Biology subjects

Xie, Q.

Publications and source records attributed to Xie, Q..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome-wide analysis of the annexin gene family in alfalfa (Medicago sativa L.) and functional characterization of MsANN22 under drought stress

Annexins are a conserved family of calcium-dependent phospholipid-binding proteins that participate in diverse plant stress responses. However, the annexin gene family in Medicago sativa and the molecular mechanisms underlying annexin-mediated drought tolerance remain largely unexplored. In this study, 73 MsANN genes were identified in the alfalfa genome. Their phylogenetic relationships, synteny, conserved motifs, gene structures, and promoter cis-acting elements were systematically analyzed. Synteny analysis revealed 75 segmental/whole-genome duplication-associated gene pairs but no tandem duplications, indicating that large-scale duplication was the major driver of MsANN family expansion. The MsANN promoters contained ABA- and drought-responsive elements. Expression profiling identified MsANN22 as a strongly drought-responsive gene encoding a cytoplasm-localized protein. MsANN22 overexpression enhanced drought tolerance in both Arabidopsis thaliana and alfalfa: in A. thaliana, MsANN22 overexpression promoted seed germination under osmotic stress, whereas in alfalfa it enhanced antioxidant capacity, with superoxide dismutase and peroxidase activities reaching 1.38- and 1.70-fold those of wild-type plants, respectively. Consistently, MsANN22-overexpressing alfalfa accumulated approximately 20% less malondialdehyde than wild-type plants and exhibited reduced H2O2 and O2*- accumulation, as indicated by DAB and NBT staining. Yeast two-hybrid and luciferase complementation assays further supported physical interactions of MsANN22 with MsIAA and MsPR-10. These findings provide insight into the molecular mechanism by which MsANN22 mediates drought tolerance and offer a candidate gene for improving drought tolerance in alfalfa.

plant biology↗

bioRxiv2026

Temporal TOC1-CLV3-WUS cascade controls organogenesis and rhythmic stem development

Coordinated organogenesis and rhythmic stem development determine shoot morphogenesis, which governs distinct plant architecture and crop yield. The shoot apical meristem (SAM) drives organ initiation and stem growth, with its activity sustained by the spatially defined CLAVATA3 (CLV3)-WUSCHEL (WUS) feedback loop. However, how the temporal CLV3-WUS module modulates SAM activity remains elusive. Here, we demonstrate that both CLV3 and WUS are required for circadian stem elongation. The clock component TIMING OF CAB EXPRESSION1 (TOC1) directly activates and sustains the circadian expression of CLV3, and this regulatory process is controlled by the APETALA1-CIRCADIAN CLOCK-ASSOCIATED1 module. Notably, the rhythmic expression pattern of CLV3, rather than its transcript abundance, plays a more important role in regulating WUS expression and sustaining SAM activity, and CLV3 confers circadian rhythmicity to WUS protein accumulation. Transcriptome and ChIP seq analyses reveal that WUS modulates multiple developmental programs, including meristem homeostasis, phytohormone responses, and cell wall remodeling. Mechanistically, WUS directly activates PECTIN METHYLESTERASE5 expression to promote stem elongation. Collectively, our findings uncover a spatiotemporal regulatory cascade that fine-tunes SAM activity to coordinate rhythmic organogenesis and stem development, thereby establishing optimal phyllotactic patterning in plants.

plant biology↗

bioRxiv2026

Joint modeling of multi-timepoint spatial observations for time-resolved spatial-unit-specific gene regulatory network inference

