There is compelling evidence that senescent cells, through the senescence-associated secretory phenotype (SASP), can promote malignant transformation and invasion. IL-1 is a key mediator of this cytokine network, but the control of its activity in the senescence program has not been elucidated. IL-1 signalling is regulated by IL-1RA, which has four variants. Here, we show that expression of intracellular IL-1RA type 1 (icIL-1RA1), which competitively inhibits binding of IL-1 to its receptor, is progressively lost during oral carcinogenesis ex vivo and that the pattern of expression is associated with keratinocyte replicative fate in vitro. We demonstrate icIL-1RA1 is an important regulator of the SASP in mortal cells, as CRISPR-CAS9 mediated icIL-1RA1 knockdown in normal and mortal dysplastic oral keratinocytes is followed by increased IL-6 and IL-8 secretion, and rapid senescence following release from ROCK inhibition. Thus, we suggest that downregulation of icIL-1RA1 in early stages of the carcinogenesis process can enable the development of a premature and de-regulated SASP, creating a pro-inflammatory state in which cancer is more likely to arise.

A novel and universal CRISPR/Cas-derived precision genome editing technology named prime editing was developed. However, low prime editing efficiency was shown in transgenic rice lines. We reasoned that enhancing pegRNA expression would be able to improve prime editing efficiency. In this report, we used two strategies to enhance pegRNA expression and constructed a prime editing vector harboring two pegRNA variants for W542L and S621I double mutations in ZmALS1 and ZmALS2. As compared with previous reports in rice, we achieved much higher prime editing efficiency in maize. Our results are inspiring and provide a direction for optimization of plant prime editors.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. In nature, adaptation to severe environmental change, such as encountered following colonization of caves, has dramatically altered genomes of species over varied time spans. Genomic sequencing approaches have identified mutations associated with troglomorphic trait evolution, but the functional impacts of these mutations remain poorly understood. The Mexican Tetra, Astyanax mexicanus, is abundant in the surface waters of northeastern Mexico, and also inhabits at least 30 different caves in the region. Cave-dwelling A. mexicanus morphs are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses for pigmentation and eye size and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability that includes an analysis of the impact on protein-coding genes across cavefish populations to gain insight into this potential source of cave adaptation. The new surface fish genome reference now provides a more complete resource for comparative, functional, developmental and genetic studies of drastic trait differences within a species.

The pathogenesis of Tuberculous meningitis (TBM) is poorly understood, but contemporary molecular biology technologies have allowed for recent improvements in our understanding of TBM. For instance, neutrophils appear to play a significant role in the immunopathogenesis of TBM, and either a paucity or an excess of inflammation can be detrimental in TBM. Further, severity of HIV-associated immunosuppression is an important determinant of inflammatory response; patients with the advanced immunosuppression (CD4+ T-cell count of 150 cells/μL. Host genetics may also influence outcomes with LT4AH genotype predicting inflammatory phenotype, steroid responsiveness and survival in Vietnamese adults with TBM. Whist in Indonesia, CSF tryptophan level was a predictor of survival, suggesting tryptophan metabolism may be important in TBM pathogenesis. These varying responses mean that we must consider whether a “one-size-fits-all” approach to anti-bacillary or immunomodulatory treatment in TBM is truly the best way forward. Of course, to allow for proper treatment, early and rapid diagnosis of TBM must occur. Diagnosis has always been a challenge but the field of TB diagnosis is evolving, with sensitivities of at least 70% now possible in less than two hours with GeneXpert MTB/Rif Ultra. In addition, advanced molecular techniques such as CRISPR-MTB and metagenomic next generation sequencing may hold promise for TBM diagnosis. Host-based biomarkers and signatures are being further evaluated in childhood and adult TBM as adjunctive biomarkers as even with improved molecular assays, cases are still missed. A better grasp of host and pathogen behaviour may lead to improved diagnostics, targeted immunotherapy, and possibly biomarker-based, patient-specific treatment regimens.

