Biology subjects

Fu, L.

Publications and source records attributed to Fu, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Proteome-wide Mapping of Cysteine Persulfidation in Deep-Sea Hyperthermophilic Archaea

Protein persulfidation, a primary post-translational modification mediated by the gaseous signaling molecule hydrogen sulfide (H2S), regulates diverse physiological processes in eukaryotes and bacteria. However, its existence and functional roles in archaea, the third domain of life, remain completely unexplored. Here, we investigate this in the deep-sea hyperthermophilic archaeon Thermococcus aciditolerans SY113, which thrives in sulfur rich hydrothermal vents and endogenously produces substantial H2S. Our profiling not only delineated the unique reactivity landscape of these modifications but also enabled quantitative analysis of its dynamic regulation by H2S. A total of 204 persulfidation sites on 171 proteins were identified, over 65% of which were dynamically regulated by H2S. Further functional analysis suggests that persulfidation represents an ancient and conserved regulatory mechanism in primordial life including the regulation of catalytic activity, maintenance of protein conformation, and mediation of protein-protein interactions. Our findings provide a valuable dataset and theoretical foundation for understanding the role of persulfidation in the physiological regulation of deep-sea hyperthermophilic archaea.

microbiology↗

bioRxiv2025

Structural Insights into Single-Stranded DNA Recognition and Modified Substrate Tolerance in an Engineered Terminal Deoxynucleotidyl Transferase

Terminal deoxynucleotidyl transferase (TdT) is the cornerstone enzyme for de novo enzymatic DNA synthesis (EDS), promising to overcome the length and sustainability limitations of traditional phosphoramidite chemistry. We previously identified a highly active TdT ortholog from Zonotrichia albicollis (ZaTdT) and engineered a variant (R335L/K337G) capable of efficiently incorporating 3-aminooxy (3-ONH2) reversible terminators. However, the atomic-level mechanism by which these engineered mutations alter substrate specificity has remained elusive. Here, we present the high-resolution (2.20 [A]) crystal structure of the engineered ZaTdT in complex with a single-stranded DNA primer. Structural analysis reveals a conserved catalytic core anchored by a hydrophobic platform (Phe110/Phe257/Trp308) that stabilizes the primer. Crucially, comparative modeling with the homologous murine TdT ternary complex demonstrates that the engineered mutations (corresponding to L190/G192 in the crystal structure) disrupt a rigid salt-bridge network at the active site entrance. This electrostatic remodeling not only reduces local positive charge but also expands the catalytic pocket depth from 3.8 [A] to 5.8 [A]. This specific spatial expansion provides the structural rationale for the accommodation of bulky 3-blocking groups, validating our rational design strategy and paving the way for next-generation long-read DNA synthesis.

biochemistry↗

bioRxiv2025

Structural Mechanism of Specific Nucleobase Recognition by a Monoclonal Antibody in CoolMPSTM Sequencing

Massively parallel sequencing (MPS) has revolutionized genomics, yet traditional methods utilizing fluorescently labeled nucleotides suffer from "scarring" effects that limit read accuracy. The CoolMPS technology overcomes this by using unlabeled reversible terminators (RTs) with a 3-O-azidomethyl blocking group, detected by highly specific fluorescent antibodies. However, the atomic-level mechanism by which these antibodies discriminate between modified and natural nucleotides within a DNA strand remains unclear. Here, we report the crystal structures of the "A-fab" antibody fragment in complex with the monomeric antigen 3-O-azidomethyl-dATP (dATP-N3) at 2.50 [A] resolution, and with an one strand of the duplex carries an azide-modified deoxyadenosine nucleotide at its 3' terminus (dsDNA-dATP-N3) at 1.99 [A] resolution. Isothermal titration calorimetry (ITC) revealed that A-fab binds dATP-N3 with nanomolar affinity (KD{approx}17.14 nM), while binding to natural dATP is negligible (KD{approx}7.51{micro}M), representing a [~]440-fold specificity. Structural analysis reveals a conserved hydrophobic pocket formed by CDR residues (e.g., Trp79, Tyr53, Leu67) that specifically accommodates the 3-azidomethyl group. This interaction is critical for affinity and is maintained in the dsDNA-ATP-N3 complex, validating the antibodys function in a sequencing context. These findings provide the structural rationale for the high fidelity of the CoolMPS platform.

