CRISPR Interference Screens Research Articles

CRISPRi/a screens in human iPSC-cardiomyocytes identify glycolytic activation as a druggable target for doxorubicin-induced cardiotoxicity.

Doxorubicin is limited in its therapeutic utility due to its life-threatening cardiovascular side effects. Here, we present an integrated drug discovery pipeline combining human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iCMs), CRISPR interference and activation (CRISPRi/a) bidirectional pooled screens, and a small-molecule screening to identify therapeutic targets mitigating doxorubicin-induced cardiotoxicity (DIC) without compromising its oncological effects. The screens revealed several previously unreported candidate genes contributing to DIC, including carbonic anhydrase 12 (CA12). Genetic inhibition of CA12 protected iCMs against DIC by improving cell survival, sarcomere structural integrity, contractile function, and calcium handling. Indisulam, a CA12 antagonist, can effectively attenuate DIC in iCMs, engineered heart tissue, and animal models. Mechanistically, doxorubicin-induced CA12 potentiated a glycolytic activation in cardiomyocytes, contributing to DIC by interfering with cellular metabolism and functions. Collectively, our study provides a roadmap for future drug discovery efforts, potentially leading to more targeted therapies with minimal off-target toxicity.

Large DNA deletions occur during DNA repair at 20-fold lower frequency for base editors and prime editors than for Cas9 nucleases.

When used to edit genomes, Cas9 nucleases produce targeted double-strand breaks in DNA. Subsequent DNA-repair pathways can induce large genomic deletions (larger than 100 bp), which constrains the applicability of genome editing. Here we show that Cas9-mediated double-strand breaks induce large deletions at varying frequencies in cancer cell lines, human embryonic stem cells and human primary T cells, and that most deletions are produced by two repair pathways: end resection and DNA-polymerase theta-mediated end joining. These findings required the optimization of long-range amplicon sequencing, the development of a k-mer alignment algorithm for the simultaneous analysis of large DNA deletions and small DNA alterations, and the use of CRISPR-interference screening. Despite leveraging mutated Cas9 nickases that produce single-strand breaks, base editors and prime editors also generated large deletions, yet at approximately 20-fold lower frequency than Cas9. We provide strategies for the mitigation of such deletions.

Expanding the therapeutic horizon in acute myeloid leukemia: The role of phosphoinositide-3-kinase-γ inhibition

Abstract Acute myeloid leukemia (AML) remains a formidable challenge due to its high relapse rate and limited therapeutic options. This study investigates the role of phosphoinositide-3-kinase-γ (PI3Kγ) in AML and explores the therapeutic potential of the PI3Kγ inhibitor, eganelisib, in preclinical models. Using genome-wide CRISPR interference screening, we identified PI3Kγ as a critical survival pathway in certain AML subsets. We demonstrated that inhibition of PI3Kγ with eganelisib disrupts oxidative phosphorylation, thereby impeding AML cell survival and proliferation. Notably, the combination of eganelisib with cytarabine showed enhanced efficacy, suggesting a synergistic interaction that improves treatment outcomes. These findings indicate that PI3Kγ is a viable therapeutic target in AML and that PI3Kγ inhibitors could be pivotal in overcoming treatment resistance and reducing relapse.

A co-culture model to study modulators of tumor immune evasion through scalable arrayed CRISPR-interference screens.

Cancer cells effectively evade immune surveillance, not only through the well-known PD-1/PD-L1 pathway but also via alternative mechanisms that impair patient response to immune checkpoint inhibitors. We present a novel co-culture model that pairs a reporter T-cell line with different melanoma cell lines that have varying immune evasion characteristics. We developed a scalable high-throughput lentiviral arrayed CRISPR interference (CRISPRi) screening protocol to conduct gene perturbations in both T-cells and melanoma cells, enabling the identification of genes that modulate tumor immune evasion. Our study functionally validates the co-culture model system and demonstrates the performance of the CRISPRi-screening protocol by modulating the expression of known regulators of tumor immunity. Together, our work provides a robust framework for future research aimed at systematically exploring mechanisms of tumor immune evasion.

In vivo CRISPRi screen identified lncRNA portfolio crucial for cutaneous squamous cell carcinoma tumor growth.

Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of all skin cancer deaths globally, making it the second-highest subtype of skin cancer. The prevalence of cSCC in humans, as well as the poor capacity for an efficient prognosis, highlights the need to uncover alternative actors and mechanisms at the foundation of skin cancer development. Significant advances have been made to better understand some key factors in cSCC progression. However, little is known about the role of noncoding RNAs, particularly of a specific category called long noncoding RNA (lncRNA). By performing pseudobulk analysis of single-cell sequencing data from normal and cSCC human skin tissues, we determined a global portfolio of lncRNAs specifically expressed in keratinocyte subpopulations. Integration of CRISPR interference screens in vitro and the xenograft model identified several lncRNAs impacting the growth of cSCC cancer lines both in vitro and in vivo. Among these, we further validated LINC00704 and LINC01116 as proliferation-regulating lncRNAs in cSCC lines and potential biomarkers of cSCC progression. Taken together, our study provides a comprehensive signature of lncRNAs with roles in regulating cSCC progression.

Linking candidate causal autoimmune variants to T cell networks using genetic and epigenetic screens in primary human T cells.

Genetic variants associated with autoimmune diseases are highly enriched within putative cis -regulatory regions of CD4 + T cells, suggesting that they alter disease risk via changes in gene regulation. However, very few genetic variants have been shown to affect T cell gene expression or function. We tested >18,000 autoimmune disease-associated variants for allele-specific expression using massively parallel reporter assays in primary human CD4 + T cells. The 545 expression-modulating variants (emVars) identified greatly enrich for likely causal variants. We provide evidence that many emVars are mediated by common upstream regulatory conduits, and that putative target genes of primary T cell emVars are highly enriched within a lymphocyte activation network. Using bulk and single-cell CRISPR-interference screens, we confirm that emVar-containing T cell cis -regulatory elements modulate both known and novel target genes that regulate T cell proliferation, providing plausible mechanisms by which these variants alter autoimmune disease risk.

Deciphering regulation of FOXP3 expression in human conventional T cells.

FOXP3 is a lineage-defining transcription factor that controls differentiation and maintenance of suppressive function of regulatory T cells (Tregs). Foxp3 is exclusively expressed in Tregs in mice. However, in humans, FOXP3 is not only constitutively expressed in Tregs; it is also transiently expressed in stimulated CD4+CD25- conventional T cells (Tconvs)1-3. Mechanisms governing the expression of FOXP3 in human Tconvs are not understood. Here, we performed CRISPR interference (CRISPRi) screens using a 15K-member gRNA library tiling 39 kb downstream of the FOXP3 transcriptional start site (TSS) to 85 kb upstream of the TSS in Treg and Tconvs. The FOXP3 promoter and conserved non-coding sequences (CNS0, CNS1, CNS2 and CNS3), characterized as enhancer elements in murine Tregs, were required for maintenance of FOXP3 in human Tregs. In contrast, FOXP3 in human Tconvs depended on regulation at CNS0 and a novel Tconv-specific noncoding sequence (TcNS+) located upstream of CNS0. Arrayed validations of these sites identified an additional repressive cis-element overlapping with the PPP1R3F promoter (TcNS-). Pooled CRISPR knockouts revealed multiple transcription factors required for proper expression of FOXP3 in Tconvs, including GATA3, STAT5, IRF4, ETS1 and DNA methylation-associated regulators DNMT1 and MBD2. Analysis of ChIP-seq and ATAC-seq paired with knock-out (KO) of GATA3, STAT5, IRF4, and ETS1 revealed regulation of CNS0 and TcNS+ accessibility. Collectively, this work identified Treg-shared and Tconv-specific cis-elements and the trans-factors that interact with them, building a network of regulators controlling FOXP3 expression in human Tconvs.

An engineered trafficking biosensor reveals a role for DNAJC13 in DOR downregulation.

Trafficking of G protein-coupled receptors (GPCRs) through the endosomal-lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and the large number of cellular proteins involved in the process. Here, we developed a high-sensitivity biosensor for GPCR expression and agonist-induced trafficking to the lysosome by leveraging the ability of the engineered peroxidase APEX2 to activate the fluorogenic substrate Amplex UltraRed (AUR). We used the GPCR-APEX2/AUR assay to perform a genome-wide CRISPR interference screen focused on identifying genes regulating expression and trafficking of the δ-opioid receptor (DOR). We identified 492 genes consisting of both known and new regulators of DOR function. We demonstrate that one new regulator, DNAJC13, controls trafficking of multiple GPCRs, including DOR, through the endosomal-lysosomal pathway by regulating the composition of the endosomal proteome and endosomal homeostasis.

