vivo (human/animal-derived organoids). B preparation of CRISPR gRNA library. C experimental design of CRISPR screening, identification of the desired gRNAs or phenotypic response. D CRISPR-Cas targeting of the identified targets, with Cas9 gene editing system (Knockout, base editing, prime editing), RNA modulation (modulation of RNA splicing, RNA base editing, RNA interference), and epigenomic edits and CRISPR interference/activation using dead Cas9 (dCas9) (Bock et al. 2022b) [ABSTRACT FROM AUTHOR]

Abstract Background Immune checkpoint therapy (ICT) provides durable responses in select cancer patients, yet resistance remains a significant challenge, prompting the exploration of underlying molecular mechanisms. Tyrosylprotein sulfotransferase-2 (TPST2), known for its role in protein tyrosine O-sulfation, has been suggested to modulate the extracellular protein-protein interactions, but its specific role in cancer immunity remains largely unexplored. Methods To explore tumor cell-intrinsic factors influencing anti-PD1 responsiveness, we conducted a pooled loss-of-function genetic screen in humanized mice engrafted with human immune cells. The responsiveness of cancer cells to interferon-γ (IFNγ) was estimated by evaluating IFNγ-mediated induction of target genes, STAT1 phosphorylation, HLA expression, and cell growth suppression. The sulfotyrosine-modified target gene of TPST2 was identified by co-immunoprecipitation and mass spectrometry. The in vivo effects of TPST2 inhibition were evaluated using mouse syngeneic tumor models and corroborated by bulk and single-cell RNA sequencing analyses. Results Through in vivo genome-wide CRISPR screening, TPST2 loss-of-function emerged as a potential enhancer of anti-PD1 treatment efficacy. TPST2 suppressed IFNγ signaling by sulfating IFNγ receptor 1 at Y397 residue, while its downregulation boosted IFNγ-mediated signaling and antigen presentation. Depletion of TPST2 in cancer cells augmented anti-PD1 antibody efficacy in syngeneic mouse tumor models by enhancing tumor-infiltrating lymphocytes. RNA sequencing data revealed TPST2’s inverse correlation with antigen presentation, and increased TPST2 expression is associated with poor prognosis and altered cancer immunity across cancer types. Conclusions We propose TPST2’s novel role as a suppressor of cancer immunity and advocate for its consideration as a therapeutic target in ICT-based treatments.

Comparative studies of great apes provide a window into our evolutionary past, but the extent and identity of cellular differences that emerged during hominin evolution remain largely unexplored. We established a comparative loss-of-function approach to evaluate whether human cells exhibit distinct genetic dependencies. By performing genome-wide CRISPR interference screens in human and chimpanzee pluripotent stem cells, we identified 75 genes with species-specific effects on cellular proliferation. These genes comprised coherent processes, including cell-cycle progression and lysosomal signaling, which we determined to be human-derived by comparison with orangutan cells. Human-specific robustness to CDK2 and CCNE1 depletion persisted in neural progenitor cells and cerebral organoids, supporting the G1-phase length hypothesis as a potential evolutionary mechanism in human brain expansion. Our findings demonstrate that evolutionary changes in human cells reshaped the landscape of essential genes and establish a platform for systematically uncovering latent cellular and molecular differences between species.

Abstract Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy. In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.

Abstract Oncolytic viruses (OVs) offer a novel approach to treat solid tumors; however, their efficacy is frequently suboptimal due to various limiting factors. To address this challenge, we engineered an OV containing targets for neuron-specific microRNA-124 and Granulocyte-macrophage colony-stimulating factor (GM-CSF), significantly enhancing its neuronal safety while minimally compromising its replication capacity. Moreover, we identified PARP1 as an HSV-1 replication restriction factor using genome-wide CRISPR screening. In models of glioblastoma (GBM) and triple-negative breast cancer (TNBC), we showed that the combination of OV and a PARP inhibitor (PARPi) exhibited superior efficacy compared to either monotherapy. Additionally, single-cell RNA sequencing (scRNA-seq) revealed that this combination therapy sensitized TNBC to immune checkpoint blockade, and the incorporation of an immune checkpoint inhibitor (ICI) further increased the survival rate of tumor-bearing mice. The combination of PARPi and ICI synergistically enhanced the ability of OV to establish durable tumor-specific immune responses. Our study effectively overcomes the inherent limitations of OV therapy, providing valuable insights for the clinical treatment of TNBC, GBM, and other malignancies.