One of the major challenges facing researchers is using an efficient homology-directed DNA repair (HDR) to replace the targeted fragment on the genome of tree species with the desired DNA fragment. In the past, researches have been conducted on genetic modifications in mammals using HDR effector proteins to guide double-stranded breaks (DSBs) more precisely. Here, we targeted XRCC4, a cofactor for DNA ligase IV that is a key role in nonhomologous end-joining (NHEJ), to enhance HDR. XRCC4 deficient incorporated with efficient homologous recombination factors CtIP and MRE11 promoted HDR efficiency in poplar tree lines up to 10% and dramatically decremented polymorphisms. We also could introduce exogenous bleomycin to the poplar genome and generate stable lines resistant to the zeocin antibiotic.

Abstract Base editors are chimeric ribonucleoprotein complexes consisting of a DNA-targeting CRISPR-Cas module and a single-stranded DNA deaminase. They enable conversion of C•G into T•A base pairs and vice versa on genomic DNA. While base editors have vast potential as genome editing tools for basic research and gene therapy, their application has been hampered by a broad variation in editing efficiencies on different genomic loci. Here we perform an extensive analysis of adenine- and cytosine base editors on thousands of lentivirally integrated genetic sequences and establish BE-DICT, an attention-based deep learning algorithm capable of predicting base editing outcomes with high accuracy. BE-DICT is a versatile tool that in principle can be trained on any novel base editor variant, facilitating the application of base editing for research and therapy.

ABSTRACT The programmable nuclease technology CRISPR/Cas9 has revolutionized gene editing in the last decade. Due to the risk of off-target editing, accurate and sensitive methods for off-target characterization are crucial prior to applying CRISPR/Cas9 therapeutically. Here, we utilized a rhesus macaque model to ask whether CIRCLE-Seq (CS), an in vitro off-target prediction method, more accurately identifies off-targets compared to in silico prediction (ISP) based solely on genomic sequence comparisons. We use AmpliSeq HD error-corrected sequencing to validate off-target sites predicted by CIRCLE-Seq and ISP for guide RNAs designed against TET2 and CD33 genes. A gRNA targeting TET2 designed using modern algorithms and predicted to have low off-target risk by both ISP and CIRCLE-Seq created no detectable mutations at off-target sites in hematopoietic cells following transplantation, even when applying highly sensitive error-corrected sequencing. In contrast, a CD33 gRNA designed using less robust algorithms with over 10-fold more off-targets sites predicted by both ISP and CIRCLE-Seq, however there was poor correlation between the sites predicted by the two methods. When almost 500 sites identified by each method were searched for in hematopoietic cells following transplantation, 19 detectable mutations in off-target sites were detected via error-corrected sequencing. Of these 19 sites, 8 sites were predicted in the top 500 sites by both methods, 8 by CIRCLE-Seq only, and 3 by ISP only. Cells with off-target editing exhibited no expansion or abnormal behavior in vivo in animals followed for up to 2 years. In conclusion, neither methodology predicted all sites, and a combination of careful gRNA design, followed by screening for predicted off-target sites in target cells by multiple methods may be required for optimizing safety of clinical development.

ABSTRACT Schizophrenia (SZ) is a common and debilitating psychiatric disorder with limited effective treatment options. Although highly heritable, risk for this polygenic disorder depends on the complex interplay of hundreds of common and rare variants. Translating the growing list of genetic loci significantly associated with disease into medically actionable information remains an important challenge. Thus, establishing platforms with which to validate the impact of risk variants in cell-type-specific and donor-dependent contexts is critical. Towards this, we selected and characterize a collection of twelve human induced pluripotent stem cell (hiPSC) lines derived from control donors with extremely low and high SZ polygenic risk scores (PRS). These hiPSC lines are publicly available at the California Institute for Regenerative Medicine (CIRM). The suitability of these extreme PRS hiPSCs for CRISPR-based isogenic comparisons of neurons and glia was evaluated across three independent laboratories, identifying 9 out of 12 meeting our criteria. We report a standardized resource of publicly available hiPSCs, with which we collectively commit to conducting future CRISPR-engineering, in order to facilitate comparison and integration of functional validation studies across the field of psychiatric genetics.

