Biology subjects

Zhou, L.

Publications and source records attributed to Zhou, L..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Calorie Restriction Induces Degeneration of Neurons with Mitochondrial DNA Depletion by Altering ER-Mitochondria Calcium Transfer

Mitochondrial DNA (mtDNA) mutations and/or depletion are implicated in epilepsy and many neurodegenerative diseases. However, systematic investigation into how mtDNA alterations relate to epilepsy and neural degeneration is needed. Here, we established a mouse model where in mtDNA depletion induced by Herpes Simplex Virus Type 1 (HSV-1) protein-UL12.5 in the brain led to an epileptic phenotype characterized by abnormal electroencephalography (EEG) patterns and increased neural excitability in hippocampus. We also found that UL12.5 mediated mtDNA depletion in neurons in vitro (rho-) causes epilepsy-like abnormal EEG. Caloric restriction (CR) is a strategy proven to reduce epileptic activity, however CR mimetic 2-deoxy-D-glucose (2-DG), induced degeneration in mtDNA depleted neurons. Mechanistically, mtDNA depletion increased mitochondria-endoplasmic reticulum (ER) contacts, facilitating CR-induced mitochondrial calcium overload. Rho- neurons did not show changes in mitochondrial motility or membrane potential. Our study revealed an unexpected axis of mtDNA depletion, ER-mitochondrial contacts, and calcium overload in the rho- neuron model. This is the first description of animal and neuronal models of mitochondrial epilepsy. Our findings with these models suggest that CR may not be a viable clinical intervention in patients with mtDNA depletion.

cell biology↗

bioRxiv2024

Elevated phagocytic capacity directs innate spinal cord repair

Immune cells elicit a continuum of transcriptional and functional states after spinal cord injury (SCI). In mammals, inefficient debris clearance and chronic inflammation impede recovery and overshadow pro-regenerative immune functions. We found that, unlike mammals, zebrafish SCI elicits transient immune activation and efficient debris clearance, without causing chronic inflammation. Single-cell transcriptomics and inducible genetic ablation showed zebrafish macrophages are highly phagocytic and required for regeneration. Cross-species comparisons between zebrafish and mammalian macrophages identified transcription and immune response regulator (tcim) as a macrophage-enriched zebrafish gene. Genetic deletion of zebrafish tcim impairs phagocytosis and regeneration, causes aberrant and chronic immune activation, and can be rescued by transplanting wild-type immune precursors into tcim mutants. Conversely, genetic expression of human TCIM accelerates debris clearance and regeneration by reprogramming myeloid precursors into activated phagocytes. This study establishes a central requirement for elevated phagocytic capacity to achieve innate spinal cord repair.

neuroscience↗

bioRxiv2024

Three-dimensional chromatin mapping of sensory neurons reveals that cohesin-dependent genomic domains are required for axonal regeneration

The in vivo three-dimensional genomic architecture of adult mature neurons at homeostasis and after medically relevant perturbations such as axonal injury remains elusive. Here we address this knowledge gap by mapping the three-dimensional chromatin architecture and gene expression programme at homeostasis and after sciatic nerve injury in wild-type and cohesin-deficient mouse sensory dorsal root ganglia neurons via combinatorial Hi-C and RNA-seq. We find that cohesin is required for the full induction of the regenerative transcriptional program, by organising 3D genomic domains required for the activation of regenerative genes. Importantly, loss of cohesin results in disruption of chromatin architecture at regenerative genes and severely impaired nerve regeneration. Together, these data provide an original three-dimensional chromatin map of adult sensory neurons in vivo and demonstrate a role for cohesin-dependent chromatin interactions in neuronal regeneration.

neuroscience↗

bioRxiv2024

A chemically defined and xeno-free hydrogel system for regenerative medicine.

Biofabricated scaffolds facilitate bona fide cellular interactions, cell type specification, and the formation of three-dimensional tissue architecture from human pluripotent stem cells (hPSCs). However, xenogenic biomaterials are poorly defined, and synthetic biomaterials remain underdeveloped and understudied, hindering regulatory approval for clinical use and preventing the translation of lab-grown therapies. Here, we describe a protein screen-based hydrogel system biofabricated from clinical-grade human components. We show that "Alphagel", a base hydrogel comprising human embryonic matrices, supports the trilineage differentiation of hPSCs into neural, cardiac, and liver tissue. Alphagel is also shown to be biocompatible and biodegradable in vivo. Further, upon adding select proteins from maturing human foetal liver to Alphagel, we show that the resulting hydrogel (termed "Hepatogel") enhances the differentiation of hPSC-derived hepatocytes (H-iHeps) compared with Matrigel. Importantly, when injected into mice livers, Hepatogel significantly improves the retention of H-iHeps compared to standard aqueous cell injections. Altogether, our results provide proof of concept that customisable and organ-specific hydrogel systems are a valuable tool for developing clinically translatable therapies for regenerative medicine and tissue engineering.

bioengineering↗

bioRxiv2024

Sympathetic neuron-derived NPY protects from obesity by sustaining the mural progenitors of thermogenic adipocytes.