Abstract Background Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. Results We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy. In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy. [ABSTRACT FROM AUTHOR]

Oncolytic viruses (OVs) offer a novel approach to treat solid tumors; however, their efficacy is frequently suboptimal due to various limiting factors. To address this challenge, we engineered an OV containing targets for neuron-specific microRNA-124 and Granulocyte-macrophage colony-stimulating factor (GM-CSF), significantly enhancing its neuronal safety while minimally compromising its replication capacity. Moreover, we identified PARP1 as an HSV-1 replication restriction factor using genome-wide CRISPR screening. In models of glioblastoma (GBM) and triple-negative breast cancer (TNBC), we showed that the combination of OV and a PARP inhibitor (PARPi) exhibited superior efficacy compared to either monotherapy. Additionally, single-cell RNA sequencing (scRNA-seq) revealed that this combination therapy sensitized TNBC to immune checkpoint blockade, and the incorporation of an immune checkpoint inhibitor (ICI) further increased the survival rate of tumor-bearing mice. The combination of PARPi and ICI synergistically enhanced the ability of OV to establish durable tumor-specific immune responses. Our study effectively overcomes the inherent limitations of OV therapy, providing valuable insights for the clinical treatment of TNBC, GBM, and other malignancies. [ABSTRACT FROM AUTHOR]

Background: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods: We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. Results: We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions: Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors. [ABSTRACT FROM AUTHOR]

UNLABELLED: The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction. ONE-SENTENCE SUMMARY: This mini-review describes experimental and computational approaches for functional genomic screening using CRISPR technologies in non-conventional microbes.

Abstract Background Long non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources). Methods INFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo. Results The INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo. Conclusions Our approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

Abstract Apatinib was the first anti-angiogenic agent approved for treatment of metastatic gastric cancer (GC). However, the emergence of resistance was inevitable. Thus investigating new and valuable off-target effect of apatinib directly against cancer cells is of great significance. Here, we identified extra spindle pole bodies-like 1 (ESPL1) was responsible for apatinib resistance in GC cells through CRISPR genome-wide gain-of-function screening. Loss of function studies further showed that ESPL1 inhibition suppressed cell proliferation, migration and promoted apoptosis in vitro, and accordingly ESPL1 knockdown sensitized GC cells to apatinib. In addition, we found ESPL1 interacted with mouse double minute 2 (MDM2), a E3 ubiquitin protein ligase, and the combination of MDM2 siRNA with apatinib synergistically ameliorated the resistance induced by ESPL1 overexpression. In summary, our study indicated that ESPL1 played a critical role in apatinib resistance in GC cells. Inhibition of MDM2 could rescue the sensitivity of GC cells to apatinib and reverse ESPL1-mediated resistance.

Abstract CRISPR genome editing approaches theoretically enable researchers to define the function of each human gene in specific cell types, but challenges remain to efficiently perform genetic perturbations in relevant models. In this work, we develop a library cloning protocol that increases sgRNA uniformity and greatly reduces bias in existing genome-wide libraries. We demonstrate that our libraries can achieve equivalent or better statistical power compared to previously reported screens using an order of magnitude fewer cells. This improved cloning protocol enables genome-scale CRISPR screens in technically challenging cell models and screen formats.

Background: Long non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources). Methods: INFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo. Results: The INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo. Conclusions: Our approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine. [ABSTRACT FROM AUTHOR]

Apatinib was the first anti-angiogenic agent approved for treatment of metastatic gastric cancer (GC). However, the emergence of resistance was inevitable. Thus investigating new and valuable off-target effect of apatinib directly against cancer cells is of great significance. Here, we identified extra spindle pole bodies-like 1 (ESPL1) was responsible for apatinib resistance in GC cells through CRISPR genome-wide gain-of-function screening. Loss of function studies further showed that ESPL1 inhibition suppressed cell proliferation, migration and promoted apoptosis in vitro, and accordingly ESPL1 knockdown sensitized GC cells to apatinib. In addition, we found ESPL1 interacted with mouse double minute 2 (MDM2), a E3 ubiquitin protein ligase, and the combination of MDM2 siRNA with apatinib synergistically ameliorated the resistance induced by ESPL1 overexpression. In summary, our study indicated that ESPL1 played a critical role in apatinib resistance in GC cells. Inhibition of MDM2 could rescue the sensitivity of GC cells to apatinib and reverse ESPL1-mediated resistance. [ABSTRACT FROM AUTHOR]

Organ transplantation is the gold standard therapy for end-stage organ failure. However, the shortage of available grafts and long-term graft dysfunction remain the primary barriers to organ transplantation. Exploring approaches to solve these issues is urgent, and CRISPR/Cas9-based transcriptome editing provides one potential solution. Furthermore, combining CRISPR/Cas9-based gene editing with an ex vivo organ perfusion system would enable pre-implantation transcriptome editing of grafts. How to determine effective intervention targets becomes a new problem. Fortunately, the advent of high-throughput CRISPR screening has dramatically accelerated the effective targets. This review summarizes the current advancements, utilization, and workflow of CRISPR screening in various immune and non-immune cells. It also discusses the ongoing applications of CRISPR/Cas-based gene editing in transplantation and the prospective applications of CRISPR screening in solid organ transplantation. [ABSTRACT FROM AUTHOR]

Abstract The CRISPR genome editing technology has revolutionized the way gene function is studied. Genome editing can be achieved in single genes or for thousands of genes simultaneously in sensitive genetic screens. While conventional genetic screens are limited to bulk measurements of cell behavior, recent developments in single-cell technologies make it possible to combine CRISPR screening with single-cell profiling. In this way, cell behavior and gene expression can be monitored simultaneously, with the additional possibility of including data on chromatin accessibility and protein levels. Moreover, the availability of various Cas proteins leading to inactivation, activation, or other effects on gene function further broadens the scope of such screens. The integration of single-cell multi-omics approaches with CRISPR screening open the path to high-content information on the impact of genetic perturbations at single-cell resolution. Current limitations in cell throughput and data density need to be taken into consideration, but new technologies are rapidly evolving and are likely to easily overcome these limitations. In this review, we discuss the use of bulk CRISPR screening in hematology research, as well as the emergence of single-cell CRISPR screening and its added value to the field.

