Biology subjects

Doench, J. G.

Publications and source records attributed to Doench, J. G..

At least 19 recordsLinked to original sources

bioRxiv2026

Combinatorial CRISPR/Cas12a excises targeted loci in the mammalian genome at very low efficiency

The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potential of combinatorial CRISPR/Cas12a to excise targeted loci in the mammalian genome by inducing double-strand breaks (DSBs) upstream and downstream of targeted exons. We designed a library of gRNA pairs targeting introns flanking 54 exons of 18 essential genes and 17 exons of 10 nonessential genes, aiming to assess the efficiency of Cas12 in excising targeted loci. While single gRNAs directly targeting essential exons produced expected fitness defects in cells, pairs of guides targeting flanking intronic regions resulted in only trivial viability effects, suggesting that excision rarely occurred. Further analysis showed no meaningful correlation between gRNA efficiency, orientation, GC content, or cut site geometry and the success of locus excision. These findings indicate that, under the conditions tested, the Cas12a system does not yield locus excision at the efficiency required for sensitive negative selection assays.

systems biology↗

bioRxiv2026

Phenotypic screens identify biologic regulators of nanoparticle uptake in diffuse midline glioma

Nanoparticle drug delivery systems hold considerable promise for locoregional administration to central nervous system tumors, yet the biological determinants of nanoparticle-cancer cell interactions remain poorly understood. Using patient-derived histone-mutant diffuse midline glioma (DMG) models, we performed a pooled CRISPR-Cas9 perturbation screen to systematically identify regulators of liposomal nanoparticle delivery. The screen identified candidate genes spanning endocytosis, vesicle transport, and metabolic signaling, revealing that nanoparticle delivery is governed by a broader landscape than previously appreciated. Among these, CTNNB1, or {beta}-catenin, emerged as a common negative regulator across two independent DMG models and two distinct nanoparticle surface chemistries. Transcriptomic profiling of CTNNB1-depleted DMG cells revealed upregulation of membrane remodeling and extracellular matrix gene programs, accompanied by reduced cell stiffness measured by a microfluidic acoustic scattering assay. This resulted in a shift in endocytic activity characterized by decreased bulk-phase macropinocytosis and increased receptor-mediated endocytosis. We further identified MAPK and mTOR pathway members as nanoparticle trafficking modulators, and demonstrated concordance between genetic and pharmacologic perturbations in modulating the liposomal nanoparticle interactions in pediatric DMG cells. These findings establish a biology-first screening approach for identifying previously unappreciated regulators with potential relevance to nanoparticle-based therapeutic strategies in pediatric brain tumors.

bioengineering↗

bioRxiv2026

Dynamic inositol pyrophosphate synthesis is a targetable therapeutic opportunity in ovarian cancer.

We previously reported that the phosphate exporter XPR1 is required to prevent toxic phosphate accumulation in ovarian cancer cells. To guide therapeutic development, we sought to systematically compare potential strategies to inhibit XPR1: directly targeting the phosphate efflux channel, targeting its partner protein KIDINS220, or inhibiting the synthesis of inositol pyrophosphates (PP-InsPs), metabolites which activate XPR1. We evaluated functional domains in XPR1 and KIDINS220 using mutational scanning and found that loss of function mutations in XPR1 clustered in distinct regions throughout the protein, with the most deleterious mutations in the PP-InsP-binding domain. In contrast, loss of function mutations in KIDINS220 were infrequent and altered the localization of XPR1, consistent with a scaffolding role for KIDINS220. These data highlight the functional relevance of PP-InsPs, which we confirmed by inhibiting their synthesis using IP6K inhibitors. We demonstrate that IP6K inhibition phenocopies XPR1 inhibition across hundreds of cancer cell lines, with the mechanism of sensitivity solely due to inhibition of cellular phosphate efflux. Finally, we show that IP6K inhibitors decrease tumor burden in xenograft models of ovarian cancer, but that the rapid resynthesis of PP-InsPs requires high exposures to achieve efficacy. This study comprehensively evaluates the XPR1-dependent phosphate efflux network and reinforces the concept of directly targeting XPR1 as a precision medicine strategy to benefit patients with ovarian cancer.