Neuropeptide Y (NPY) is secreted by sympathetic nerves1,2, but its direct impact on thermogenic adipocytes is unknown. Here we uncover the mechanism by which peripheral NPY protects from obesity. Our imaging of cleared murine brown and white adipose tissue (BAT and WAT) established that NPY+ sympathetic axons are only a minority that mostly maps to the peri-vasculature; our analysis of single-cell RNA-sequencing datasets identifies mural cells as the main NPY-responsive cells in adipose tissues. We show that NPY sustains mural cells, which are known to be a source of beige cells in both BAT and WAT3-5 and that NPY facilitates the differentiation to thermogenic adipocytes. We found that diet-induced-obesity leads to neuropathy of NPY+ axons and concomitant depletion of the mural cell pool of beige fat progenitors. This defect is replicated in conditional knockout (cKO) mice with NPY specifically abrogated from sympathetic neurons. These cKO mice have whitened BAT with reduced thermogenic ability and lower energy expenditure even before the onset of obesity; they develop adult-onset obesity on a regular chow diet and are more susceptible to diet induced obesity without increasing food consumption. Our results indicate that, relative to central NPY, peripheral NPY produced by the sympathetic nerves has the opposite effect on body weight homeostasis by sustaining the proliferation of the mural cell progenitors of thermogenic adipocytes.

physiology↗

bioRxiv2024

Harnessing Data-Intelligence-Intensive Multi-Agent System for Life Science Research

Advancements in high-throughput sequencing technologies and artificial intelligence offer unprecedented opportunities for groundbreaking discoveries in bioinformatics research. However, the challenges of exponential growth of omics data and the rapid development of artificial intelligence technologies require automated big biological data analysis capability and interdisciplinary knowledge-driven scientific insight. Here we propose a data-intelligence-intensive bioinformatics copilot (Bio-Copilot) system that synergizes AI capabilities with human expertise to facilitate hypothesis-free exploratory research and inspire novel scientific insights in large-scale omics studies. Bio-Copilot forms high-quality intensive intelligence through close collaboration between multiple agents, driven by large language models (LLMs), and human experts. To augment the capabilities of Bio-Copilot, this study devises an agent group management strategy, an effective human-agent interaction mechanism, a shared interdisciplinary knowledge database, and continuous learning strategies for the agents. We comprehensively compare Bio-Copilot against GPT-4o and several leading AI agents across diverse bioinformatics tasks, using a broad range of evaluation metrics. Bio-Copilot achieves the overall state-of-the-art performance across all tasks, while showcases exceptional task completeness. Furthermore, in the application of constructing a large-scale human lung cell atlas, Bio-Copilot not only reproduces the intricate data integration process detailed in a seminal study but also introduces a hierarchical annotation strategy to capture the continuous nature of cellular states and uncovers the characteristics of rare cell types, highlighting its potential to unravel hidden complexities in biological systems. Beyond the technical achievements, this study also underscores the profound implications of integrating AI capabilities with expert knowledge in accelerating impactful biological discoveries and exploring uncharted territories in life sciences.