ABSTRACT Advances in precision oncology have transformed cancer therapy from broadly-applied cytotoxic therapy to personalized treatments based on each tumor’s unique molecular alterations. Here we investigate the oncogene-specific dependencies conferred to lung cancer cells by differing cancer driver variants of KRAS, EGFR , and RIT1 . Integrative analysis of genome-wide CRISPR screens in isogenic cell lines together with small molecule drug sensitivity profiling identified a unique vulnerability of RIT1 -mutant cells to loss of spindle assembly checkpoint regulators, such as Aurora kinase A and USP9X. We find that oncogenic RIT1 M90I alters mitotic timing, suggesting that RIT1 -mutant cells have a weakened spindle assembly checkpoint. In addition, we uncovered a specific cooperation of mutant RIT1 with loss of Hippo pathway genes. In human cancer, RIT1 mutations and amplifications frequently co-occur with loss of Hippo pathway gene expression. These results provide the first genome-wide atlas of oncogenic RIT1 -cooperating factors and genetic dependencies and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1 -mutant cancer.

Abstract Spinocerebellar Ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. Actually, there are no treatments available to delay the onset, stop or slow down the progression of this pathology. Many approaches developed over the years involve the use of siRNAs and antisense oligonucleotides (ASOs). Here we develop and validate a CRISPR/Cas9 therapeutic strategy in fibroblasts isolated from SCA1 patients. We started from the screening of 10 different sgRNAs able to recognize regions upstream and downstream the CAG repeats, in exon 8 of ATXN1 gene. The two most promising sgRNAs, G3 and G8, whose efficiency was evaluated with an in vitro system, significantly downregulated the ATXN 1 protein expression. This downregulation was due to the introduction of indels mutations into the ATXN1 gene. Notably, with an RNA-seq analysis, we demonstrated minimal off-target effects of our sgRNAs. These preliminary results support CRISPR/Cas9 as a promising approach for treated polyQ-expanded diseases.

Abstract Extensive testing is essential to break the transmission of the new coronavirus SARS-CoV-2, which causes the ongoing COVID-19 pandemic. Recently, CRISPR-based diagnostics have emerged as attractive alternatives to quantitative real-time PCR due to their faster turnaround time and their potential to be used in point-of-care testing scenarios. However, existing CRISPR-based assays for COVID-19 have not considered viral genome mutations and RNA editing in human cells. Here, we present the VaNGuard (Variant Nucleotide Guard) test that is not only specific and sensitive for SARS-CoV-2, but can also detect the virus when its genome or transcriptome has evolved or has been edited by deaminases in infected human cells. We show that an engineered AsCas12a enzyme is more tolerant of mismatches than wildtype LbCas12a and that multiplexed Cas12a targeting can overcome the presence of single nucleotide variations. Our assay can be completed in 30 minutes with a dipstick for a rapid point-of-care test.

ABSTRACT The mammalian molecular clock is based on a transcription-translation feedback loop (TTFL) containing Period1, 2 ( Per1, 2 ), Cryptochrome1, 2 ( Cry1, 2 ), and Brain and Muscle ARNT-Like 1 ( Bmal1 ). TTFL robustness is endowed by genetic complementation between these components; therefore, multiple genes must be knocked out to physiologically investigate the molecular clock, which requires extensive research resources. To facilitate molecular clock disruption, we developed a CRISPR-Cas9-based single adeno-associated viral (AAV) system targeting the circadian clock (CSAC) for Pers, Crys , or Bmal1 . First, we designed single guide RNAs (sgRNAs) targeting individual clock genes using an in silico approach and validated their efficiency in Neuro2a cells. To target multiple genes, multiplex sgRNA plasmids were constructed using Golden Gate assembly and expressed in viral vectors. CSAC efficiency was demonstrated by decreased protein expression in vitro and ablated molecular oscillation ex vivo . We also measured locomotor activity and body temperature in Cas9-expressing mice injected with CSAC at the suprachiasmatic nucleus. Circadian rhythm disruption was observed under free-running conditions, indicating that CSAC can efficiently and robustly disrupt molecular circadian clock. Thus, CSAC is a simple and powerful tool for investigating the physiological role of the molecular clock in vivo .