cancer biology↗

bioRxiv2026

Direct comparison of CRISPR knockout and interference with Perturb-seq

CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) are two workhorse technologies for loss-of-function studies, yet direct comparisons between the two are scant relative to their widespread adoption. Here, we establish benchmarking libraries for Cas9-based CRISPRko and CRISPRi screens using Perturb-seq as the read-out. For both modalities, we observe consistent transcriptional signatures among cells with the same genes perturbed, strong evidence of on-target signal. We also examine tradeoffs between modalities: while CRISPRi guides demonstrate heightened rates of off-target activity, we also observe artifacts stemming from the cellular response to double-stranded breaks with the use of CRISPRko. The libraries and analyses presented here will be a useful benchmarking and de-risking resource for any group preparing for a large-scale Perturb-seq screen.

genomics↗

bioRxiv2026

Discovery of Tcf7 regulators with clonally-resolved CRISPR screens identifies Trim28 as a mediator of CD8 T cell differentiation in tumors

Stem-like TCF7+ CD8 T cells sustain anti-tumor responses and support immune checkpoint blockade. We systematically identified regulators of this cell state using genome-wide CRISPR screens in primary T cells in vitro. Using random barcodes to link clonal relationships with guide identity and transcriptional states in single cells, we inferred differentiation trajectories and differentiation rates of CD8 T cells in tumors, while mitigating confounding clonal bias. We found that Trim28-deficient T cells in tumors were enriched in the TCF7+ cell state, depleted in cycling and terminal effector states, and uniquely generated a tissue-resident memory (TRM)-like state with increased chromatin accessibility at known TRM loci as well as repeat elements. Despite the increase in TCF7+ CD8 T cells, loss of Trim28 did not improve tumor control, likely because of reduced effector differentiation, highlighting the need for tuning the balance and dynamics of stem-like versus effector states for effective tumor clearance.

immunology↗

bioRxiv2026

CD164 is an endolysosomal host factor for entry of Clade A New World Arenaviruses

Arenaviruses are divided into Old World (OW) and New World (NW) groups. OW arenaviruses enter cells through a pH-dependent receptor switch from a plasma-membrane factor to an endolysosomal receptor for subsequent membrane fusion, whereas clade B NW arenaviruses use transferrin receptor 1 without a secondary receptor. Using a vesicular stomatitis virus (VSV) chimera expressing the glycoprotein complex (GPC) of the clade A NW arenavirus Pichinde virus, we performed a genome-wide CRISPR loss-of-function screen and identified the endolysosomal sialomucin CD164 as an essential host factor. CD164 knockout cells were resistant to VSV chimeras bearing the GPCs of Pichinde, Parana, and Flexal viruses, and to authentic Pichinde and Parana virus, with susceptibility restored by complementation. The requirement mapped to the cysteine-rich domain of CD164, which bound GP1 in a pH-dependent manner through main-chain interactions. These findings define CD164 as an endolysosomal receptor for clade A NW arenaviruses expanding the receptor switching paradigm.

microbiology↗

bioRxiv2026

KDM6 Enzymes are the Mechanistic Targets of Mutant IDH that Dictate Replication Stress Sensitivity

Cancer-associated isocitrate dehydrogenase (IDH) mutations sensitize gliomas to replication stress, although the underlying mechanisms are unclear. IDH-mutant enzymes synthesize (R)-2-hydroxyglutarate (R2HG), which broadly inhibits 2-oxoglutarate-dependent enzymes. We performed forward genetic screens targeting all 2-oxoglutarate-dependent enzymes and discovered that KDM6 histone demethylases play a vital role in protecting cells from replication stress. Genetic or R2HG-mediated repression of KDM6 catalytic activity sensitized glioma cells to disparate replication stress-inducing drugs, including Ataxia-telangiectasia and Rad3-related (ATR) and dihydroorotate dehydrogenase (DHODH) inhibitors. This liability is generalizable because KDM6A loss-of-function mutations commonly observed in urothelial carcinomas sensitized bladder cancer cells to DHODH inhibition, thereby phenocopying IDH mutations in glioma. To exploit these oncogene-induced replication stress vulnerabilities, we developed an effective, on-target, and well-tolerated DHODH inhibitor, GLIO-1, that is poised for clinical translation. Collectively, we reveal KDM6 activity as a fundamental determinant of replication stress sensitivity and nominate pan-cancer, mechanism-based biomarkers of ATR and DHODH inhibitor efficacy. STATEMENT OF SIGNIFICANCEWe discovered that the KDM6 enzymes are the mechanistic targets of R2HG that mediate mutant IDH-induced replication stress hypersensitivity. We report a promising new DHODH inhibitor, GLIO-1, and nominate KDM6 and IDH mutations as predictive biomarkers for the antitumor effects of GLIO-1 and other replication stress inducers.