BackgroundHow gene regulatory programs reorganize across space and time is central to development and disease, but current methods infer regulatory structure from single snapshots. The emergence of spatiotemporal transcriptomics calls for methods that resolve regulation along both axes. ResultsWe introduce SpaTemGRN, which jointly models observations from multi-timepoint slides in spatiotemporal transcriptomics to infer time-resolved spatial-unit-specific putative GRNs. Compared with existing methods, SpaTemGRN allows later-stage units to borrow statistical strength from spatially proximate and temporally preceding units. In the AppNL-G-F mouse model of Alzheimers disease, SpaTemGRN revealed region- and age-dependent strengthening of complement-glia regulatory coupling. An early, broadly distributed complement signature precedes later, spatially focal coupling with astrocytic and microglial responses. In the developing mouse embryonic brain, SpaTemGRN identified progressively sharpening and spatially segregated regulatory programs as the early neural tube regionalizes. Across simulated datasets, SpaTemGRN recovered regulatory edges more robustly than four existing methods and maintained the most stable performance across stages. ConclusionsSpaTemGRN provides a flexible hypothesis-generating framework for investigating how spatially localized gene-gene dependencies are remodeled across biological stages. All source code for SpaTemGRN is available at https://github.com/yibingjiang/SpaTemGRN.

bioinformatics↗

bioRxiv2026

LIFU-Responsive Nanocomplexes Deliver PDGF-BB mRNA for Plaques Stabilization via Neovascularization Modulation

BackgroundPathological intraplaque neovascularization, vascular leakage, and fibrous cap thinning contribute to vulnerable atherosclerotic plaque rupture. Platelet- derived growth factor-BB (PDGF-BB) has been shown to promote pericyte recruitment, thereby stabilizing the microvascular structure, and to induce phenotypic modulation of vascular smooth muscle cells (VSMCs), which enhances fibrous cap thickness and reinforces plaque stability. Nevertheless, systemic protein delivery is limited by rapid clearance and potential off-target effects. MethodsWe developed PDGF-BB mRNA-loaded lipid nanoparticle-poly(lactic-co- glycolic acid) nanobubble complexes (LNPmRNA@PLGA) and used low-intensity focused ultrasound (LIFU) to enhance plaque-targeted delivery. Cellular uptake, PDGF- BB expression, vascular mural-cell responses, plaque histology, hemodynamics, and proteomic changes were evaluated in vitro and in ApoE-/-Fbn1C1041G+/- mice. ResultsLIFU enhanced nanocomplex uptake and PDGF-BB expression, promoted vascular smooth muscle cell proliferation, migration, and phenotypic switching, and increased pericyte coverage. In vivo, LIFU plus LNPmRNA@PLGA reduced the plaque vulnerability index by 78.2% and the neovascularization area by 67.3% compared with controls, while increasing collagen deposition and improving carotid hemodynamics. ConclusionsLIFU-responsive delivery of PDGF-BB mRNA stabilized vulnerable plaques by promoting neovessel maturation and strengthening the fibrous cap. This strategy provides a spatially controlled framework for therapeutic remodeling of high-risk atherosclerotic plaques. Research Perspective What New Question Does This Study Raise?O_LICan spatially controlled PDGF-BB mRNA delivery simultaneously mature intraplaque neovessels and reinforce the fibrous cap without the systemic effects associated with recombinant PDGF-BB? C_LI What Question Should Be Addressed Next?O_LIFuture studies should define the therapeutic window, durability, and long-term safety of LIFU-triggered PDGF-BB mRNA delivery in large-animal models that more closely reproduce human plaque rupture. C_LI

pharmacology and toxicology↗

bioRxiv2026

Synthesis of Ultra-Large Fibrous Proteins from Bacteria via a Looped-Translation System

High molecular weight fibrous proteins such as silk, elastin, and collagens, are fundamental for providing shape to macroscopic biological structures, yet their recombinant production remains challenging because of their extreme size and sequence repetitiveness. Here, we report a circular RNA-based ribosome translation platform that enables iterative ribosome synthesis of fibrous proteins through continuously "looped" translation. To promote efficient circularization of repetitive fibrous protein transcripts, we combined a synonymous codon locker sequence strategy with RNA circularization chaperones. Guided by a ribosome traffic model, we further optimized the translation bottlenecks within the circular RNA, substantially improving translation yields. The established looped translation platform is applicable to at least six classes of fibrous proteins and generated products with molecular weight exceeding titin at 3.8 MDa. The synthesized polypeptides were characterized through electron microscopy, bulk material fabrication, and mechanical analysis, demonstrating properties associated with ultra-high molecular weight polypeptides. Finally, we coupled looped translation to secretion through a programmed ribosomal frameshift, enabling export of fibrous protein across cellular membranes in both Escherichia coli and Bacillus subtilis. We envision that the genetic tools presented here could find a range of applications in bioplastics and engineered living materials.