ABSTRACT CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here we tackle these deficiencies by building upon our previous CRISPR-mediated tissue restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in (HACK). In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila . These improvements may be also applied to other model systems.

Abstract Trans -acting DNA variants may specifically affect mRNA or protein levels of genes located throughout the genome. However, prior work compared trans -acting loci mapped in studies performed separately or with limited statistical power. Here, we developed a CRISPR-based system for simultaneous quantification of mRNA and protein of a given gene via dual fluorescent reporters in single, live cells of the yeast Saccharomyces cerevisiae . In large populations of recombinant cells from a cross between two genetically divergent strains, we mapped 86 trans -acting loci affecting the expression of ten genes. Less than 20% of these loci had concordant effects on mRNA and protein of the same gene. Most loci influenced protein but not mRNA of a given gene. One such locus harbored a premature stop variant in the YAK1 kinase gene that had specific effects on protein or mRNA of dozens of genes. These results demonstrate complex, post-transcriptional genetic effects on gene expression. One sentence summary A CRISPR-based dual reporter assay enables genetic mapping of DNA variants that specifically affect mRNA or protein levels in trans .

Abstract We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous C. elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans : the small fragment can quickly and easily be fused to almost any protein of interest and can be detected wherever the large fragment is expressed and complemented. There is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet, and generate transgenic C. elegans lines to allow easy single-color labeling in muscles and dual-color labeling in somatic cells. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for an easy, cloning-free method for CRISPR/Cas9 editing.

ABSTRACT Vasopressin regulates osmotic water transport in the renal collecting duct by PKA-mediated control of the water channel aquaporin-2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA-Cα) and PKA catalytic β (PKA-Cβ). To identify the roles of these two protein kinases, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA-Cα or PKA-Cβ was deleted using CRISPR-Cas9-based genome editing. Controls were cells carried through the genome editing procedure, but without deletion of PKA. TMT mass tagging was used for protein mass spectrometric quantification. Of the 4635 phosphopeptides that were quantified 67 were significantly altered in abundance with PKA-Cα deletion, while 21 were significantly altered in abundance with PKA-Cβ deletion. However, only four sites were changed in both. The target proteins identified in PKA-Cα-null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA-Cβ-null cells were largely associated with the actin cytoskeleton and cell junctions. In contrast, in vitro incubation of mpkCCD proteins with recombinant PKA-Cα and PKA-Cβ resulted in virtually identical phosphorylation changes. In addition, analysis of total protein abundances in the in vivo samples showed that PKA-Cα deletion resulted in a near disappearance of AQP2 protein, while PKA-Cβ deletion did not decrease AQP2 abundance. We conclude that PKA-Cα and PKA-Cβ serve substantially different functions in renal collecting duct cells and that differences in phosphorylation targets may be due to differences in protein interactions, e.g. mediated by AKAP, C-KAP or PDZ binding.

Abstract Regulatory sRNAs in photosynthetic cyanobacteria have been reported, but the lack of plausible RNA chaperones involved in this regulation appears enigmatic. Here, we analyzed the full ensemble of cellular RNAs and cofractionating proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803, a cyanobacterium rich in internal membrane systems. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished based on the in-gradient protein distribution supporting the validity of the approach. We determined the sedimentation profiles of ~67.3% of all annotated proteins and ~62.2% of all transcripts. A clustering approach of the in-gradient distribution profiles supported the mutually exclusive association of either RpoZ or the 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes. Focusing on ribonucleoprotein complexes, the data suggest the existence of previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. The resulting short list of potential RNA chaperones contains a YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The dataset is available at https://trex90.shinyapps.io/GradSeqExplorer_SynPCC6803/ , providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism.

Summary CRISPR-Cas defense systems have been coopted multiple times in nature for guide RNA-directed transposition by Tn7-like elements. Prototypic Tn7 uses dedicated proteins for two targeting pathways, one targeting a neutral and conserved attachment site in the chromosome and a second directing transposition into mobile plasmids facilitating cell-to-cell transfer. We show that Tn7-CRISPR-Cas elements evolved a system of guide RNA categorization to accomplish the same two-pathway lifestyle. Selective regulation of specialized guide RNAs allows long-term memory for access to chromosomal sites upon entry into a new host, while conventional CRISPR features maintain the ability to continually acquire guide RNAs to new plasmid and phage targets. Transposon-encoded guide RNAs are also privatized to be recognized only by the transposon-adapted system working with selective regulation to guard against toxic self-targeting by endogenous CRISPR-Cas defense systems. This information reveals new avenues to engineer guide RNAs for enhanced CRISPR-Cas functionality for genome modification.