cancer biology↗

bioRxiv2026

A pooled CRISPR screen reveals genes critical for erythroblast enucleation

Terminal erythroid differentiation involves dramatic cellular remodeling that culminates in the expulsion of the nucleus, a process known as enucleation. While enucleation is conserved across mammals and is crucial for the generation of fully functional erythrocytes, the mechanisms governing this process have remained largely unknown, in part because the absence of genetic material in mature, enucleated red blood cells hinders genetic experimentation. Here, we performed a pooled, forward-genetic CRISPR-Cas9 screen in enucleated red blood cells derived from primary human hematopoietic stem cells to identify genes required for enucleation. We found that Chloride Intracellular Channel 3 (CLIC3) and Vesicle-associated membrane protein 8 (VAMP8) are both necessary for terminal erythroid differentiation, yet likely act through different mechanisms. Knockdown of CLIC3 led to a delay in erythroblast differentiation, culminating in impaired enucleation. We found that the knockdown cells had increased p53 and p21 and exhibited cell cycle alterations, suggesting CLIC3 plays a crucial role in coordinating cell cycle progression during erythropoiesis. In comparison, VAMP8-depleted cells initially appear to undergo accelerated differentiation but then display a specific defect in enucleation. Transcriptional analysis of the VAMP8-knockdown cells suggested dysregulation of pathways for vesicle trafficking and actin binding, and imaging of late-stage erythroblasts revealed impaired nuclear polarization and disorganized actin. This work provides a new approach for functional genomics in enucleated cells and reveals novel factors important for terminal erythroid differentiation and enucleation. Key pointsO_LIA CROPseq-based CRISPR-Cas9 screen enables functional genomics in enucleated primary human red blood cells. C_LIO_LIChloride Intracellular Channel 3 (CLIC3) and Vesicle Associated Membrane Protein 8 (VAMP8) were identified as critical for terminal erythroid differentiation and enucleation, likely acting through two distinct mechanisms. C_LI

cell biology↗

bioRxiv2026

Optimized parameters for Cas9 CRISPR interference library design

CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific sgRNAs for the design of compact libraries. While several genome-wide Cas9 CRISPRi libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed sequence patterns. We leverage these findings to design an optimized Cas9 CRISPRi library, Katsano, and validate its performance with genome-wide viability screens.

bioengineering↗

bioRxiv2025

Host cyclophilin-mediated maturation of an obligate intracellular bacterial surface virulence factor

Adaptation of obligate intracellular bacteria to the eukaryotic cytoplasm creates dependencies on host factors that comprise unique infection mechanisms and vulnerabilities compared to facultative intracellular species. Here, we identify the peptidylprolyl isomerase cyclophilin A (PPIA/CypA) as a convergent host dependency in obligate intracellular bacterial pathogens. Using a host-directed functional genetic screen in the emerging tick-borne pathogen Rickettsia parkeri, we find that PPIA promotes infection and actin-based motility through an unprecedented inter-kingdom surface protein maturation event that facilitates transmembrane translocation of the actin-nucleating virulence factor Sca2. PPIA is required for efficient R. parkeri infection across human and tick cells, and pharmacological inhibition of PPIA phenocopies the loss of Sca2 in vitro and in vivo. This dependency extends to Chlamydia trachomatis and Orientia tsutsugamushi but appears absent in multiple facultative intracellular species. Targeting of host PPIA may thus represent a shared infection dependency across obligate intracellular bacterial pathogens with unmet therapeutic needs.

microbiology↗

bioRxiv2025

Genome-wide CRISPR interference screen identifies Clip2 as a novel regulator of osteocyte maturation and morphology

Osteocytes play critical roles in bone, making them attractive targets for therapeutics to improve bone mass and strength. The genes driving osteocyte maturation and function are not fully understood. Here we aimed to identify novel genes responsible for osteocyte differentiation and dendrite development by performing a genome-wide CRISPR-interference (CRISPRi) screen in the Ocy454 osteocyte-like cell line. We identify CD61 (integrin {beta}3) as a marker of osteocyte maturation: surface CD61 expression increases during osteocyte maturation, and CD61high cells express higher levels of osteocyte marker genes. We then developed a flow cytometry-based assay to quantify surface CD61 protein levels as a phenotypic endpoint for functional genomic screening. In a genome-wide screen, we identified Clip2, which encodes a microtubule binding protein, as one of dozens of genes necessary for CD61 expression. Clip2 inhibition decreased surface CD61 expression, reduced expression of osteocyte-specific genes Dmp1 and Sost, and impaired dendrite morphology in vitro. Together, these results highlight the utility of surface CD61 as a marker of osteocyte maturity and identify a role of the microtubule cytoskeleton for osteocyte differentiation, form, and function.