The placenta serves as the interface between the mother and fetus, facilitating the exchange of gases and nutrients between their separate blood circulation systems. Trophoblasts in the placenta play a central role in this process. Our current understanding of mammalian trophoblast development relies largely on mouse models. However, given the diversification of mammalian placentas, findings from the mouse placenta cannot be readily extrapolated to other mammalian species, including humans. To fill this knowledge gap, we performed CRISPR knockout screening in human trophoblast stem cells (hTSCs). We targeted genes essential for mouse placental development and identified more than 100 genes as critical regulators in both human hTSCs and mouse placentas. Among them, we further characterized in detail two transcription factors, DLX3 and GCM1, and revealed their essential roles in hTSC differentiation. Moreover, a gene function–based comparison between human and mouse trophoblast subtypes suggests that their relationship may differ significantly from previous assumptions based on tissue localization or cellular function. Notably, our data reveal that hTSCs may not be analogous to mouse TSCs or the extraembryonic ectoderm (ExE) in which in vivo TSCs reside. Instead, hTSCs may be analogous to progenitor cells in the mouse ectoplacental cone and chorion. This finding is consistent with the absence of ExE-like structures during human placental development. Our data not only deepen our understanding of human trophoblast development but also facilitate cross-species comparison of mammalian placentas. [ABSTRACT FROM AUTHOR]

Purpose: Tissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been suggested that many drugs need active or defined transporters to pass the cell membrane. In contrast to efflux components induced after anti-cancer drugs reach the intracellular compartment, drug importers are required for initial drug responses. Furthermore, tissue-specific uptake of anti-cancer drugs may directly impact the side effects of many drugs when they accumulate in healthy tissues. Therefore, linking anti-cancer drugs to their respective drug import transporters would directly help to predict drug responses, whilst minimizing side effects. Methods: To identify drug transporters of the commonly used anti-cancer drug doxorubicin, we performed focused CRISPR activation and knockout genetic screens targeting all potential membrane-associated transporters and proteins. We monitored the direct uptake of doxorubicin by fluorescence-activated cell sorting (FACS) as the screening readout for identifying transporters/proteins directly involved in doxorubicin uptake. Results: Integrating the data from these comprehensive CRISPR screenings, we confirmed previously indicated doxorubicin exporters such as ABCB1 and ABCG2 genes, and identified novel doxorubicin importer gene SLC2A3 (GLUT3). Upregulation of SLC2A3 led to higher doxorubicin uptake and better cell killing, indicating SLC2A3 could be a new marker to predict doxorubicin drug response and minimize side effects for the personalized application of this conventional chemotherapeutic drug. Conclusions: Our study provides a comprehensive way for identifying drug transporters, as exemplified by the commonly used anti-cancer drug doxorubicin. The newly identified importers may have direct clinical implications for the personalized application of doxorubicin in treating distinct tumors. Our results also highlight the necessity of combining both CRISPR knockout and CRISPR activation genetic screens to identify drug transporters. [ABSTRACT FROM AUTHOR]

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors. [ABSTRACT FROM AUTHOR]

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system is a powerful tool that enables precise and efficient gene manipulation. In a relatively short time, CRISPR has risen to become the preferred gene-editing system due to its high efficiency, simplicity, and programmability at low costs. Furthermore, in the recent years, the CRISPR toolkit has been rapidly expanding, and the emerging advancements have shown tremendous potential in uncovering molecular mechanisms and new therapeutic strategies for human diseases. In this review, we provide our perspectives on the recent advancements in CRISPR technology and its impact on precision medicine, ranging from target identification, disease modeling, and diagnostics. We also discuss the impact of novel approaches such as epigenome, base, and prime editing on preclinical cancer drug discovery. [ABSTRACT FROM AUTHOR]

The CRISPR genome editing technology has revolutionized the way gene function is studied. Genome editing can be achieved in single genes or for thousands of genes simultaneously in sensitive genetic screens. While conventional genetic screens are limited to bulk measurements of cell behavior, recent developments in single-cell technologies make it possible to combine CRISPR screening with single-cell profiling. In this way, cell behavior and gene expression can be monitored simultaneously, with the additional possibility of including data on chromatin accessibility and protein levels. Moreover, the availability of various Cas proteins leading to inactivation, activation, or other effects on gene function further broadens the scope of such screens. The integration of single-cell multi-omics approaches with CRISPR screening open the path to high-content information on the impact of genetic perturbations at single-cell resolution. Current limitations in cell throughput and data density need to be taken into consideration, but new technologies are rapidly evolving and are likely to easily overcome these limitations. In this review, we discuss the use of bulk CRISPR screening in hematology research, as well as the emergence of single-cell CRISPR screening and its added value to the field. [ABSTRACT FROM AUTHOR]

