Biology subjects

Wang, Q.

Publications and source records attributed to Wang, Q..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

The Rise and Fall of SARM1 Base-Exchange Inhibitors

The sterile alpha and TIR motif containing 1 (SARM1) enzyme is a key driver of axonal degeneration in response to injury, making it an attractive target for treating chemotherapy-induced peripheral neuropathy (CIPN) and other nervous system diseases. In this study, we identified and optimised a new class of base-exchange inhibitors (BEXi) targeting SARM1 and explored their molecular interactions and conformational effects using cryo-EM, HDX-MS and SAXS. Although BEXi produced robust inhibition across all biochemical and cellular assay formats, application at sub-inhibitory concentrations consistently led to paradoxical SARM1 activation, and in neuronal assays, accelerated neurite degeneration. Further analysis showed that BEXi only delayed, rather than prevented, neurite degeneration when applied to primary neuronal cells, even at exceedingly high inhibitor concentrations. These results prompted us to discontinue BEXi development in favour of alternative strategies, underscoring the complexity of SARM1 as a therapeutic target and the need for comprehensive, mechanistically informed screening cascades.

neuroscience↗

bioRxiv2025

Spatiotemporal control of PIWI compartmentalization by mitochondrial scaffolds defines pachytene piRNA pathway organization

Pachytene piRNAs are the least understood class of piRNAs in the mammalian male germ line. During meiosis, their biogenesis occurs near mitochondrial outer membrane in germ granules known as intermitochondrial cement (IMC). However, how mitochondrial factors regulate the trafficking of PIWI proteins into and out of the IMC remain poorly understood. Here we show that the cytoplasmic PIWI proteins MILI and MIWI are recruited for pachytene piRNA biogenesis via distinct mitochondrial membrane proteins. Loss of the mitochondrial scaffold protein ASZ1 during meiosis in mice disrupts multiple downstream biogenesis steps, leading to misregulation of MILI and MIWI, failure of IMC formation, and a near-complete loss of mature pachytene piRNAs. Strikingly, despite the drastic depletion of pachytene piRNAs, LINE1 transposon silencing remains unaffected. We identify three classes of pachytene piRNA pathway components that coordinate piRNA production and compartmentalization. Our findings reveal that chromatoid body precursors serve as a central hub for the accumulation of pachytene PIWI-piRNA complexes, thus establishing a connection between IMC-based biogenesis and downstream piRNA function.

developmental biology↗

bioRxiv2025

Long-term Locus Coeruleus Stimulation Exacerbates Tau Pathology in PS19 Mice

BackgroundAlzheimers disease (AD) is the most common form of dementia, characterized by the accumulation of amyloid-{beta} (A{beta}) plaques and hyperphosphorylated Tau tangles. The locus coeruleus (LC) is among the first brain regions to show degeneration and Tau pathology during the early stages of AD. Previous studies have demonstrated that short-term chemogenetic LC stimulation can improve memory performance in the TgF344-AD rat model, while long-term norepinephrine (NE) reuptake inhibition can worsen memory deficits in the ADLPTau mouse model. However, the effects of long-term LC stimulation in Tau mouse models on memory, synaptic plasticity, and tauopathy remain unclear. ObjectiveTo evaluate the impact of long-term locus coeruleus stimulation on memory, synaptic plasticity, and tauopathy in PS19 mice using behavioral paradigms, electrophysiological recordings, and immunohistochemical analysis. MethodsThe radial arm water maze and fear conditioning test were conducted to assess memory performance in PS19 mice with and without long-term LC stimulation. Hippocampal long-term potentiation was recorded to evaluate the effect of long-term LC stimulation on synaptic plasticity. Immunohistochemistry was employed to examine Tau phosphorylation, neurodegeneration, and neuroinflammation. ResultsLong-term LC stimulation in PS19 mice exacerbated spatial memory deficits in the water maze, impaired contextual fear memory, reduced hippocampal LTP, and increased AEP expression, Tau hyperphosphorylation, and astrocyte activation. ConclusionLong-term LC stimulation may exacerbate memory deficits in PS19 mice by impairing synaptic plasticity and increasing neural degeneration in the hippocampus. Elevated norepinephrine levels resulting from long-term LC stimulation may increase AEP expression, contributing to Tau hyperphosphorylation in the LC.