cell biology↗

bioRxiv2025

Balancing off-target and on-target considerations for optimized Cas9 CRISPR knockout library design

The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations yield library deprecation over time, further motivating an updated genome-wide design effort. Recently, we have developed an enhanced model, Rule Set 3, which leveraged an expansive training set and feature space to predict guide efficacy. However, the benefit of such advances to library design is limited by current approaches to balance predictions of on-target activity with off-target considerations. Here we present a guide selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides. We pair this model with strategic design choices to create Jacquere, an updated, optimized, and validated Cas9 CRISPR knockout (CRISPRko) genome-wide library for the human genome.

genomics↗

bioRxiv2025

Discovery of chromatin-based determinants of azacytidine and decitabine anti-cancer activity

The DNA-incorporating nucleoside analogs azacytidine (AZA) and decitabine (DEC) have clinical efficacy in blood cancers, yet the precise mechanism by which these agents kill cancer cells has remained unresolved - specifically, whether their anti-tumor activity arises from conventional DNA damage or DNA hypomethylation via DNA methyltransferase 1 (DNMT1) inhibition. This incomplete mechanistic understanding has limited their broader therapeutic application, particularly in solid tumors, where early clinical trials showed limited efficacy. Here, through the assessment of drug sensitivity in over 600 human cancer models and comparison to a non-DNA-damaging DNMT1 inhibitor (GSK-3685032), we establish DNA hypomethylation, rather than DNA damage, as the primary killing mechanism of AZA and DEC across diverse cancer types. In further support of an epigenetic killing mechanism, CRISPR drug modifier screens identified a core set of chromatin regulators, most notably the histone deubiquitinase USP48, as AZA and DEC protective factors. We show that USP48 is recruited to newly hypomethylated CpG islands and deubiquitinates non-canonical histones, establishing USP48 as a key molecular link between the two components of epigenetic gene regulation: DNA methylation and chromatin modification. Furthermore, loss of USP48, which occurs naturally through biallelic deletions in human cancers, sensitized both hematologic and solid tumors to DNMT1 inhibition in vitro and in vivo. Our findings elucidate the epigenetic mechanism of action of AZA and DEC and identify a homeostatic link between DNA methylation and chromatin state, revealing new therapeutic opportunities for DNMT1 inhibitors in solid tumors.

cancer biology↗

bioRxiv2025

Sialylated CD43 is a glyco-immune checkpoint for macrophage phagocytosis

Macrophages in the tumor microenvironment exert potent anti-tumorigenic activity through phagocytosis. Yet therapeutics that enhance macrophage phagocytosis have not improved outcomes in clinical trials for patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). To systematically identify regulators of phagocytosis, we performed genome-scale CRISPR knockout screens in human leukemia cells co-cultured with human monocyte-derived macrophages. Surprisingly, we found that whereas the classic "dont eat me" signal CD47 inhibited mouse macrophages, it did not inhibit phagocytosis by human macrophages. In contrast, the O-linked glycosylation and sialylation pathways were strong negative regulators of phagocytosis. In AML, the cell surface O-linked glycoprotein CD43 was the major effector of the O-linked glycosylation and sialylation pathways. Genetic deletion or antibody blockade of CD43 enhanced macrophage phagocytosis. This work highlights the importance of using human platforms to identify immune checkpoints, and nominates CD43 as a glyco-immune regulator of human macrophage phagocytosis.

cancer biology↗

bioRxiv2025

CREB5 promotes immunotherapy resistance via tumor-intrinsic collagenmatrix deposition