bioinformatics↗

bioRxiv2024

hESCs-derived organoids achieve liver zonation by LSEC modulation

Liver zonation, essential for diverse physiological functions, is lacking in existing organoid models, hindering their ability to recapitulate liver development and pathogenesis. Addressing this gap, we explored the feasibility of achieving zonated organoid by co-culturing human embryonic stem cells (hESCs) derived hepatocytes with hESCs derived liver sinusoidal endothelial cells (LSECs) exhibiting characteristics of either the liver lobules pericentral (PC) or periportal (PP) regions. Introducing zonated LSECs with variable WNT2 signaling subtly regulate hepatocyte zonation, resulting in noticeable metabolic function changes. Considering the lipid metabolism variations in PC and PP organoids, we constructed biomimetic zonated non-alcoholic fatty liver disease (NAFLD) organoids and revealed that glucagon-like peptide-1 receptor agonist (GLP-1RA) target LSECs, but not hepatocytes, indicating potential therapeutic mechanisms of GLP-1RA in NAFLD alleviation. This study highlights the crucial role of non-parenchymal cells in organoids for recapitulating niche heterogeneity, offering further insights for drug discovery and in vitro modeling of organ heterogeneity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=184 SRC="FIGDIR/small/592562v1_figu1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1907c9eorg.highwire.dtl.DTLVardef@e5a76borg.highwire.dtl.DTLVardef@152a1dcorg.highwire.dtl.DTLVardef@981f_HPS_FORMAT_FIGEXP M_FIG Graphical AbstractSchematic of hESCs-derived liver zonation organoids C_FIG

bioengineering↗

bioRxiv2024

High Refractive Index Imaging Buffer for Dual Color 3D SMLM Imaging of Thick Samples

The current limitations of single-molecule localization microscopy (SMLM) in deep tissue imaging, primarily due to depth-dependent aberrations caused by refractive index (RI) mismatch, present a significant challenge in achieving high-resolution images at greater depths. To extend the imaging depth, we optimized the imaging buffer of SMLM with RI matched to the objective immersion medium, and systematically evaluated five different RI matched buffers, focusing on their impact on the blinking behavior of red-absorbing dyes and the quality of reconstructed super-resolution images. Particularly, we found clear unobstructed brain imaging cocktails (CUBIC) based imaging buffer could match the RI of oil and was able to clear the tissue samples. With the help of RI matched imaging buffer, we showed high quality dual color 3D SMLM images with imaging depth ranging from few microns to tens of microns in both cultured cells and sectioned tissue samples. This advancement offers a practical and accessible method for high-resolution imaging at greater depths without specialized optical equipment or expertise.

biophysics↗

bioRxiv2024

Toll-Like-Receptor 5 protects against pulmonary fibrosis by reducing lung dysbiosis

Idiopathic pulmonary fibrosis (IPF) is a devastating pulmonary disease with no curative treatment other than lung transplantation. IPF results from maladaptive responses to lung epithelial injury, but the underlying mechanisms remain unclear. Here, we show that deficiency in the innate immune receptor, toll-like receptor 5 (TLR5), is associated with IPF in humans and with increased susceptibility to epithelial injury and experimental fibrosis in mice, while activation of lung epithelial TLR5 through a synthetic flagellin analogue protects from experimental fibrosis. Mechanistically, epithelial TLR5 activation induces antimicrobial gene expression and ameliorates dysbiosis after lung injury. In contrast, TLR5 deficiency in mice and IPF patients is associated with lung dysbiosis. Elimination of the microbiome in mice through antibiotics abolishes the protective effect of TLR5 and reconstitution of the microbiome rescues the observed phenotype. In aggregate, TLR5 deficiency is associated with IPF and dysbiosis in humans and in the murine model of pulmonary fibrosis. Furthermore, TLR5 protects against pulmonary fibrosis in mice and this protection is mediated by effects on the microbiome. One-sentence summaryDeficiency in the innate immune receptor TLR5 is a risk factor for pulmonary fibrosis, because TLR5 prevents microbial dysbiosis after lung injury.

immunology↗

bioRxiv2024

Targeting circadian transcriptional programs in triple negative breast cancer through a cis-regulatory mechanism

Circadian clock genes are emerging targets in many types of cancer, but their mechanistic contributions to tumor progression are still largely unknown. This makes it challenging to stratify patient populations and develop corresponding treatments. In this work, we show that in breast cancer, the disrupted expression of circadian genes has the potential to serve as biomarkers. We also show that the master circadian transcription factors (TFs) BMAL1 and CLOCK are required for the proliferation of metastatic mesenchymal stem-like (mMSL) triple-negative breast cancer (TNBC) cells. Using currently available small molecule modulators, we found that a stabilizer of cryptochrome 2 (CRY2), the direct repressor of BMAL1 and CLOCK transcriptional activity, synergizes with inhibitors of proteasome, which is required for BMAL1 and CLOCK function, to repress a transcriptional program comprising circadian cycling genes in mMSL TNBC cells. Omics analyses on drug-treated cells implied that this repression of transcription is mediated by the transcription factor binding sites (TFBSs) features in the cis-regulatory elements (CRE) of clock-controlled genes. Through a massive parallel reporter assay, we defined a set of CRE features that are potentially repressed by the specific drug combination. The identification of cis-element enrichment might serve as a new concept of defining and targeting tumor types through the modulation of cis-regulatory programs, and ultimately provide a new paradigm of therapy design for cancer types with unclear drivers like TNBC.