pathology↗

bioRxiv2025

Elevated plasma cholesterol causally improves sepsis outcome by promoting hepatic metabolic reprogramming

RationaleSepsis is a life-threatening condition with high mortality and limited therapeutic options. While hypocholesterolemia is associated with poor outcomes, the causal role of cholesterol and its underlying mechanisms remain unclear. ObjectivesTo determine whether elevated plasma cholesterol improves sepsis survival and to elucidate the mechanistic basis of this effect. MethodsWe analyzed septic patients from the MIMIC-IV database. Total cholesterol levels were compared between 28-day survivors and non-survivors using Wilcoxon rank-sum tests. Cox proportional hazards regression assessed the association between cholesterol levels and mortality, adjusting for age, sex, race, SOFA score, and Charlson comorbidity index. To establish causality, C57BL/6J mice were randomized to receive either a high-cholesterol diet (HCD) or regular diet (RD) for three days prior to cecal ligation and puncture (CLP) to induce sepsis. Measurements and Main ResultsSurvivors had significantly higher cholesterol levels than non-survivors (median, 135 vs. 126 mg/dL, p < 0.001). High cholesterol ([≥] 133 mg/dL) was independently associated with reduced 28-day mortality (adjusted HR = 0.80, 95% CI: 0.67-0.95, p = 0.012). In mice, HCD elevated plasma cholesterol and significantly improved survival (52.5% to 90%), independent of immune modulation.Hepatic transcriptomics revealed metabolic reprogramming, including upregulation of oxidative phosphorylation and glutathione-mediated antioxidant pathways, and suppression of endoplasmic reticulum proteostasis. Notably, inhibition of mitochondrial respiration abolished the survival benefit. ConclusionsElevated plasma cholesterol causally improves sepsis outcomes by promoting hepatic metabolic reprogramming. These findings offer mechanistic insight into clinical observations and suggest hepatic bioenergetics as a novel therapeutic target in sepsis.

physiology↗

bioRxiv2025

Enhancing HIPEC for Ovarian Cancer using Adjunctive Biomaterials

Ovarian cancer is one of the most lethal gynecological malignancies, with high mortality rates primarily due to late-stage diagnoses and extensive peritoneal metastases. Despite improvements in surgical and chemotherapeutic treatments, the prognosis for advanced ovarian cancer remains poor, highlighting the urgent need for innovative therapeutic approaches. Hyperthermic intraperitoneal chemotherapy (HIPEC) has emerged as a promising treatment, delivering heated chemotherapeutic agents directly into the peritoneal cavity post-cytoreductive surgery. However, HIPEC adoption is limited by three critical complications: suboptimal therapeutic efficacy in resistant tumors; abdominal adhesion formation; and systemic toxicity, including cisplatin-induced nephrotoxicity. This study investigates carbon monoxide gas- entrapping materials (CO-GEMs) as a novel multifunctional adjunctive therapy to address the three HIPEC limitations simultaneously. CO-GEMs effectively encapsulate and deliver carbon monoxide, leveraging the differential effects of CO in cancerous where it has been shown to reduce tumor burden. In ovarian cancer models, CO-GEMs significantly enhanced cisplatin efficacy, reducing the metastatic tumor burden by 46.6% through the downregulation of drug resistance pathways, including the IL-17, TNF, and NF-{kappa}B pathways; ECM receptor interaction, and VEGF signaling. CO-GEMs also prevented peritoneal adhesion formation by suppressing inflammatory cell infiltration and collagen deposition, with a significant reduction in adhesion severity scores. Additionally, enteral CO-GEMs provided significant nephroprotection against cisplatin-induced acute kidney injury, as demonstrated by reduced blood urea nitrogen levels. CO-GEMs represent a promising innovation that simultaneously improves HIPEC therapeutic efficacy, prevents surgical complications, and reduces systemic toxicity. This multifunctional approach addresses multiple clinical limitations of HIPEC, potentially transforming treatment outcomes for patients with advanced ovarian cancer through an enhanced therapeutic index and improved safety profile.

bioengineering↗

bioRxiv2025

Replication-competent SIVcpz CRISPR screen identifies barriers to successful cross-species transmission