biochemistry↗

bioRxiv2025

Tibetan near-complete pangenome reveals complex variants underlying high-altitude adaptation

Genomic diversity in indigenous populations offers critical insights into human evolution and population-specific adaptation. Here, we generated 70 near-complete, fully phased genome assemblies from 35 high-altitude Tibetan trios and identified 4.55 million small variants and 63,031 structural variants (SVs) absent from the current global pangenome reference. Comparative analyses with a 595 globally diverse long-read genome dataset revealed 207 previously uncharacterized Tibetan-enriched SVs spanning 275 genes implicated in high-altitude physiological adaptation. Genotyping these SVs in 1,164 Tibetan and Han individuals with 88 physiological traits reveals five variants significantly associated with cardiopulmonary and hematological phenotypes central to hypoxia adaptation. We further map 2.45 Gb of archaic hominin sequences in Tibetans and uncover 18 previously unrecognized introgressed regions enriched in Tibetans with genes related to hematopoiesis and lung function. These regions harbor five Tibetan-enriched SVs, indicating that archaic introgression contributed SVs relevant to human adaptation. Together, these findings reveal substantial uncharacterized genomic diversity in Tibetans and highlight a previously underappreciated role of SVs and archaic SVs in shaping human evolution and high-altitude adaptation.

genomics↗

bioRxiv2025

Developmental regulation of intestinal best4+ cells

best4+/CFTR-high expressing cells are a recently described intestinal epithelial cell type potentially altered in inflammatory bowel disease and colorectal cancer. However, their developmental origin, developmental regulation, and functions remain undefined. This study identifies their conserved transcriptional program and uses zebrafish to dissect their developmental regulation in vivo. Lineage tracing identified that best4+ cells arise from atoh1b+ secretory progenitors. We identify that Notch signaling, mediated by dll4, specifies best4+ cells at the expense of enterochromaffin cells. Downstream of Notch, meis1b confers best4+ cell identity. best4+ cells then exhibit regionalized gene expression, regulated by pbx3a. Additionally, this study demonstrates a system where best4+ cells can be manipulated, observed, and removed in an organismal context. Live imaging and electron microscopy of best4+ cells identified dynamic cellular projections, suggesting a sensory or communicative function. Removal of best4+ cells in vivo eliminated previously proposed functions: they are not required to restore intestinal pH following acidic challenge and do not absorb nutrients. However, we identify region-specific intracellular pH differences that suggest potential functional heterogeneity. Altogether, this study presents a comprehensive description of best4+ cell development from birth to spatial regulation that will be instrumental to understand how best4+ cells change in disease or might be therapeutically manipulated and presents the tools to dissect their function in vivo.

developmental biology↗

bioRxiv2025

De Novo Hepatic Pyrimidine Synthesis Regulates Systemic Energy Homeostasis

Fasting blood uridine is increased in obesity and type 2 diabetes (T2D), but the significance of hepatic uridine biosynthesis to the etiology of both remains elusive. We found that de novo pyrimidine synthesis in the liver is reduced by fasting and diet-induced obesity, while suppression of hepatic pyrimidine synthesis promotes obesity and insulin resistance. The metabolic sequalae of hepatic pyrimidine synthesis suppression, however, is not associated with altered plasma uridine concentration. Instead, it is associated with an increased hepatic glucose production and a decreased hepatic insulin clearance, two key functions of hepatocytes in regulating systemic energy homeostasis. We found that enhanced gluconeogenesis is the primary reason for increased hepatic glucose production. Moreover, uridine, which was maintained stable in the circulation by adipose tissue and the liver, preferentially shut down pyrimidine synthesis in hepatocytes but not adipocytes at blood concentrations that occur with fasting. Remarkably, uridine, at fasting levels, increases gluconeogenesis further in hepatocytes when de novo pyrimidine synthesis is suppressed, indicating a synergistical action of uridine and its biosynthesis pathway in promoting hepatic glucose production, a mechanism highly relevant to the pathophysiology of insulin resistance in obesity. Theologically, maintenance of blood uridine within the narrow range protects mammals from high-rate spontaneous tumorigenesis. Since obesity promotes an increase in blood uridine from adipocytes, suppressing uridine synthesis in hepatocytes becomes a critical response to lower spontaneous tumorigenesis. Pyrimidine synthesis suppression in hepatocytes, however, promotes gluconeogenesis and ultimately triggers obesity and T2D. These findings suggest a new paradigm for the etiology of metabolic deterioration in diet-induced obesity, in which perturbations in uridine promotes obesity and T2D.