cancer biology↗

bioRxiv2024

Fish CDK2 recruits Dtx4 to degrade TBK1 through ubiquitination in the antiviral response

Although the classical biological protein cell cycle protein kinase CDK2 has been extensively studied in higher vertebrates, its function in lower vertebrates beyond the regulation of mitosis remains unknown. In this study, we report a distinct mechanism whereby IFN expression is negatively regulated in fish by CDK2. After infection with the spring viremia of carp virus (SVCV), fish CDK2 expression significantly increased in tissues and cells. Moreover, antiviral resistance was improved in cdk2-/- homozygotes, and the antiviral cytokine interferon (IFN) expression was significantly higher. At the cellular level, CDK2 overexpression reduced IFN expression, while cdk2 knockdown increased the ability of cells to produce IFN. Subsequently, it was discovered that fish CDK2 binds and degrades TBK1, resulting in reduced IFN. CDK2 increases the K48-linked ubiquitination of TBK1, causing its degradation, while E3 ubiquitin ligase Dtx4 was found to be involved in this process following the significant enhancement of TBK1 K48-linked ubiquitination. Protein mass spectrometry and immunoblot analysis confirmed that the K567 site on TBK1 is essential for CDK2 to engage with Dtx4 and degrade TBK1; thus, after mutating the K567 site, K48-linked ubiquitination of TBK1 was not enhanced by Dtx4, and TBK1 was not degraded by CDK2. Our data demonstrate that fish CDK2 recruits the E3 ubiquitin ligase Dtx4 to target the K567 site of TBK1 and promote its degradation. These results suggest that CDK2 in lower vertebrates is implicated in a specialized role for antiviral innate immunity.

immunology↗

bioRxiv2024

Organ specificity and commonality of epigenetic aging in low- and high-running capacity rats

BackgroundEpigenetic drift, which are gradual age-related changes in DNA methylation patterns, plays a significant role in aging and age-related diseases. However, the relationship between exercise, epigenetics, and aging and the molecular mechanisms underlying their interactions are poorly understood. The aim of this study was to investigate the relationship between cardiorespiratory fitness (CRF), epigenetic aging, and promoter methylation of individual genes across multiple organs in selectively bred low- and high-capacity runner (LCR and HCR) aged rats. MethodsIn this study, we performed reduced representation bisulfite sequencing (RRBS) on LCR and HCR aged rats, focusing on the hippocampus, heart, soleus muscle, and large intestine samples. We calculated various epigenetic indicators, including rat epigenetic clocks, global mean DNA methylation (GMM), mean methylation entropy (MME), and gene-specific promoter methylation. ResultsEpigenetic clocks, trained on available rat blood-derived RRBS data did not reflect differences in CRF between LCR and HCR rats across all four organs. However, we observed organ specific differences in GMM and MME between LCR and HCR rats. The direction of these differences was the opposite compared to the age-related changes in the rat blood suggesting that a high CRF may mitigate age-related epigenetic changes in an organ-specific manner. Notably the soleus muscle, exhibited the most pronounced differences in promoter methylation due to CRF. We also identified seven genes whose promoter methylation was consistently influenced by CRF in all four organs. Moreover, we found that age acceleration of the soleus muscle was significantly higher compared to the heart and the hippocampus, and significantly lower compared to the large intestine. Finally, we found that the age acceleration was not consistent across organs. ConclusionsOur data suggest that CRF associates with epigenetic aging in an organ-specific manner and regulates the promoter methylation of individual genes in an organ-specific and organ-common manner. Our findings provide important insights into the biology of aging and emphasize the need to validate rejuvenation strategies in the context of the organ-specific nature of epigenetic aging.

physiology↗

bioRxiv2024

Tumor suppressor p73 transcriptionally regulates c-FLIP to impede its priming of extrinsic apoptosis while a switcher compound degrades c-FLIP protein