Simian immunodeficiency viruses (SIVs) have crossed from apes to humans at least four times, but only one event gave rise to the AIDS pandemic. The host barriers that pandemic HIV-1 group M (major) strains overcame to spread efficiently in humans remain poorly understood. To identify such barriers, we performed CRISPR-Cas9 screens driven by the replication efficiency of SIVcpz, the chimpanzee precursor of HIV-1. Guide RNA libraries targeting more than 500 human genes encoding potential antiviral factors were inserted into the replication-competent SIVcpz MB897 molecular clone, which is phylogenetically closely related to HIV-1 group M strains. Propagation in Cas9-expressing human SupT1 T cells significantly enriched for sgRNAs targeting ADAR, AXIN1, CEACAM3, CD72, EHMT2, GRN, HMOX1, HMGA1, ICAM2, CD72, IFITM2, MEFV, PCED1B, SGOL2, SMARCA4, SUMO1 and TMEM173. These hits only partially overlapped with those identified in analogous HIV-1-based screens, indicating virus-specific restriction profiles. Functional analyses confirmed that IFITM2 (interferon-induced transmembrane protein 2), PCED1B (PC-esterase domain-containing protein 1B), MEFV (Mediterranean fever protein, pyrin/TRIM20), and AXIN1 (Axis inhibition protein 1), restrict replication of SIVcpz but not of HIV-1 group M strains in primary human CD4 T cells. These findings reveal previously unrecognized host factors that limit SIVcpz replication in human cells and highlight barriers that HIV-1 likely overcame during its adaptation for pandemic spread. One Sentence SummaryCRISPR screens with replication-competent SIVcpz identify human antiviral factors limiting efficient viral replication after zoonotic transmission.

immunology↗

bioRxiv2025

YAP1 defines an emergent, plastic population of relapsed small cell lung cancer

Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid onset of chemoresistance and poor clinical outcomes. Transcriptional heterogeneity among treatment-naive SCLC tumors underlies four transcriptional subtypes, each with distinct clinical vulnerabilities. Though previously hypothesized to delineate a distinct subtype, expression of YAP1 is largely absent from treatment-naive, pure SCLC. To characterize relapsed SCLC, circulating tumor DNA, circulating tumor cells, and core needle biopsies from SCLC patients and preclinical models following resistance to standard-of-care therapies were analyzed. In contrast to treatment-naive SCLC, these analyses reveal an emergent YAP1-positive cell population that coincides with treatment resistance. These YAP1-positive cells exhibit characteristics of drug tolerant persister cells, including senescence, stemness, and plasticity, as YAP1 positive cells largely abandon features characteristic of SCLC to adopt those of large-cell neuroendocrine carcinoma (LCNEC). As a result of this SCLC-like to LCNEC-like evolution, YAP1-positive cells lack several clinically relevant SCLC surface targets (i.e., DLL3, SEZ6), but are enriched for others (i.e., B7-H3, TROP2). We propose a model where YAP1 expressing cells emerge with SCLC treatment resistance and characterize a tenacious subpopulation capable of diverging from the treatment naive lineage and adopting features to evade therapeutic response.

cancer biology↗

bioRxiv2025

β2 adrenergic receptors orchestrate neutrophil demargination and recruitment to the ischemic heart following myocardial infarction.

Neutrophils play a crucial role in instigating inflammation as well as its resolution post-myocardial infarction (MI). Although granulopoiesis in the bone marrow (BM) is the major source of cardiac neutrophils post-MI, infiltration of neutrophils to the heart occurs much quicker than peak granulopoiesis. These observations suggest that sources other than granulopoiesis may supply neutrophils to the heart during the early hours post-MI. Using a combination of flow cytometry, BM ablation of hematopoietic stem cells, confocal microscopy and multiple proteomics analysis, we found that the first wave of neutrophils recruited to the ischemic heart is exclusively sourced from vasculature and not from granulopoiesis in the BM/ spleen. The MI-evoked neutrophilia during the early hours bore all hallmarks of demargination induced by classical demarginating agents such as dexamethasone/ norepinephrine (NE). Various pharmacological and genetic strategies aimed at suppressing NE synthesis or disruption of {beta}-AR signaling reduced both neutrophil demargination as well as recruitment to the heart. Interestingly, however, despite a marked reduction in cardiac neutrophil burden only short-term inhibition of {beta}-ARs improved cardiac remodeling and function. Our findings support a pharmacological strategy to contain the initial onslaught of neutrophils on the ischemic heart using {beta}2-AR blockers to regulate the otherwise runaway inflammatory response.