synthetic biology↗

bioRxiv2026

Critical reassessment of lipophilic dye labeling reveals negligible incorporation into small extracellular vesicles derived form serum-free cultured cells

Lipophilic dyes are widely used to track extracellular vesicles (EVs), yet their labeling efficiency toward bona fide small EVs (sEVs) remains poorly defined. Using a serum-free HEK293F system that produces endogenously fluorescent protein-tagged sEVs (sEVs-FPT) as an unambiguous reference while minimizing interference from dye-binding non-vesicular particles, we reassessed this efficiency with two orthogonal methods: nanoflow cytometry and fluorescence microscopy. PKH26, PKH67, and DiD labeled less than 0.5% of sEVs-FPT, regardless of vesicle heterogeneity. In vivo, dye-derived signals were far less abundant than tag-derived signals and failed to colocalize with them. Mechanistic evidence indicates that this failure stems from the inability of sEVs, as acellular structures, to actively internalize dye aggregates. These findings reveal a fundamental limitation of lipophilic dye-based EV tracking and underscore the need for orthogonally validated labeling strategies. One Sentence SummaryLipophilic dyes fail to label bona fide small extracellular vesicles, calling dye-based vesicle tracking into question.

bioengineering↗

bioRxiv2026

Natural compounds magnolol and dicoumarol enhance adipogenesis for future food applications

Edible adipose tissue can enhance the sensory and nutritional qualities of cultivated and plant-based meats, yet efficient adipogenic differentiation remains a major bottleneck and synthetic PPAR{gamma} agonists are not approved for use in food production. Here, we report a natural compound screen in 3T3-L1 adipocytes that identifies magnolol and dicoumarol as enhancers of adipogenesis; this combination also robustly promotes lipid accumulation in primary porcine dedifferentiated fat cells and ovine preadipocytes. Transcriptomic analyses show that magnolol and dicoumarol induce adipogenesis in murine and porcine cell systems through canonical adipogenic pathways with a narrower transcriptional footprint than the potent PPAR{gamma} agonist rosiglitazone. These findings support the potential of naturally occurring compounds magnolol and dicoumarol as enhancers of adipogenesis for both mechanistic studies and food-relevant applications. More broadly, our findings establish a generalizable screening framework and identify small-molecule combinations that accelerate adipose tissue engineering across murine, porcine, and ovine culture systems.

bioengineering↗

bioRxiv2026

Ciliary integrity defines a central canal repair checkpoint linking microtubule stabilization to spinal cord regeneration

Microtubule-stabilizing agents consistently improve functional recovery after spinal cord injury (SCI), yet the structural mechanism underlying their shared therapeutic effects remains unclear. Here, we find that chemically distinct stabilizers converge on preservation of ciliary integrity within central canal-associated cells, including ependymal cells and cerebrospinal fluid-contacting neurons. Using complementary SCI models, including complete transection and crush injury, we observe that maintenance of ciliary architecture is associated with reduced glial scarring, improved tissue continuity, and enhanced locomotor recovery. Single-cell transcriptomic analysis further identifies these cell populations as prominent responders to microtubule stabilization, with ciliogenesis-related programs selectively preserved. Importantly, pharmacological disruption of cilia-associated signaling attenuates recovery, whereas promoting ciliogenesis partially recapitulates therapeutic effects, identifying ciliary integrity as a critical cilia-associated structural dependency that contributes to microtubule-stabilizer-mediated spinal cord repair. Together, these findings identify a cilia-dependent central canal regenerative niche as a candidate structural checkpoint linking microtubule stabilization to functional recovery after SCI and identify ciliogenesis as a therapeutically actionable target for SCI. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/733052v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@320578org.highwire.dtl.DTLVardef@5240dorg.highwire.dtl.DTLVardef@181f251org.highwire.dtl.DTLVardef@4bacd1_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2026