The tumor suppressor p73 is a member of the p53 family and transcriptionally activates multiple p53-targets involved in cell cycle regulation and apoptosis. In addition to pro- apoptotic signaling, outcomes of p73 activation include cell survival signals. Thus, p73 activity and targets may provide insight in cell fate outcomes between cell survival and apoptosis following cellular stress. We report that cellular FLICE inhibitory protein (c- FLIP), a master antiapoptotic factor, is a transcriptional target of p73. The activation of p73 ( and {beta} isoforms) transcriptionally upregulates c-FLIP-L/S expression in cancer cells. The cell fate decision following p73 activation is determined by the adjustment of the balance of outcomes of p73 activation between p73-induced pro-apoptotic signaling and c-FLIP-L/S expression in cancer cells. p73 primes extrinsic apoptosis via an autocrine death ligand-DR5 axis, and the priming appears to be titrated at the level of c-FLIP-L/S. The p73-upregulation of c-FLIP-L/S increases the threshold of extrinsic apoptosis. Cells with poor priming levels convert to cell cycle arrest and survival. Depletion of c-FLIP-L/S increases the p73-priming levels towards extrinsic apoptosis and sensitizes cancer cells to p73-primed extrinsic apoptosis. We further identified a small-molecule CB-7587351 ("switcher compound") that alters p73 activation outcomes through c-FLIP-L/S protein degradation. Therapeutic activation of p73 can restore p53- signaling in mutant p53-expressing cancer cells effectively bypassing the p53 deficiency in cancer cells. Our discovery of p73 transcriptional upregulation of c-FLIP provides a promising strategy for depleting c-FLIP to improve antitumor efficacy of p73-targeting cancer therapy for p53-mutant tumors.

cancer biology↗

bioRxiv2024

Knowledge Graph-based Thought: a knowledge graph enhanced LLMs framework for pan-cancer question answering

BackgroundIn recent years, Large Language Models (LLMs) have shown promise in various domains, notably in biomedical sciences. However, their real-world application is often limited by issues like erroneous outputs and hallucinatory responses. ResultsWe developed the Knowledge Graph-based Thought (KGT) framework, an innovative solution that integrates LLMs with Knowledge Graphs (KGs) to improve their initial responses by utilizing verifiable information from KGs, thus significantly reducing factual errors in reasoning. The KGT framework demonstrates strong adaptability and performs well across various open-source LLMs. Notably, KGT can facilitate the discovery of new uses for existing drugs through potential drug-cancer associations, and can assist in predicting resistance by analyzing relevant biomarkers and genetic mechanisms. To evaluate the Knowledge Graph Question Answering (KGQA) task within biomedicine, we utilize a pan-cancer knowledge graph to develop a pan-cancer question answering benchmark, named the Pan-cancer Question Answering (PcQA). ConclusionsThe KGT framework substantially improves the accuracy and utility of LLMs in the biomedical field. This study serves as a proof-of-concept, demonstrating its exceptional performance in biomedical question answering. Key PointsO_LIWe introduce a framework combining LLMs with KGs to improve factual accuracy in LLM reasoning. C_LIO_LIOur system is a flexible architecture that seamlessly integrates various LLMs. C_LIO_LIUtilizing a pan-cancer knowledge graph, we have proposed the first KGQA benchmark in the field of biomedicine. C_LIO_LICase studies reveal our method enhanced LLMs in addressing biomedical challenges such as drug repositioning, resistance research, individualized treatment, and biomarker analysis. C_LIO_LIThe method performs favorably in comparison to existing methods. C_LI

bioinformatics↗

bioRxiv2024

Translational control of microglial inflammatory and neurodegenerative responses

In Alzheimers Disease (AD), activation of the mechanistic target of rapamycin (mTOR) pathway is essential for microglia neuroprotective roles, but it is unclear which mTOR effectors promote these neuroprotective functions. The mTOR complex 1 (mTORC1) inactivates the translation suppressors eukaryotic translation Initiation Factor 4E (eIF4E)-Binding Proteins (4E-BP) to promote mRNA translation. We show that 4E-BP1 inactivation is impaired in microglia under AD-relevant conditions. Depleting 4E-BPs in microglia increases mitochondrial metabolism, suppresses the pro-inflammatory profile, and mitigates amyloid-induced apoptosis. Furthermore, in the cerebrospinal fluid of patients with amyloid pathology, there was a positive association between microglia activation and neurodegeneration, which increases along 4E-BP1 levels. Thus, we propose the engagement mTORC1-4E-BP1 axis as a neuroprotective mechanism and a therapeutic target or biomarker for microglia modulation in AD.