immunology↗

bioRxiv2025

IRG1/itaconate/NRF2/GSH axis in tumor-associated macrophages drives therapy resistance and immune evasion in BRCA1-deficient breast cancer

Tumor-associated macrophages (TAMs) are major contributors to immunosuppression and therapeutic resistance, including resistance to PARP inhibitors (PARPi) in BRCA1-deficient breast cancer. However, the mechanisms underlying TAM-mediated PARPi resistance remain unclear. Here, we demonstrate that TAM-derived glutathione (GSH) impairs the efficacy of PARPi by protecting tumor cells from DNA damage and ferroptosis while suppressing STING-mediated immune activation. Mechanistically, STAT5-driven upregulation of the IRG1/itaconate axis in TAMs rewires mitochondrial metabolism and activates NRF2-dependent GSH biosynthesis. GSH is subsequently released into the tumor microenvironment, where it is taken up by tumor cells, protecting them from PARPi-induced cytotoxicity and dampening immune responses. Pharmacological inhibition of IRG1 reverses these effects, restoring PARPi sensitivity and enhancing anti-tumor immunity in BRCA1-deficient tumor models. Collectively, these findings uncover a TAM-specific immunometabolic program that limits PARPi efficacy and highlight the IRG1/NRF2/GSH axis as a promising therapeutic target to improve treatment outcomes in BRCA1-associated breast cancer.

cancer biology↗

bioRxiv2025

Kinase KEY1 controls pyrenoid condensate size throughout the cell cycle by disrupting phase separation interactions

Biomolecular condensates spatially organize cellular functions, but the regulation of their size, number, dissolution, and re-condensation is poorly understood. The pyrenoid, an algal biomolecular condensate that mediates one-third of global CO2 fixation, typically exists as one large condensate per chloroplast, but during cell division it transiently dissolves and reconfigures into multiple smaller condensates. Here, we identify a kinase, KEY1, in the model alga Chlamydomonas reinhardtii that regulates pyrenoid condensate size and number dynamics throughout the cell cycle and is necessary for normal pyrenoid function and growth. Unlike wild type, key1 mutant cells have multiple smaller condensates throughout the cell cycle that fail to dissolve during cell division. We show that KEY1 localizes to the condensates and promotes their dissolution by disrupting interactions between their core constituents, the CO2-fixing enzyme Rubisco and its linker protein EPYC1, through EPYC1 phosphorylation. We develop a biophysical model that recapitulates KEY1-mediated condensate size and number regulation and suggests a mechanism for controlling condensate position. These data provide a foundation for the mechanistic understanding of the regulation of size, number, position, and dissolution in pyrenoids and other biomolecular condensates.

cell biology↗

bioRxiv2025

Quantitative Prevalence, Chemical Speciation, and ECM-Embedded Networks of Biogenic Silicon in Animal Tissues Revealed by Refined Analytical Methods

Silicon, abundant in Earths biosphere, plays crucial roles in many organisms. However, its biological functions in animals remain inadequately characterized, primarily constrained by methodological limitations in quantifying, identifying, and visualizing biogenic silicon in tissues. Here, we refined the molybdenum blue colorimetric method (MB) for more accurate silicon quantification, developed integrated mass spectrometry techniques to delineate chemical natures of biogenic silicon, and optimized micro-X-ray fluorescence (micro-XRF) imaging for spatial mapping in animals. Employing these methodologies, we demonstrated that silicon abundance in animals substantially exceeds prior estimates--surpassing essential elements like iron. We also revealed the chemical architectures of diverse silicon compounds and visualized tissue-specific distributions in mice and humans, indicating its predominant localization within extracellular matrix (ECM) and suggesting the formation of silicified networks. These findings establish silicon as a structurally and quantitatively significant element with profound biological implications, warranting its recognition as an essential factor in human physiology and pathology.

biochemistry↗

bioRxiv2025

Comparative population genomics reveals adaptive convergence in two Drosophila species across global environments