Treatment with immune checkpoint inhibitors induces remarkable clinical responses in several cancer types. However, most cancer patients fail to respond to immunotherapy, and patients who initially respond often exhibit acquired resistance. Understanding the universe of immune evasion strategies will enable design of more effective immunotherapies. Here, we identify genes that drive immune evasion using genome-scale in vivo CRISPR gain-of-function screens in tumors treated with anti-PD-1 antibodies and found that the transcription factor CREB5 drives immune checkpoint blockade resistance. Using transcriptional profiling and functional studies, we show that CREB5 promotes a mesenchymal-like phenotype in melanoma characterized by upregulation of extracellular matrix genes including collagen and collagen-stabilizing factors. Using engineered tumor models and knockout mice, we found that immunotherapy resistance is functionally mediated by tumor-intrinsic collagen deposition. Collagen is the major ligand for the inhibitory receptor LAIR1, broadly expressed on T cells, B cells, NK cells, and myeloid cells. Deletion of LAIR1 in mice or overexpression of the decoy receptor LAIR2 in tumors abrogated the resistance induced by CREB5 overexpression, demonstrating that collagen-LAIR1 inhibitory signaling drives resistance to immune checkpoint inhibitors. These observations define a transcriptional program that remodels the tumor microenvironment to promote immunotherapy resistance via extracellular matrix deposition and indicates that targeting this pathway may enhance immunotherapy efficacy. One-Sentence Summary: In vivo gain-of-function screening in immunotherapy-treated mice reveals a transcription factor, Creb5, that drives the mesenchymal state in melanoma and facilitates immune escape by promoting tumor-intrinsic collagen matrix deposition.

cancer biology↗

bioRxiv2025

A transcriptional biosensor reveals mechanisms of α-ketoglutarate signaling to chromatin

Alpha-ketoglutarate (KG) is required for chromatin demethylation but mechanisms controlling KG abundance in the nucleus are poorly defined. Therefore, we designed a biosensor system to monitor this metabolite pool in human cells using an KG-responsive cyanobacterial transcription factor, NtcA. We then coupled this system with a genetic screen to identify genes that regulate KG in the nucleus, defining an inter-organelle pathway in which sequential mitochondrial activities of the GPT2 transaminase and SLC25A11 transporter supply nuclear KG. Using a mouse model of GPT2 deficiency, a human inborn error of metabolism, we found that this pathway controls chromatin methylation in the developing brain. Our work provides a tool to assess KG signaling to chromatin and a framework for leveraging forward genetics to study nuclear metabolite pools.

synthetic biology↗

bioRxiv2025

HKU5 bat merbecoviruses use divergent mechanisms to engage bat and mink ACE2 as entry receptors

Identifying receptors for bat coronaviruses is critical for spillover risk assessment, countermeasure development, and pandemic preparedness. While Middle East respiratory syndrome coronavirus (MERS-CoV) uses DPP4 for entry, the receptors of many MERS-related betacoronaviruses remain unknown. The bat merbecovirus HKU5 was previously shown to have an entry restriction in human cells. Using both pseudotyped and full-length virus, we show that HKU5 uses Pipistrellus abramus bat ACE2 but not human ACE2 or DPP4 as a receptor. Cryo-electron microscopy (cryo-EM) analysis of the virus-receptor complex and structure-guided mutagenesis reveal a spike and ACE2 interaction that is distinct from other ACE2-using coronaviruses. MERS-CoV vaccine sera poorly neutralize HKU5 informing pan-merbecovirus vaccine design. Notably, HKU5 can also engage American mink and stoat ACE2, revealing mustelids as potential intermediate hosts. These findings highlight the versatility of merbecovirus receptor use and underscore the need for continued surveillance of bat and mustelid species.

microbiology↗

bioRxiv2024

Morphological map of under- and over-expression of genes in human cells

Cell Painting images offer valuable insights into a cells state and enable many biological applications, but publicly available arrayed datasets only include hundreds of genes perturbed. The JUMP (Joint Undertaking in Morphological Profiling) Cell Painting Consortium perturbed roughly 75% of the protein-coding genome in human U-2 OS cells, generating a rich resource of single-cell images and extracted features. These profiles capture the phenotypic impacts of perturbing 15,243 human genes, including overexpressing 12,609 genes (using open reading frames, ORFs) and knocking out 7,975 genes (using CRISPR-Cas9). We mitigated technical artifacts by rigorously evaluating data processing options and validated the datasets robustness and biological relevance. Analysis of phenotypic profiles revealed novel gene clusters and functional relationships, including those associated with mitochondrial function, cancer, and neural processes. The JUMP Cell Painting genetic dataset is a valuable resource for exploring gene relationships and uncovering novel functions.

bioinformatics↗