An enterococcal phage protein inhibits type IV restriction enzymes involved in antiphage defense

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in vancomycin-resistant E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

CRISPR screening uncovers a long-range enhancer for ONECUT1 in pancreatic differentiation and links a diabetes risk variant

Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, ∼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

Ciliary biology intersects autism and congenital heart disease.

Autism spectrum disorder (ASD) commonly co-occurs with congenital heart disease (CHD), but the molecular mechanisms underlying this comorbidity remain unknown. Given that children with CHD come to clinical attention by the newborn period, understanding which CHD variants carry ASD risk could provide an opportunity to identify and treat individuals at high risk for developing ASD far before the typical age of diagnosis. Therefore, it is critical to delineate the subset of CHD genes most likely to increase the risk of ASD. However, to date there is relatively limited overlap between high confidence ASD and CHD genes, suggesting that alternative strategies for prioritizing CHD genes are necessary. Recent studies have shown that ASD gene perturbations commonly dysregulate neural progenitor cell (NPC) biology. Thus, we hypothesized that CHD genes that disrupt neurogenesis are more likely to carry risk for ASD. Hence, we performed an in vitro pooled CRISPR interference (CRISPRi) screen to identify CHD genes that disrupt NPC biology similarly to ASD genes. Overall, we identified 45 CHD genes that strongly impact proliferation and/or survival of NPCs. Moreover, we observed that a cluster of physically interacting ASD and CHD genes are enriched for ciliary biology. Studying seven of these genes with evidence of shared risk (CEP290, CHD4, KMT2E, NSD1, OFD1, RFX3, TAOK1), we observe that perturbation significantly impacts primary cilia formation in vitro. While in vivo investigation of TAOK1 reveals a previously unappreciated role for the gene in motile cilia formation and heart development, supporting its prediction as a CHD risk gene. Together, our findings highlight a set of CHD risk genes that may carry risk for ASD and underscore the role of cilia in shared ASD and CHD biology.

An enterococcal phage protein broadly inhibits type IV restriction enzymes involved in antiphage defense.

The prevalence of multidrug resistant (MDR) bacterial infections continues to rise as the development of antibiotics needed to combat these infections remains stagnant. MDR enterococci are a major contributor to this crisis. A potential therapeutic approach for combating MDR enterococci is bacteriophage (phage) therapy, which uses lytic viruses to infect and kill pathogenic bacteria. While phages that lyse some strains of MDR enterococci have been identified, other strains display high levels of resistance and the mechanisms underlying this resistance are poorly defined. Here, we use a CRISPR interference (CRISPRi) screen to identify a genetic locus found on a mobilizable plasmid from Enterococcus faecalis involved in phage resistance. This locus encodes a putative serine recombinase followed by a Type IV restriction enzyme (TIV-RE) that we show restricts the replication of phage phi47 in E. faecalis. We further find that phi47 evolves to overcome restriction by acquiring a missense mutation in a TIV-RE inhibitor protein. We show that this inhibitor, termed type IV restriction inhibiting factor A (tifA), binds and inactivates diverse TIV-REs. Overall, our findings advance our understanding of phage defense in drug-resistant E. faecalis and provide mechanistic insight into how phages evolve to overcome antiphage defense systems.

A whole-genome CRISPR screen identifies the spindle accessory checkpoint as a locus of nab-paclitaxel resistance in a pancreatic cancer cell line

Pancreatic adenocarcinoma is one of the most aggressive and lethal forms of cancer. Chemotherapy is the primary treatment for pancreatic cancer, but resistance to the drugs used remains a major challenge. A genome-wide CRISPR interference and knockout screen in the PANC-1 cell line with the drug nab-paclitaxel has identified a group of spindle assembly checkpoint (SAC) genes that enhance survival in nab-paclitaxel. Knockdown of these SAC genes (BUB1B, BUB3, and TTK) attenuates paclitaxel-induced cell death. Cells treated with the small molecule inhibitors BAY 1217389 or MPI 0479605, targeting the threonine tyrosine kinase (TTK), also enhance survival in paclitaxel. Overexpression of these SAC genes does not affect sensitivity to paclitaxel. These discoveries have helped to elucidate the mechanisms behind paclitaxel cytotoxicity. The outcomes of this investigation may pave the way for a deeper comprehension of the diverse responses of pancreatic cancer to therapies including paclitaxel. Additionally, they could facilitate the formulation of novel treatment approaches for pancreatic cancer.