Lung cancer is an extremely aggressive and highly prevalent disease worldwide, and it is one of the leading causes of cancer death. Deciphering intrinsic genetic mechanism, finding new targets, and overcoming drug resistance are the key to lung cancer treatment. High-throughput CRISPR screening has been extensively used to obtain the genes related to cancers including lung cancer. This review describes CRISPR/Cas9 or CRISPR/dCas9-based technologies for high-throughput screening. We summarize the applications of CRISPR screening technology in exploring the mechanism of lung cancer development in vivo or in vitro, overcoming drug resistance, improving the effect of immunotherapy, and discovering new therapeutic targets. This review highlights the potential of CRISPR screening in combination with tumor barcoding and high-throughput sequencing (Tuba-seq) to precisely quantify the impact of alterations in many tumor suppressor genes on lung cancer.

We sought to explore the hypothesis that host factors required for HIV-1 replication also play a role in latency reversal. Using a CRISPR gene library of putative HIV dependency factors, we performed a screen to identify genes required for latency reactivation. We identified several HIV-1 dependency factors that play a key role in HIV-1 latency reactivation including ELL, UBE2M, TBL1XR1, HDAC3, AMBRA1, and ALYREF. The knockout of Cyclin T1 (CCNT1), a component of the P-TEFb complex that is important for transcription elongation, was the top hit in the screen and had the largest effect on HIV latency reversal with a wide variety of latency reversal agents. Moreover, CCNT1 knockout prevents latency reactivation in a primary CD4+ T cell model of HIV latency without affecting the activation of these cells. RNA sequencing data showed that CCNT1 regulates HIV-1 proviral genes to a larger extent than any other host gene and had no significant effects on RNA transcripts in primary T cells after activation. We conclude that CCNT1 function is non-essential in T cells but is absolutely required for HIV latency reversal. [ABSTRACT FROM AUTHOR]

Protocols to differentiate human pluripotent stem cells have advanced in terms of cell type specificity and tissue-level complexity over the past 2 decades, which has facilitated human disease modeling in the most relevant cell types. The ability to generate induced PSCs (iPSCs) from patients further enables the study of disease mutations in an appropriate cellular context to reveal the mechanisms that underlie disease etiology and progression. As iPSC-derived disease models have improved in robustness and scale, they have also been adopted more widely for use in drug screens to discover new therapies and therapeutic targets. Advancement in genome editing technologies, in particular the discovery of CRISPR-Cas9, has further allowed for rapid development of iPSCs containing disease-causing mutations. CRISPR-Cas9 technologies have now evolved beyond creating single gene edits, aided by the fusion of inhibitory (CRISPRi) or activation (CRISPRa) domains to a catalytically dead Cas9 protein, enabling inhibition or activation of endogenous gene loci. These tools have been used in CRISPR knockout, CRISPRi, or CRISPRa screens to identify genetic modifiers that synergize or antagonize with disease mutations in a systematic and unbiased manner, resulting in identification of disease mechanisms and discovery of new therapeutic targets to accelerate drug discovery research. However, many technical challenges remain when applying large-scale functional genomics approaches to differentiated PSC populations. Here we review current technologies in the field of iPSC disease modeling and CRISPR-based functional genomics screens and practical considerations for implementation across a range of modalities, applications, and disease areas, as well as explore CRISPR screens that have been performed in iPSC models to-date and the insights and therapies these screens have produced.

The infection of mammalian cells by viruses and innate immune responses to infection are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation of the transcription factor interferon regulatory factor 3 (IRF3) and consequent induction of host immune responses to RNA viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes in the subcellular localization of host or viral proteins. Here, we increased the throughput of our optical pooled screening technology by over fourfold. This allowed us to carry out a genome-wide CRISPR knockout screen using high-resolution multiparameter imaging of cellular responses to Sendai virus infection coupled with in situ cDNA sequencing by synthesis (SBS) to identify 80,408 single guide RNAs (sgRNAs) in 10,366,390 cells—over an order of magnitude more genomic perturbations than demonstrated previously using an in situ SBS readout. By ranking perturbations using human-designed and deep learning image feature scores, we identified regulators of IRF3 translocation, Sendai virus localization, and peroxisomal biogenesis. Among the hits, we found that ATP13A1, an ER-localized P5A-type ATPase, is essential for viral sensing and is required for targeting of mitochondrial antiviral signaling protein (MAVS) to mitochondrial membranes where MAVS must be localized for effective signaling through retinoic acid-inducible gene I (RIG-I). The ability to carry out genome-wide pooled screens with complex high-resolution image-based phenotyping dramatically expands the scope of functional genomics approaches. [ABSTRACT FROM AUTHOR]

The genome is the blueprint for life, and over the past decade, CRISPR has become a very powerful method for editing our genetic makeup. In this article, we will explore the importance of CRISPR in developing breakthrough therapies for monogenic conditions and neurodegenerative diseases, and for enhancing the effectiveness of immuno-oncology.

Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large‐scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence‐profiled 524 American Joint Committee on Cancer Stage I–III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co‐mutated, and an absence of co‐occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole‐genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 (IRF4) as a melanoma‐associated dependency. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.

Abstract Background Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

The advent of cellular immunotherapy in the clinic has entirely redrawn the treatment landscape for a growing number of human cancers. Genetically reprogrammed immune cells, including chimeric antigen receptor (CAR)-modified immune effector cells as well as T cell receptor (TCR) therapy, have demonstrated remarkable responses across different hard-to-treat patient populations. While these novel treatment options have had tremendous success in providing long-term remissions for a considerable fraction of treated patients, a number of challenges remain. Limited in vivo persistence and functional exhaustion of infused immune cells as well as tumor immune escape and on-target off-tumor toxicities are just some examples of the challenges which restrain the potency of today's genetically engineered cell products. Multiple engineering strategies are being explored to tackle these challenges. The advent of multiplexed precision genome editing has in recent years provided a flexible and highly modular toolkit to specifically address some of these challenges by targeted genetic interventions. This class of next-generation cellular therapeutics aims to endow engineered immune cells with enhanced functionality and shield them from immunosuppressive cues arising from intrinsic immune checkpoints as well as the hostile tumor microenvironment (TME). Previous efforts to introduce additional genetic modifications into immune cells have in large parts focused on nuclease-based tools like the CRISPR/Cas9 system or TALEN. However, nuclease-inactive platforms including base and prime editors have recently emerged and promise a potentially safer route to rewriting genetic sequences and introducing large segments of transgenic DNA without inducing double-strand breaks (DSBs). In this review, we discuss how these two exciting and emerging fields--cellular immunotherapy and precision genome editing--have co-evolved to enable a dramatic expansion in the possibilities to engineer personalized anti-cancer treatments. We will lay out how various engineering strategies in addition to nuclease-dependent and nuclease-inactive precision genome editing toolkits are increasingly being applied to overcome today's limitations to build more potent cellular therapeutics. We will reflect on how novel information-rich unbiased discovery approaches are continuously deepening our understanding of fundamental mechanisms governing tumor biology. We will conclude with a perspective of how multiplexed-engineered and gene edited cell products may upend today's treatment paradigms as they evolve into the next generation of more potent cellular immunotherapies. [ABSTRACT FROM AUTHOR]

Simple Summary: NRAS mutations constitutively activate cell proliferation signaling in malignant tumors. The elucidation of NRAS downstream signaling genes may lead to the control of NRAS-mutant tumors. Genome-wide CRISPR activation screening was performed on THP-1 B11, in which NRAS expression and cell proliferation could be regulated by doxycycline. Multiple candidate genes were identified from clones that survived in NRAS-off, and three genes—DOHH, HIST1H2AC, and TAF6—were finally identified as being downstream of NRAS signaling and confirmed to contribute to the proliferation of THP-1 cells. These molecules are promising new therapeutic targets for NRAS-mutant leukemia. Mutations in NRAS constitutively activate cell proliferation signaling in malignant neoplasms, such as leukemia and melanoma, and the clarification of comprehensive downstream genes of NRAS might lead to the control of cell-proliferative signals of NRAS-driven cancers. We previously established that NRAS expression and proliferative activity can be controlled with doxycycline and named as THP-1 B11. Using a CRISPR activation library on THP-1 B11 cells with the NRAS-off state, survival clones were harvested, and 21 candidate genes were identified. By inducting each candidate guide RNA with the CRISPR activation system, DOHH, HIST1H2AC, KRT32, and TAF6 showed higher cell-proliferative activity. The expression of DOHH, HIST1H2AC, and TAF6 was definitely upregulated with NRAS expression. Furthermore, MEK inhibitors resulted in the decreased expression of DOHH, HIST1H2AC, and TAF6 proteins in parental THP-1 cells. The knockdown of DOHH, HIST1H2AC, and TAF6 was found to reduce proliferation in THP-1 cells, indicating that they are involved in the downstream proliferation of NRAS. These molecules are expected to be new therapeutic targets for NRAS-mutant leukemia cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Clustered regularly interspaced short palindromic repeats (CRISPR) were discovered in prokaryotes, and the technology can also be used to edit the genome in mammalian cells. The discovery was awarded the Nobel Prize in 2020, as CRISPR has opened up new possibilities to edit the human genome. CRISPR has been applied to study cancer because the method allows for many new ways to model the disease. This includes the development of pre-clinical models of cancer, where CRISPR is used to generate mutations that are found in human cancer. Therefore, unique mutations can be studied in a physiologically relevant setting, and CRISPR technology has accelerated the engineering of these models. This review focuses on exploring the current knowledge of CRISPR editing in adult tissues for generating pre-clinical models to study cancer. Clustered regularly interspaced short palindromic repeats (CRISPR) are widely used in cancer research to edit specific genes and study their functions. This applies both to in vitro and in vivo studies where CRISPR technology has accelerated the generation of specific loss- or gain-of-function mutations. This review focuses on CRISPR for generating in vivo models of cancer by editing somatic cells in specific organs. The delivery of CRISPR/Cas to designated tissues and specific cell compartments is discussed with a focus on different methods and their advantages. One advantage of CRISPR/Cas is the possibility to target multiple genes simultaneously in the same cell and therefore generate complex mutation profiles. This complexity challenges the interpretation of results and different methods to analyze the samples discussed herein. CRISPR-induced tumors are also different from classical tumors in pre-clinical models. Especially the clonal evolution of CRISPR-induced tumors adds new insight into cancer biology. Finally, the review discusses future perspectives for CRISPR technology in pre-clinical models with a focus on in vivo screening, CRISPR activation/inhibition, and the development of prime/ base-editing for the introduction of specific gene editing. [ABSTRACT FROM AUTHOR]