The extent to which evolution is predictable across lineages remains unclear. To address this question, we investigated convergent adaptation in a pair of globally distributed sibling species, Drosophila melanogaster and D. simulans. We integrated whole-genome data from approximately 2,000 strains sampled across major continents, and revealed a more recent global colonization of D. simulans relative to D. melanogaster. Using a suite of complementary selection scans, we quantified signatures of positive selection across evolutionary timescales and genomic contexts. Despite substantial divergence, approximately 9-13% of adaptively evolving genes were shared between species across methods, revealing widespread convergence at the gene and pathway levels. Convergence was particularly pronounced for insecticide resistance genes, and was also evident in oxidative stress experiment. This study provides a quantitative, multiscale framework for dissecting molecular convergence, offering insights into the predictability of evolution, the constraints imposed by genomic architecture, and the dynamics of adaptation under global environmental change.

evolutionary biology↗

bioRxiv2025

Role of tankyrase scaffolding in the β-catenin destruction complex and WNT signaling

Aberrant WNT/{beta}-catenin signaling drives tumorigenesis and metastasis in cancer. Although enzymatic inhibitors of tankyrase (TNKS) effectively block AXIN degradation and stabilize the {beta}-catenin destruction complex (DC), they have demonstrated limited efficacy in various cancer models. Here we demonstrate that, unexpectedly, the induction of AXIN puncta represents a major barrier to achieving therapeutic efficacy. Mechanistically, catalytic inhibition of TNKS prevents TNKS turnover and drives its accumulation in the DC, wherein the scaffolding function of TNKS induces AXIN puncta formation, rigidifies the DC, and impedes {beta}-catenin turnover. Chemically induced degradation of TNKS overcomes this limitation by stabilizing AXIN without puncta formation, providing a deeper suppression of the WNT/{beta}-catenin pathway activity and the proliferation of colorectal cancer cells harboring dysfunctional APC mutations. Collectively, these findings provide an explanation for the unsatisfactory outcomes of drugging the WNT/{beta}-catenin signaling pathway by TNKS inhibitors and highlight TNKS degradation as a promising approach to treat WNT/{beta}-catenin-driven cancers.

cancer biology↗

bioRxiv2025

PI-FC: Pre-training Individual-specific Functional Connectome through State-invariant Contrastive Learning

Functional MRI enables non-invasive mapping of brain connectivity, yet its clinical translation remains hindered by uncontrolled state-dependent variability that obscures individual-specific signatures during routine scanning. Here we introduce PI-FC -- a deep learning framework leveraging state-invariant contrastive learning to extract stable individual brain signatures across diverse arousal levels, cognitive states, and temporal scales spanning tens of seconds to hours. PI-FC achieves equivalent phenotypic prediction accuracy using substantially reduced scanning time, and eliminates state-dependent effects varying task demands and brain states. Trained on 36,119 subjects across 8 independent datasets, our model demonstrates superior cross-site generalization and outperforms traditional functional connectome (FC) in predicting neuropsychiatric conditions including schizophrenia, autism, depression, and anxiety. Furthermore, PI-FC enables zero-shot inference of brain age, biological sex, and cognitive ability without site-specific retraining. Overall, PI-FC represents a robust, clinically scalable framework that overcomes fundamental barriers to real-world deployment of precision functional neuroimaging.

neuroscience↗

bioRxiv2025

Convergent utilization of APN receptor by hedgehog merbecoviruses

Dipeptidyl peptidase-4 (DPP4) and angiotensin-converting enzyme 2 (ACE2) are well-established receptors for merbecoviruses, yet the receptor usage of merbecoviruses of European and Asian hedgehogs (EriCoVs) remains unknown. Here, by testing hedgehog orthologs of known coronavirus receptors, we identify hedgehog aminopeptidase N (APN) as a functional receptor for EriCoVs. Analysis of 94 APN orthologs indicates that EriCoVs have a limited host range, primarily utilizing hedgehog APN and, to a lesser extent, APN from certain felids, shaped by specific determinants at the virus-receptor interface. Cryo-EM reveals an APN-binding mode distinct from those used by alpha- and deltacoronaviruses. Functional assays indicate that hedgehog transmembrane serine protease 2 (TMPRSS2) enhances spike activation and promotes pseudovirus entry. Neutralizing antibodies targeting RBD and APN were developed and could effectively block EriCoV pseudovirus entry and propagation. These findings reveal an unexpected convergent evolution of APN utilization among merbecovirus, establishing a foundation for risk assessment and countermeasure development. Graphic abstractConvergent utilization of hedgehog APN by EriCoVs O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/676546v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ea334aorg.highwire.dtl.DTLVardef@cd53bforg.highwire.dtl.DTLVardef@352102org.highwire.dtl.DTLVardef@a1eed_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Condensate-based shells and scaffolds via interfacial liquid-to-solid transition of disordered peptides