Abstract Plasmids are a foundational tool for basic and applied research across all subfields of biology. Increasingly, researchers in synthetic biology are relying on and developing massive libraries of plasmids as vectors for directed evolution, combinatorial gene circuit tests, and for CRISPR multiplexing. Verification of plasmid sequences following synthesis is a crucial quality control step that creates a bottleneck in plasmid fabrication workflows. Crucially, researchers often elect to forego the cumbersome verification step, potentially leading to reproducibility and— depending on the application—security issues. In order to facilitate plasmid verification to improve the quality and reproducibility of life science research, we developed a fast, simple, and open source pipeline for assembly and verification of plasmid sequences from Illumina reads. We demonstrate that our pipeline, which relies on de novo assembly, can also be used to detect contaminating sequences in plasmid samples. In addition to presenting our pipeline, we discuss the role for verification and quality control in the increasingly complex life science workflows ushered in by synthetic biology.

Abstract Genome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9 (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated, isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty of the sixty-four hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-Photoswitchable Screening (AI-PS) offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

Abstract Activated Ezrin-Radixin-Moesin (ERM) proteins link the plasma membrane to the actin cytoskeleton to generate domain specific structures, such as microvilli. Among many kinases implicated in ERM activation are the homologs LOK and SLK. CRISPR/Cas9 was used to knockout all ERM proteins or LOK/SLK in human cells. LOK/SLK knockout eliminates all ERM activating phosphorylation. The apical domain of cells lacking LOK/SLK or ERMs is strikingly similar, with loss of microvilli, alterations in junctions, induction of myosin-II containing apical stress-fiber-like structures with enhanced myosin-light-chain (MLC) phosphorylation, and enhanced apical tension. Both knockout lines have elevated active RhoA, revealing that active ERMs are negative regulators of RhoA. Constitutively active ezrin can reverse the phenotypes of either ERMs or LOK/SLK knockouts, showing that the major function of LOK/SLK is to activate ERMs. Thus, LOK/SLK and ERM proteins work as a module to regulate RhoA and contribute to the proper apical morphology of epithelial cells.

Abstract Background Pulmonary fibrosis is the sequela of many pulmonary diseases, such as pneumoconiosis and idiopathic pulmonary fibrosis. The principal characteristics of pulmonary fibrosis comprise myofibroblast proliferation, alveolar damage and deposition of extracellular matrix components, which causes abnormal lung structure remodeling and an irreversible decline in lung function; however, the detailed mechanisms remain unclear. The current study focused on the role of ZC3H4, a new member of the zinc finger protein family, in SiO 2 -induced pulmonary fibrosis. Methods The expression of ZC3H4 and fibroblast activation markers (COL1A1, COL3A1 and ACTA1) was measured by western blotting and immunofluorescence staining after SiO 2 exposure (50 µg/cm 2 ). The functional change in fibroblasts was studied with a scratch assay and a 3D migration assay. The CRISPR/Cas9 system was used to explore the regulatory mechanisms of ZC3H4 in pulmonary fibroblast cells. Results The expression levels of ZC3H4 and sigmar1 (a key regulator of ER stress) were increased in pulmonary fibroblast cells and were associated with fibroblast activation, as indicated by the increase in COL1A1, COL3A1 and ACTA1, as well as the migration ability. The SiO 2 -enhanced fibroblast activation was attenuated by specific knockdown of ZC3H4 and inhibition of ER stress, demonstrating that ZC3H4 activated fibroblasts via the sigmar1/ER stress pathway. Interestingly, an ER stress blockade also inhibited ZC3H4 expression, indicating the positive feedback regulatory mechanism of ER stress on ZC3H4. Conclusions Our results demonstrate that ZC3H4 and sigmar1 might act as novel therapeutic targets for silicosis, providing a reference for further pulmonary fibrosis research.