Abstract Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor. E3 ligases play important functions in glioma pathogenesis. CRISPR system offers a powerful platform for genome manipulation, while the screen of E3 ligases in GBM still remains to be explored. Here, we first constructed an E3 ligase small guide RNA (sgRNAs) library for glioma cells growth screening. After four passages, 299 significantly enriched or lost genes (SELGs) were compared with the initial state. Then the clinical significance of SELGs were validated and analyzed with TCGA glioblastoma and CGGA datasets. As RNF185 showed lost signal, decreased expression and favorable prognostic significance, we chose RNF185 for functional analysis. In vitro overexpressed cellular phenotype showed that RNF185 was a tumor suppressor in two glioma cell lines. Finally, the molecular mechanism of decreased RNF185 expression was investigated and increased miR-587 expression and DNA hypermethylation was evaluated. This study would provide a link between the molecular basis and glioblastoma pathogenesis, and a novel perspective for glioblastoma treatment.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release. [Display omitted] • CRISPR screening of mt-RBPs reveals key regulators of mt-dsRNA expression • NSUN4 installs m5C RNA modification on mitochondrial mRNAs and lncRNAs • C1QBP recognizes m5C-modified mitochondrial RNAs and recruits PNPT1 • m5C modification marks the mitochondrial RNAs for decay and cytosolic release Through CRISPR screening of 89 mitochondrial RBPs, Kim et al. find that NSUN4 downregulates mitochondrial dsRNA expression by installing 5-methylcytosine modification on light-strand RNAs. The modified RNAs are recognized by C1QBP, which recruits PNPT1 to facilitate RNA turnover, and are released to the cytosol, where they activate the immune response. [ABSTRACT FROM AUTHOR]

We sought to explore the hypothesis that host factors required for HIV-1 replication also play a role in latency reversal. Using a CRISPR gene library of putative HIV dependency factors, we performed a screen to identify genes required for latency reactivation. We identified several HIV-1 dependency factors that play a key role in HIV-1 latency reactivation including ELL, UBE2M, TBL1XR1, HDAC3, AMBRA1, and ALYREF. The knockout of Cyclin T1 (CCNT1), a component of the P-TEFb complex that is important for transcription elongation, was the top hit in the screen and had the largest effect on HIV latency reversal with a wide variety of latency reversal agents. Moreover, CCNT1 knockout prevents latency reactivation in a primary CD4+ T cell model of HIV latency without affecting the activation of these cells. RNA sequencing data showed that CCNT1 regulates HIV-1 proviral genes to a larger extent than any other host gene and had no significant effects on RNA transcripts in primary T cells after activation. We conclude that CCNT1 function is non-essential in T cells but is absolutely required for HIV latency reversal.

The advent of cellular immunotherapy in the clinic has entirely redrawn the treatment landscape for a growing number of human cancers. Genetically reprogrammed immune cells, including chimeric antigen receptor (CAR)-modified immune effector cells as well as T cell receptor (TCR) therapy, have demonstrated remarkable responses across different hard-to-treat patient populations. While these novel treatment options have had tremendous success in providing long-term remissions for a considerable fraction of treated patients, a number of challenges remain. Limited in vivo persistence and functional exhaustion of infused immune cells as well as tumor immune escape and on-target off-tumor toxicities are just some examples of the challenges which restrain the potency of today’s genetically engineered cell products. Multiple engineering strategies are being explored to tackle these challenges.The advent of multiplexed precision genome editing has in recent years provided a flexible and highly modular toolkit to specifically address some of these challenges by targeted genetic interventions. This class of next-generation cellular therapeutics aims to endow engineered immune cells with enhanced functionality and shield them from immunosuppressive cues arising from intrinsic immune checkpoints as well as the hostile tumor microenvironment (TME). Previous efforts to introduce additional genetic modifications into immune cells have in large parts focused on nuclease-based tools like the CRISPR/Cas9 system or TALEN. However, nuclease-inactive platforms including base and prime editors have recently emerged and promise a potentially safer route to rewriting genetic sequences and introducing large segments of transgenic DNA without inducing double-strand breaks (DSBs). In this review, we discuss how these two exciting and emerging fields—cellular immunotherapy and precision genome editing—have co-evolved to enable a dramatic expansion in the possibilities to engineer personalized anti-cancer treatments. We will lay out how various engineering strategies in addition to nuclease-dependent and nuclease-inactive precision genome editing toolkits are increasingly being applied to overcome today’s limitations to build more potent cellular therapeutics. We will reflect on how novel information-rich unbiased discovery approaches are continuously deepening our understanding of fundamental mechanisms governing tumor biology. We will conclude with a perspective of how multiplexed-engineered and gene edited cell products may upend today’s treatment paradigms as they evolve into the next generation of more potent cellular immunotherapies.