neuroscience↗

bioRxiv2024

An Innovative Mitochondrial-targeted Gene Therapy for Cancer Treatment

Targeting cancer cell mitochondria holds great therapeutic promise, yet current strategies to specifically and effectively destroy cancer mitochondria in vivo are limited. Here, we introduce mLumiOpto, an innovative mitochondrial-targeted luminoptogenetics gene therapy designed to directly disrupt the inner mitochondrial membrane (IMM) potential and induce cancer cell death. We synthesize a blue light-gated channelrhodopsin (CoChR) in the IMM and co-express a blue bioluminescence-emitting Nanoluciferase (NLuc) in the cytosol of the same cells. The mLumiOpto genes are selectively delivered to cancer cells in vivo by using adeno-associated virus (AAV) carrying a cancer-specific promoter or cancer-targeted monoclonal antibody-tagged exosome-associated AAV. Induction with NLuc luciferin elicits robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering cancer cell IMM permeability disruption, mitochondrial damage, and subsequent cell death. Importantly, mLumiOpto demonstrates remarkable efficacy in reducing tumor burden and killing tumor cells in glioblastoma or triple-negative breast cancer xenografted mouse models. These findings establish mLumiOpto as a novel and promising therapeutic strategy by targeting cancer cell mitochondria in vivo.

cancer biology↗

bioRxiv2024

CDK12 Loss Promotes Prostate Cancer Development While Exposing Vulnerabilities to Paralog-Based Synthetic Lethality

Biallelic loss of cyclin-dependent kinase 12 (CDK12) defines a unique molecular subtype of metastatic castration-resistant prostate cancer (mCRPC). It remains unclear, however, whether CDK12 loss per se is sufficient to drive prostate cancer development--either alone, or in the context of other genetic alterations--and whether CDK12-mutant tumors exhibit sensitivity to specific pharmacotherapies. Here, we demonstrate that tissue-specific Cdk12 ablation is sufficient to induce preneoplastic lesions and robust T cell infiltration in the mouse prostate. Allograft-based CRISPR screening demonstrated that Cdk12 loss is positively associated with Trp53 inactivation but negatively associated with Pten inactivation--akin to what is observed in human mCRPC. Consistent with this, ablation of Cdk12 in prostate organoids with concurrent Trp53 loss promotes their proliferation and ability to form tumors in mice, while Cdk12 knockout in the Pten-null prostate cancer mouse model abrogates tumor growth. Bigenic Cdk12 and Trp53 loss allografts represent a new syngeneic model for the study of androgen receptor (AR)-positive, luminal prostate cancer. Notably, Cdk12/Trp53 loss prostate tumors are sensitive to immune checkpoint blockade. Cdk12-null organoids (either with or without Trp53 co-ablation) and patient-derived xenografts from tumors with CDK12 inactivation are highly sensitive to inhibition or degradation of its paralog kinase, CDK13. Together, these data identify CDK12 as a bona fide tumor suppressor gene with impact on tumor progression and lends support to paralog-based synthetic lethality as a promising strategy for treating CDK12-mutant mCRPC.

cancer biology↗

bioRxiv2024

High-fidelity enhanced AsCas12a knock-in mice for efficient multiplexed gene editing, disease modeling and orthogonal immunogenetics

The advancement of CRISPR gene editing technology, especially the development of Cas9 knock-in mice, has significantly boosted the functional discovery of various genetics factors in diverse fields including genetics, genomics, immunology, and the biology of cancer. However, the pleiotropic effects on human disease and the complex nature of gene interaction networks require a knock-in mouse model capable of simultaneous multiplexed gene perturbation. Here, we present the generation and applications of Cre-dependent conditional and constitutive high-fidelity, enhanced AsCas12a (enAsCas12a-HF1) Rosa26-knock-in mice in the C57BL/6 background. With these mouse strains, we demonstrate highly efficient and multiplexed in vivo and ex vivo genome engineering as applied to lipid nanoparticle (LNP)-RNA-based liver protein targeting, AAV-based tumor modeling, and retrovirus-based immune cell engineering. By integrating with a dCas9-SPH CRISPR activation transgenic strain, we establish a simultaneous dual gene activation and knockout (DAKO) system that showcases the modular potential of these enAsCas12a-HF1 mice. Importantly, constitutive expression of enAsCas12a-HF1 does not lead to any discernable pathological differences as compared to the C57BL/6 background strain. These knock-in mice and the accompanying delivery methods would empower the deconvolution of complex gene interaction networks in broad areas of research.

bioengineering↗