Living organisms use biomolecular condensates to respond to dynamic environments and create functional materials with complex architectures. Exploring such phase-separated systems beyond the naturally occurring scenarios may offer valuable insights for emergent synthetic biosystems. Here, we report the self-assembly behavior of a short, disordered peptide sequence (termed CT45) derived from a protein present in the bioad-hesive system of tick ectoparasites. We show that CT45 spontaneously accumulates at polar-nonpolar interfaces, and further undergoes liquid-liquid and liquid-to-solid phase transitions to create mechanically stable structures. When encapsulated within vesicles and presented with a stable oil-water interface, CT45 rapidly forms solid shells, which can be reinforced by up-concentrating the material through osmotic imbalance. Unex-pectedly, when presented with a transient acetone-water interface, CT45 condenses at the evaporating interface and forms interconnected, porous mesoscopic scaffolds. The underlying mechanism is found to be the amphiphilic nature of CT45 leading to in-terfacial accumulation, enhancing intermolecular{pi} -based interactions to trigger phase transitions. The micron-sized shells exhibit appreciable mechanical strength and the porous scaffolds present a highly stable platform capable of retaining molecules. In conclusion, the presented condensate-based microscopic and mesoscopic scaffolds hold significance in customizable condensate architectures, with potential applications in biomedical engineering and synthetic biology.

bioengineering↗

bioRxiv2025

Beyond Ornithine Metabolism in Gyrate Atrophy: Tissue-Specific Proteomic Insights into Neonatal and Adult OAT Deficiency

Ornithine aminotransferase (OAT) links the urea cycle, TCA cycle, and amino acid metabolism by interconverting ornithine to pyrroline-5-carboxylate and glutamate. Mutations in OAT cause hyperornithinemia and predominantly affect the eye, leading to gyrate atrophy of the choroid and retina (GA), a rare inherited blinding disorder. To understand the early molecular changes that make the eye susceptible to damage, we performed quantitative proteomic and metabolomic profiling of liver, retina, and retinal pigment epithelium and choroid (RPE/Cho) from OAT-deficient (Oatrhg) mice prior to detectable vision impairment. In addition to reduced OAT expression and elevated ornithine, methylation-related metabolites such as N(6)-methyl-lysine were altered in all examined tissues of Oatrhgmice. In the liver, ornithine disposal through the urea cycle was enhanced, together with altered expression of detoxification enzymes and histone H2B proteins. In contrast, the retina had minimal proteomic changes but pronounced alterations in amino acid pathways supporting glutamate homeostasis. The RPE/Cho demonstrated the most extensive proteomic changes, particularly in mitochondrial metabolism, cytoskeleton, and extracellular matrix, along with reductions in metabolites involved energy metabolism and antioxidant capacity. Together, these findings highlight common and tissue-specific impacts of OAT on the liver and ocular tissues and provide insight into early molecular changes that contribute to the selective vulnerability of the eye in GA. Proteomics data are available via ProteomeXchange (PXD063614) and metabolomics data via MassIVE repository (MSV000101103).

biochemistry↗

bioRxiv2025

Characterization and engineering of highly efficient Cas12j genome editors

The large size of CRISPR-Cas enzymes limits their delivery for therapeutic applications. Cas12j nucleases offers hypercompact alternative but show moderate editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologues (Cas12j-11 to Cas12j-18) from viral metagenomes. All showed low editing activity in mammalian cells. We engineered T5 exonuclease-Cas12j fusions (T5Exo-Cas12j), two of which, T5Exo-Cas12j-12, and -18 exhibited up to 42% editing in HEK293T and 9% in K-562 cells, outperforming wild-type Cas12j counterparts and comparable to LbCas12a. Intriguingly, robust in cellula editing in both HEK293T and K-562 cells was strictly dependent on the presence of 5'-TAC trinucleotides within the target DNA sequence. Furthermore, we fused the Cas12j orthologues with the TadA8e deaminase and developed base editors, termed Be-(d)Cas12j. Among these, Be-(d)Cas12j-13 demonstrated efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by characterizing and engineering novel Cas12j orthologues into compact, high-efficiency genome editors.

bioengineering↗