Aflatoxin B1 (AFB1) is amongst the mycotoxins commonly affecting human and animal health, raising global food safety and control concerns. The mechanisms underlying AFB1 toxicity are poorly understood. Moreover, antidotes against AFB1 are lacking. Genome-wide CRISPR/Cas9 knockout screening in porcine kidney cells identified the transcription factor BTB and CNC homolog 1 (BACH1) as a gene required for AFB1 toxicity. The inhibition of BACH1 expression in porcine kidney cells and human hepatoma cells resulted in increased resistance to AFB1. BACH1 depletion attenuates AFB1-induced oxidative damage via the upregulation of antioxidant genes. Subsequently, virtual structural screening identified the small molecule 1-Piperazineethanol, α-[(1,3-benzodioxol-5-yloxy)methyl] -4-(2-methoxyphenyl) (M2) as an inhibitor of BACH1. M2 and its analogues inhibited AFB1-induced porcine and human cell death in vitro, while M2 administration significantly improved AFB1-induced symptoms of weight loss and liver injury in vivo. These findings demonstrate that BACH1 plays a central role in AFB1-induced oxidative damage by regulating antioxidant gene expression. We also present a potent candidate small-molecule inhibitor in developing novel treatments for AFB1 toxicity. [ABSTRACT FROM AUTHOR]

CD3-engaging bispecific antibodies (BsAbs) enable the formation of an immune synapse between T cells and tumor cells, resulting in robust target cell killing not dependent on a preexisting tumor specific T cell receptor. While recent studies have shed light on tumor cell-specific factors that modulate BsAb sensitivity, the T cell-intrinsic determinants of BsAb efficacy and response durability are poorly understood. To better clarify the genes that shape BsAb-induced T cell responses, we conducted targeted analyses and a large-scale unbiased in vitro CRISPR/Cas9-based screen to identify negative regulators of BsAb-induced T cell proliferation. These analyses revealed that CD8+ T cells are dependent on CD4+ T cell-derived signaling factors in order to achieve sustained killing in vitro. Moreover, the mammalian target of rapamycin (mTOR) pathway and several other candidate genes were identified as intrinsic regulators of BsAb-induced T cell proliferation and/or activation, highlighting promising approaches to enhancing the utility of these potent therapeutics. [ABSTRACT FROM AUTHOR]

MYC is a major oncogene that plays an important role in cell proliferation in human cancers. Therefore, the mechanism behind MYC regulation is a viable therapeutic target for the treatment of cancer. Comprehensive and efficient screening of MYC regulators is needed, and we had previously established a promoter screening system using fluorescent proteins and the CRISPR library. For the efficient identification of candidate genes, a database was used, for which mRNA expression was correlated with MYC using datasets featuring "Similar" and "Not exactly similar" contexts. INTS14 and ERI2 were identified using datasets featuring the "Similar" context group, and INTS14 and ERI2 were capable of enhancing MYC promoter activity. In further database analysis of human cancers, a higher expression of MYC mRNA was observed in the INTS14 mRNA high-expressing prostate and liver cancers. The knockdown of INTS14 in prostate cell lines resulted in decreased MYC mRNA and protein expression and also induced G0/1 arrest. This study confirmed that CRISPR screening combined with context-matched database screening is effective in identifying genes that regulate the MYC promoter. This method can be applied to other genes and is expected to be useful in identifying the regulators of other proto-oncogenes. [ABSTRACT FROM AUTHOR]

Ferroptosis has emerged as a new type of programmed cell death that can be harnessed for cancer therapy. The concept of ferroptosis was for the first time proposed in in the early 2000s, as an iron-dependent mode of regulated cell death caused by unrestricted lipid peroxidation (LPO) and subsequent plasma membrane rupture. Since the discovery and characterization of ferroptosis, a wealth of research has improved our understanding of the main pathways regulating this process, leading to both the repurposing and the development of small molecules. However, ferroptosis is still little understood and several aspects remain to be investigated. For instance, it is unclear whether specific oncogenes, cells of origin or tumor niches impose specific susceptibility/resistance to ferroptosis or if there are some ferroptosis-related genes that may be used as bona fide pan-cancer targetable dependencies. In this context, even though RAS-driven cancer cell lines seemed to be selectively sensitive to ferroptosis inducers, subsequent studies have questioned these results, indicating that in some cases mutant RAS is necessary, but not sufficient to induce ferroptosis. In this perspective, based on publicly available genomic screening data and the literature, we discuss the relationship between RAS-mutation and ferroptosis susceptibility in cancer. [ABSTRACT FROM AUTHOR]