CRISPR Screening Uncovers a Long-Range Enhancer for ONECUT1 in Pancreatic Differentiation and Links a Diabetes Risk Variant.

Functional enhancer annotation is a valuable first step for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants for investigation. However, unbiased enhancer discovery in physiologically relevant contexts remains a major challenge. To discover regulatory elements pertinent to diabetes, we conducted a CRISPR interference screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers uncovered, we focused on a long-range enhancer ∼664 kb from the ONECUT1 promoter, since coding mutations in ONECUT1 cause pancreatic hypoplasia and neonatal diabetes. Homozygous enhancer deletion in hPSCs was associated with a near-complete loss of ONECUT1 gene expression and compromised pancreatic differentiation. This enhancer contains a confidently fine-mapped type 2 diabetes associated variant (rs528350911) which disrupts a GATA motif. Introduction of the risk variant into hPSCs revealed substantially reduced binding of key pancreatic transcription factors (GATA4, GATA6 and FOXA2) on the edited allele, accompanied by a slight reduction of ONECUT1 transcription, supporting a causal role for this risk variant in metabolic disease. This work expands our knowledge about transcriptional regulation in pancreatic development through the characterization of a long-range enhancer and highlights the utility of enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.

Core regions in immunoglobulin heavy chain enhancers essential for survival of non-Hodgkin lymphoma cells are identified by a CRISPR interference screen.

Chromosomal translocations in non-Hodgkin lymphoma (NHL) result in activation of oncogenes by placing them under the regulation of immunoglobulin heavy chain (IGH) super-enhancers. Aberrant expression of translocated oncogenes induced by enhancer activity can contribute to lymphomagenesis. The role of the IGH enhancers in normal B-cell development is well established, but knowledge regarding the precise mechanisms of their involvement in control of the translocated oncogenes is limited. The goal of this project was to define the critical regions in the IGH regulatory elements and identify enhancer RNAs (eRNA). We designed a sgRNA library densely covering the IGH enhancers and performed tiling CRISPR interference screens in three NHL cell lines. This revealed three regions crucial for NHL cell growth. With chromatin-enriched RNA-Seq we showed transcription from the core enhancer regions and subsequently validated expression of the eRNAs in a panel of NHL cell lines and tissue samples. Inhibition of the essential IGH enhancer regions decreased expression of eRNAs and translocated oncogenes in several NHL cell lines. The observed expression and growth patterns were consistent with the breakpoints in the IGH locus. Moreover, targeting the Eμ enhancer resulted in loss of B-cell receptor expression. In a Burkitt lymphoma cell line, MYC overexpression partially rescued the phenotype induced by IGH enhancer inhibition. Our results indicated the most critical regions in the IGH enhancers and provided new insights into the current understanding of the role of IGH enhancers in B-cell NHL. As such, this study forms a basis for development of potential therapeutic approaches.