Spatiotemporal Cascade of AP1/CCA1-TOC1 Module Gates Stem Development

Stem development is crucial in plant vertical architecture and overall crop improvement. The molecular mechanisms underlying the initiation and circadian growth of stem remain still enigmatic. Here, we demonstrate that APETALA1 (AP1) spatially initiates circadian stem growth through a noncell-autonomous role of TIMING OF CAB EXPRESSION1 (TOC1) emanating from the floral meristem to inflorescence meristem in Arabidopsis thaliana. Mechanistically, AP1 interacts with and recruits CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) to associate TOC1. Dynamic formation of the AP1-CCA1 complex is crucial for maintaining the circadian rhythmicity and expression of TOC1. Tissue-specific expression of TOC1 in floral meristems rescues the developmental defects of toc1 mutants at both functional and transcriptional levels. Furthermore, TOC1 directly activates the circadian expression of pectin methylesterase gene family, which is critical for its role in stem development. AP1 homologs regulate circadian stem elongation and plant height in wheat and rice, underscoring the conserved mechanism across flowering plants. Our findings uncovered a spatiotemporal regulatory cascade gating stem development and identified candidate genes for crop improvement.

plant biology↗

bioRxiv2026

Senescent cells induce vascular MHC II to recruit CD4+ T cells and drive inflammation in aging adipose tissue

Adipose tissue exhibits pronounced inflammation during aging, yet the mechanisms sustaining this chronic state are not well understood. By creating an atlas integrating histology, single-nucleus transcriptomics and flow cytometry across the murine lifespan, we find that age-associated inflammation is distinct from the obesity-like inflammatory profile observed at mid-life. Specifically, age-associated inflammation is characterized by a potent interferon-gamma (IFN{gamma}) response signature and the accumulation of T cells. We demonstrate that senescent cells act as an upstream trigger, indirectly initiating an IFN{gamma} response that upregulates vascular MHC II to promote extravasation of CD4+ T cells into the aging tissue. These recruited T cells then act as a critical source of IFN{gamma}, thereby perpetuating a positive feedback loop that maintains chronic immune infiltration and tissue inflammation. These results provide a multi-modal view of adipose aging and identify a mechanism that sustains its age-associated inflammation.

immunology↗

bioRxiv2026

Aerosol delivery-based serial femtosecond crystallography

Serial femtosecond crystallography (SFX) has revolutionised structural biology by enabling direct visualization of conformational dynamics of biomacromolecules at near-physiological temperatures. However, conventional SFX sample delivery at X-ray free-electron laser (XFEL) facilities introduces a significant X-ray scattering background, which may limit the data quality and resolution. Here, we introduce an aerosol delivery-based method that drastically reduces the back-ground scattering by minimising the liquid environment surrounding the nanocrystals. We validate our method by solving the structure of Cydia pomonella granulovirus nanocrystals at 1.9 [A] resolution, achieving orders-of-magnitude lower background scattering compared to a liquid jet-based method. Structural comparison with the liquid jet-based model revealed similar overall structure, suggesting that the native structure is largely preserved despite dehydration during aerosolisation. Our method enables efficient SFX studies, particularly pump-probe time-resolved SFX on protein nanocrystals with enhanced signal-to-noise ratios, as well as high-throughput small molecule SFX (smSFX) applications for pharmaceuticals and functional materials.

biophysics↗

bioRxiv2026

Unravelling genome-wide mosaic microsatellite mutations at single-cell resolution

Short tandem repeats (STRs), or microsatellites, are highly mutable genomic elements that modulate gene regulations and are implicated in a range of human diseases. However, detecting mosaic STR mutations at single-cell resolution remains challenging due to both technical and biological complexities. To address this, we developed BayesMonSTR, a robust algorithm that enables accurate detection of mosaic STR mutations. Using this tool in single-cell analysis of human tissues, we reveal an accumulation of longer mosaic STR insertions and deletions (indels) in aging mitotic and post-mitotic cells. Strikingly, prefrontal cortex (PFC) neurons accumulate a higher burden of STR mutations than B cells or lung epithelium, with aged neurons exhibiting a particularly pronounced increase in longer STR deletions. These mutations are enriched at transcription start sites (TSSs) and active enhancers of highly expressed genes. Our work establishes a foundation for genome-wide, hypothesis-free discovery of disease-associated mosaic STR mutations and reveals a previously unexplored landscape of mosaic STR variation in development and aging.