Objective: Disruption of the circadian rhythm is associated with cancer occurrence, response to chemotherapy, and poor prognosis. Thus, using internal clock-based chronotherapy to optimize the administration time may improve the therapeutic effects of anticancer drugs while reducing the side effects. Chronotherapy with 5-fluorouracil (5-FU) has been observed in colorectal cancer (CRC) for a long time, but its effect is under controversial and the mechanism remains unclear. Methods: Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening and RNA-sequencing were combined to identify the potential genes or pathways involved in 5-FU chronochemotherapy. Genetic deletion or overexpression of pyrimidine metabolic pathway genes were conducted to examine cellular viability with or without 5-FU via flow cytometry. Western blotting, qPCR, chromatin immunoprecipitation, gain-of-function and loss-of-function assays of several CRC cell lines in vitro and in vivo were used to elaborate and validate the mechanism of 5-FU chronotherapeutic effects. Results: Chronochemotherapeutic effects of 5-FU on CRC in vivo were verified. Furthermore, 5-FU chronochemotherapy related genes such as UPP2, UCK2 and UMPS in the pyrimidine metabolic pathway were identified. Disturbance in these genes, especially UMPS, perturbs 5-FU treatment outcomes in CRC cells. Mechanistically, the core circadian gene, brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1), extensively regulate gene expression in pyrimidine metabolic pathway by binding to E-box element in the promoter region of key genes such as UMPS and perturb their enzymatic activities, thereby maintain diurnal efficacy of 5-FU in CRC cells. Conclusion: This study uncovered a new mechanism by which a core circadian gene BMAL1 increases the effectiveness of 5-FU by enhancing the expression and enzymatic activities of key genes in the pyrimidine metabolic pathway in CRC cells. The findings suggest a novel strategy for CRC chemotherapy by targeting chrono-modulated genes of the 5-FU metabolic pathway. [ABSTRACT FROM AUTHOR]

Recently approved RET tyrosine kinase inhibitors (TKIs) have shown promising therapeutic effects against RET-rearranged non-small cell lung cancer (NSCLC) or RET-mutated thyroid cancer. However, resistance develops, limiting long-term efficacy. Although many RET-TKI resistance mechanisms, such as secondary mutations in RET or activation of bypass pathways, are known, some primary or acquired resistance mechanisms are unclear. Here, human genome-wide CRISPR/Cas9 screening was performed to identify genes related to drug-tolerant persister cells. Patient-derived cells with RET-fusion were introduced genome-wide sgRNA library and treated with RET-TKI for 9 days, resulting in the discovery of several candidate genes. Knockout of MED12 or MIG6 significantly increased residual drug-tolerant persister cells under RET-TKI treatment. MIG6 loss induced significant EGFR activation even with low concentrations of EGFR ligands and led to resistance to RET-TKIs. EGFR inhibition with afatinib or cetuximab in combination with RET TKIs was effective in addressing drug persistence. By contrast, a KIF5B-RET positive cells established from a RET-rearranged NSCLC patient, showed significant resistance to RET-TKIs and high dependence on EGFR bypass signaling. Consistently, knocking out EGFR or RET led to high sensitivity to RET or EGFR inhibitor respectively. Here, we have provided a comprehensive analysis of adaptive and acquired resistance against RET-rearranged NSCLC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests.R. Katayama received research grants from Chugai, and TOPPAN. The other authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Tuberculosis (TB), the leading cause of death from bacterial infections worldwide, results from infection with Mycobacterium tuberculosis (Mtb). The antitubercular agents delamanid (DLM) and pretomanid (PMD) are nitroimidazole prodrugs that require activation by an enzyme intrinsic to Mtb; however, the mechanism(s) of action and the associated metabolic pathways are largely unclear. Profiling of the chemical-genetic interactions of PMD and DLM in Mtb using combined CRISPR screening reveals that the mutation of rv2073c increases susceptibility of Mtb to these nitroimidazole drugs both in vitro and in infected mice, whereas mutation of rv0078 increases drug resistance. Further assays show that Rv2073c might confer intrinsic resistance to DLM/PMD by interfering with inhibition of the drug target, decaprenylphophoryl-2-keto-b-D-erythro-pentose reductase (DprE2), by active nicotinamide adenine dinucleotide (NAD) adducts. Characterization of the metabolic pathways of DLM/PMD in Mtb using a combination of chemical genetics and comparative liquid chromatography-mass spectrometry (LC-MS) analysis of DLM/PMD metabolites reveals that Rv0077c, which is negatively regulated by Rv0078, mediates drug resistance by metabolizing activated DLM/PMD. These results might guide development of new nitroimidazole prodrugs and new regimens for TB treatment., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)