Abstract PR04: Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma

Abstract We sought to identify mechanisms that activate MYC expression in germinal center B cell-(GCB) diffuse large B-cell lymphomas (DLBCL) in the absence of MYC rearrangement. High-throughput CRISPR-interference screens in MYC-rearranged (n=4) and non-MYC-rearranged (n=2) GCB-DLBCL cell lines revealed a distal enhancer complex 436 kb downstream of MYC, GME-1, that was uniquely essential in non-rearranged (MYC-intact) cell lines. CRISPR-interference studies confirmed growth inhibition and MYC downregulation upon GME-1 repression in 4 DLBCL cell lines. ChIP-Seq showed that GME-1 subunits were bound by OCT2, OCA-B, and MEF2B, which are known to activate germinal center-specific enhancers, and knockdown of the corresponding genes reduced MYC expression. GME-1 subunits showed GCB-specific acetylation and accessibility across normal human B cell populations in public chromatin datasets. The mouse ortholog of GME-1 showed germinal center B cell-specific accessibility. GME-1 was strongly acetylated in MYC-intact but not MYC-rearranged GCB-DLBCL cell lines and patient biopsies. Hi-C revealed significant looping of GME-1 to the MYC promoter in MYC protein+, MYC-intact DLBCL biopsies (n=2), but not MYC-rearranged DLBCL (n=2). We used sequence conservation and chromatin accessibility data to define four GME-1 subunits (195A/B and196A/B), which were cloned into luciferase enhancer reporter constructs and compared to two subunits of the BCL6 distal super-enhancer complex that had previously been identified as regulated by the OCT2/OCA-B/MEF2B complex. GME-1 subunit 195B was the only strongly transcription-activating element based on luciferase assays across three MYC-intact cells lines, where it showed activity comparable to BCL6 enhancers. In contrast, GME-1 enhancers were minimally active in reporter assays performed in two MYC-rearranged GCB-DLBCL cell lines. Deletion of a conserved OCT2 binding site or knockdown of the POU2F2 and POU2AF1 genes reduced activity of GME-1. Thus GME-1 trans-regulation is divergent from that of previously characterized GCB enhancers, despite shared dependency on the OCT2 ternary complex. Whole-genome sequencing (WGS) revealed that the GME-1 dependent cell line SUDHL5 bears a 176bp tandem duplication within GME-1 195B. This altered enhancer showed stronger luciferase reporter activity across three MYC-intact cell lines. Published WGS datasets showed isolated genomic tandem copy gains of GME-1 in 2/49 biopsies from recurrent/refractory DLBCL and 1/93 unselected DLBCL. In a cohort of 18 DLBCL with the high-risk DZSig gene expression signature, GME-1 amplification was seen in two biopsies with concurrent deletion of the PVT1 promoter (PVT1-P) and no inter-chromosomal MYC locus rearrangement. CRISPRi showed that PVT1-P repression reduced growth and MYC expression in GME-1-dependent DLBCL, but not in cell lines where rearrangements removed PVT-P from its position in cis with the MYC gene. Our findings identify GME-1 as an activator of MYC expression and target of rare but recurrent genomic copy gains in high-risk DLBCL. Citation Format: Aishwarya Gurumurthy, Ashwin R. Iyer, Shih-Chun A. Chu, Marcin P. Cieslik, Russell J.H. Ryan. Characterization of an essential MYC enhancer targeted by focal genomic alterations in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR04.

Functional multiomics reveals genetic and pharmacologic regulation of surface CD38 in multiple myeloma

AbstractCD38 is a surface ectoenzyme expressed at high levels on myeloma plasma cells and is the target for the monoclonal antibodies (mAbs) daratumumab and isatuximab. Pretreatment CD38 density on tumor cells is an important determinant of mAb efficacy. Several small molecules have been found to increase tumor surface CD38, with the goal of boosting mAb efficacy in a cotreatment strategy. Numerous other CD38-targeting therapeutics are currently in preclinical or clinical development. Here, we sought to extend our currently limited insight into CD38 surface expression by using a multiomics approach. Genome-wide CRISPR interference screens integrated with patient–centered epigenetic analysis confirmed known regulators of CD38, such as RARA, while revealing XBP1 and SPI1 as other key transcription factors governing surface CD38 levels. CD38 knockdown followed by cell surface proteomics demonstrated no significant remodeling of the myeloma “surfaceome” after genetically induced loss of this antigen. Integrated transcriptome and surface proteome data confirmed high specificity of all-trans retinoic acid in upregulating CD38, in contrast to the broader effects of azacytidine and panobinostat. Finally, unbiased phosphoproteomics identified inhibition of MAP kinase pathway signaling in tumor cells after daratumumab treatment. Our work provides a resource to design strategies to enhance efficacy of CD38-targeting immunotherapies in myeloma.

CRISPRi screen of long non-coding RNAs identifies LINC03045 regulating glioblastoma invasion.

Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.

Pooled CRISPR Interference Screening Identifies Crucial Transcription Factors in Gas-Fermenting Clostridium ljungdahlii.

Gas-fermenting Clostridium species hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) in Clostridium ljungdahlii, a representative species of gas-fermenting Clostridium, with a special focus on TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth of C. ljungdahlii, respectively. We focused our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleoside triphosphates (dNTPs) supply, carbon fixation, and product synthesis in C. ljungdahlii, thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression of deoR in C. ljungdahlii by adjusting its promoter strength, leading to an improved growth rate and ethanol synthesis of C. ljungdahlii when utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermenting Clostridium species, expanding the horizons for functional genomic research in these industrially important bacteria.

Systematic decoding of cis gene regulation defines context-dependent control of the multi-gene costimulatory receptor locus in human T cells

Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.