bioinformatics↗

bioRxiv2026

Curcumin Promotes Myelin Repair after Spinal Cord Injury via Spatially Selective Regulation of CLASP2 Phosphorylation

Spinal cord injury (SCI) remains a devastating neurological disorder, where limited axonal regeneration and inefficient remyelination severely restrict recovery. Although curcumin has recognized neuroprotective properties, its mechanism in myelin repair is unclear. Here, we identify a phosphorylation-dependent cytoskeletal pathway as a key target of curcumin. In a mouse spinal cord transection model, curcumin treatment improved hindlimb motor function, reduced fibrotic scarring, and increased myelin basic protein (MBP) in the injured region. Phosphoproteomic profiling revealed cytoskeletal regulation as a major process affected by curcumin, with CLASP2 emerging as a critical target. Curcumin enhanced CLASP2 phosphorylation at Ser1025, a modification that strengthened Golgi association and increased EB1 distribution at the cell periphery, thereby promoting microtubule anchoring without altering global stability. This spatially selective regulation provides a novel mechanism by which curcumin fine-tunes cytoskeletal organization to support remyelination. Analysis of published single-cell sequencing data further showed CLASP2 enrichment in myelin-forming cells, underscoring its relevance. Unlike prior studies emphasizing anti-inflammatory or antioxidant effects, our findings reveal a defined molecular mechanism linking curcumin to cytoskeletal remodeling and myelin repair, highlighting its potential as a safe and accessible therapeutic candidate for SCI.

cell biology↗

bioRxiv2026

Joint Segmental Duplication Co-option Drives Human-specific Transcriptional Readthrough and Expression Fine-tuning of NPEPPS-TBC1D3

Segmental duplication (SD) is a major driver of functional changes in evolution and disease. Many genes embedded within SDs, such as NPEPPS and TBC1D3, display substantial copy number variation (CNV) across individuals. Yet, the precise identification of the functional copies and their transcriptional outputs remains largely unstudied. Focusing on NPEPPS and TBC1D3, we illustrate human-specific expression fine-tuning mechanisms associated with readthrough transcripts. We identified a human-specific NPEPPS-TBC1D3 digenic genomic structure that originated from a joint SD pair and became fixed across populations. Experiments demonstrate that this structure generates NPEPPS-TBC1D3 readthrough transcripts, which are the predominant isoforms of TBC1D3 expression in various cell types, fine-tuning its protein level. Furthermore, a human-specific hypomethylation signal within an upstream CpG island of NPEPPS precisely pinpoints the expressed TBC1D3 paralog. Moreover, we reveal transcriptional readthrough events are [~]3-fold enriched for joint-SD-associated transcriptional readthrough (JSDTR) and identify 109 JSDTR gene pairs, including neurodevelopmentally important pairs and clinically interesting SERF1A/B-SMN1/2. Taken together, our findings comprehensively describe an example of how a joint SD event shaped evolution and suggest that JSDTR is a broad mechanism for the emergence of new functions.

genetics↗

bioRxiv2025

Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

Maintaining sustained deoxyribonucleotide triphosphate (dNTP) pools is essential for DNA replication fidelity and genome stability. In EGFR-mutant non-small cell lung carcinoma (NSCLC), we find that disruption of dNTP homeostasis plays a critical role in determining sensitivity to the EGFR inhibitor osimertinib and in shaping mechanisms of acquired resistance. Transcriptomic and biochemical analyses revealed that osimertinib suppresses RRM2 expression, a key regulator of dNTP synthesis, through the downregulation of the transcription factor MYBL2. In response to osimertinib-mediated replication stress and dNTP depletion, cells activated a compensatory pathway involving the stress-inducible ribonucleotide reductase subunit RRM2B via a novel transcriptional regulator, TNNT3. We further identified CHK2 signaling as essential for TNNT3 nuclear translocation and RRM2B transcriptional activation. Inhibition of CHK2 or combined CHK1/2 blockade impaired RRM2B induction, exacerbated replication stress, and delayed the development of osimertinib resistance both in vitro and in xenograft models. Collectively, our findings reveal that EGFR-mutant NSCLC cells rely on a dynamic EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis to maintain dNTP pool balance under therapeutic pressure. Disruption of this axis sensitizes tumors to osimertinib and impairs the acquisition of resistance, highlighting dNTP metabolism as a critical vulnerability and actionable target in EGFR-driven lung cancer. STATEMENT OF SIGNIFICANCETargeting dNTP metabolism via the EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis sensitizes EGFR-mutant NSCLC to osimertinib treatment and delays drug resistance, revealing a novel and actionable therapeutic vulnerability in EGFR-driven lung cancer.