ABSTRACT Regulatory interactions mediated by transcription factors (TFs) make up complex networks that control cellular behavior. Fully understanding these gene regulatory networks (GRNs) offers greater insight into the consequences of disease-causing perturbations than studying single TF binding events in isolation. Chromosomal translocations of the Mixed Lineage Leukemia gene ( MLL ) produce MLL fusion proteins such as MLL-AF4, causing poor prognosis acute lymphoblastic leukemias (ALLs). MLL-AF4 is thought to drive leukemogenesis by directly binding to genes and inducing aberrant overexpression of key gene targets, including anti-apoptotic factors such as BCL-2. However, this model minimizes the potential for circuit generated regulatory outputs, including gene repression. To better understand the MLL-AF4 driven regulatory landscape, we integrated ChIP-seq, patient RNA-seq and CRISPR essentiality screens to generate a model GRN. This GRN identified several key transcription factors, including RUNX1, that regulate target genes using feed-forward loop and cascade motifs. We used CRISPR screening in the presence of the BCL-2 inhibitor venetoclax to identify functional impacts on apoptosis. This identified an MLL-AF4:RUNX1 cascade that represses CASP9, perturbation of which disrupts venetoclax induced apoptosis. This illustrates how our GRN can be used to better understand potential mechanisms of drug resistance acquisition. Graphical abstract caption A network model of the MLL-AF4 regulatory landscape identifies feed-forward loop and cascade motifs. Functional screening using CRISPR and venetoclax identified an MLL-AF4:RUNX1: CASP9 repressive cascade that impairs drug-induced cell death.

Abstract Loss-of-function mutations in KMT2D are a striking feature of the germinal centre (GC) lymphomas, resulting in decreased H3K4 methylation and altered gene expression. We hypothesised that inhibition of the KDM5 family, which demethylates H3K4me3/me2, would re-establish H3K4 methylation and restore the expression of genes repressed upon loss of KMT2D . KDM5-inhibition increased H3K4me3 levels and caused an anti-proliferative response in vitro , which was markedly greater in both endogenous and CRISPR-edited KMT2D mutant DLBCL cell lines, whilst tumour growth was inhibited in KMT2D mutant xenografts in vivo . KDM5-inhibition reactivated both KMT2D-dependent and -independent genes, resulting in diminished B-cell receptor signalling and altered expression of BCL2 family members, including BCL2 itself, allowing it to synergise with agents targeting these pathways. KDM5-inhibition may offer an effective therapeutic strategy for ameliorating KMT2D loss-of-function mutations in GC-lymphomas. Statement of significance We detail a novel way of reverting the effects of loss-of-function mutations in the histone methyltransferase KMT2D by inhibiting the KDM5 demethylase family, increasing levels of H3K4me3 and restoring expression of KMT2D regulated genes.

ABSTRACT Hypercholesterolemia is a causal and modifiable risk factor for atherosclerotic cardiovascular disease. A critical pathway regulating cholesterol homeostasis involves the receptor-mediated endocytosis of low-density lipoproteins into hepatocytes, mediated by the LDL receptor. We applied genome-scale CRISPR screening to query the genetic determinants of cellular LDL uptake in HuH7 cells cultured under either lipoprotein-rich or lipoprotein-starved conditions. Candidate LDL uptake regulators were validated through the synthesis and secondary screening of a customized library of gRNA at greater depth of coverage. This secondary screen yielded significantly improved performance relative to the primary genome-wide screen, with better discrimination of internal positive controls, no identification of negative controls, and improved concordance between screen hits at both the gene and gRNA level. We then applied our customized gRNA library to orthogonal screens that tested for the specificity of each candidate regulator for LDL versus transferrin endocytosis, the presence or absence of genetic epistasis with LDLR deletion, the impact of each perturbation on LDLR expression and trafficking, and the generalizability of LDL uptake modifiers across multiple cell types. These findings identified several previously unrecognized genes with putative roles in LDL uptake and suggest mechanisms for their functional interaction with LDLR.