cancer biology↗

bioRxiv2025

Ultrahigh-Throughput Forster Resonance Energy Transfer-Activated Droplet Sorting for Terminator Polymerase Engineering

Droplet microfluidic screening systems enable high-throughput, labor-saving enzyme directed evolution by employing fluorescence, absorbance, and Raman-activated sorting strategies for library screening. Forster resonance energy transfer (FRET) - a nanoscale technique for monitoring intramolecular/intermolecular conformational changes - is yet to be integrated into this process. We upgraded a single-channel sorter to a dual-channel one without redesigning the microscopy setup, which can monitor FRET signals during enzyme reactions in droplets at kilohertz rates. We applied this upgraded sorter to improve the incorporation efficiency of KOD DNA polymerase towards reversible terminator deoxyribonucleotides, a property crucial for its application in next-generation sequencing (NGS). Our data show that a single-round sorting can achieve 30-fold enrichment of active variants. Five KOD variants enabling 100-cycle single-end runs of DNA sequencing were identified using a novel cyclic reversible termination (CRT) substrate featuring a terminator group {approx} 5-fold bulkier than the classical azidomethyl moiety. We also engineered a metagenome-derived novel polymerase, and a variant achieving 90% terminator incorporation efficiency within 2 minutes was identified after two rounds of enrichment. In sum, we provide a practical setup for dual-channel FRET-based droplet sorting, and demonstrate its ability in terminator polymerases engineering, thereby broaden the scope of microfluidic applications.

biochemistry↗

bioRxiv2025

Lrrc55 modulates beta-cell resilience and calcium transporter expression under metabolic and physiologic stress

During pregnancy, pancreatic {beta}-cell mass and function are increased in adaptation to insulin resistance; this requires the action of prolactin receptor signaling. Recently, we discovered the Lrrc55, an auxiliary subunit of the voltage- and calcium-activated potassium channel (BK channel), is a prosurvival factor in {beta}-cell, and its expression and highly and specifically up regulated in the pancreatic islets during pregnancy. We found that overexpression of Lrrc55 protects {beta}-cells from glucolipotoxicity (GLT)-induced apoptosis. The protective effect of Lrrc55 is associated with dampening of the ER stress response and preservation of the releasable pool of calcium in the ER. Thus, we hypothesized that Lrrc55 protects {beta}-cells from GLT-induced ER stress and apoptosis by regulating ER calcium handling. Here, we report that Lrrc55 restored the GLT-mediated decrease in expression levels the insulin regulators, Pdx-1 and MafA, as well as the ER calcium regulator SERCA. Lrrc55 also attenuated the GLT-induced increase in expression of the ER calcium channel RyR2. Lrrc55 also attenuated GLT-mediated increase in protein expression of pro-apoptotic molecules CHOP and IRE1, and increased activation of the anti-apoptotic molecule Akt. However, Lrrc55 does not alter the activity levels of SERCA or IP3R under physiologic conditions. Transgenic mice with global deletion of Lrrc55 (Lrrc55-/-) are more susceptible to streptozotocin-induced diabetes. Surprisingly, Lrrc55-/- mice are more glucose tolerant and secreted more insulin during pregnancy. Together, these results suggest a role for Lrrc55 in maintaining expression of the ER calcium regulators in the presence of GLT but it may negatively regulate insulin secretion.

physiology↗

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↗