pathology↗

bioRxiv2025

Autonomous liquid-handling robotics scripting through large language models enables accessible and safe protein engineering workflows

Laboratory automation accelerates synthetic biology, but robot programming remains a barrier to broader participation. Large language model (LLM)-based automation must address four interconnected requirements in this field: sequence-level biosecurity, community standards, context-dependent biological behavior, and distributed collaboration across laboratories. Here we present LabscriptAI, an execution-aware agent framework that couples authoring and runtime control loops to translate natural-language instructions into platform-specific robot scripts and support bounded recovery under deterministic authorization and human oversight. On a 90-task liquid-handling benchmark, LabscriptAI achieved a 96.7% simulation-pass rate, the highest among the evaluated LLM-based and commercial baselines. We demonstrated its capabilities through sequence screening at designated workflow checkpoints; standardized cell-free characterization of 854 green fluorescent protein (GFP) designs from 171 student teams, coupled to data deposition; containment-aware engineering of a formaldehyde-converting enzyme, yielding 6.7-fold higher ethylene glycol titers in a two-stage cascade; and quality-assured preparation of 531 genetic parts for the 2025 iGEM Distribution Kit distributed worldwide. Together, these results establish LabscriptAI as a biosecurity-aware, community-integrated framework connecting protocol authoring, controlled execution, and traceable experimental outputs across laboratory platforms.

bioengineering↗

bioRxiv2025

An Objective Detection Method for Tinnitus using Memristors

Approximately 10-20% of the world population experiences tinnitus, which is increased with age and comorbid with frustration, annoyance, anxiety, depression, cognitive dysfunction, insomnia and stress. The underlying mechanisms of tinnitus remain largely unknown and there are no quantifiable biomarkers, making this prevalent disorder a global burden. There are no FDA-approved therapeutics and the current assessment mainly relies on self-reports (e.g., questionnaires), which underscore the need for diagnostic tools for tinnitus. Memristors, nanoscale electronic components mimicking synaptic plasticity, can monitor and control neuronal activity, hold transformative potential for detecting and characterizing tinnitus objectively. Here, we harvested the blood from human and mice with tinnitus, and engineered memristors via magnetron sputtering and made the functional layer with serum. Its core mechanism lies in regulating the reversible formation and rupture of conductive filaments which is formed by utilizing abnormally elevated glutamate concentrations in serum assisted Ag+ ions. This serum-based memristor achieves unprecedented discrimination between tinnitus and non-tinnitus individuals. Furthermore, this platform provides mechanistic insights into tinnitus pathogenesis, with potential implications in other neurological disorders.

pathology↗

bioRxiv2025

A complete and near-perfect rhesus macaque reference genome: lessons from subtelomeric repeats and sequencing bias

A truly complete, telomere-to-telomere (T2T), and error-free reference genome remains a foundational resource--and long-standing goal--for unbiased comparative and functional genomics. While recent T2T assemblies of humans and other primates have made substantial progress, most still contain thousands of base-level errors, particularly within highly repetitive regions. Here, we present T2T-MMU8v2.0, a near-perfect T2T assembly of the rhesus macaque (Macaca mulatta), representing the highest base-level accuracy reported in a primate genome to date. By employing an optimized ONT-only assembly strategy, we identify subtelomeric satellite-rich regions as the principal bottleneck to improving assembly quality, owing to technological biases in long-read platforms and limitations in current hybrid assembly frameworks. We discover 268 previously unannotated repeat families and resolve [~]8 Mbp of SATR satellite arrays, with over 99-fold enrichment in historically misassembled subtelomeric regions. These satellites form four distinct genomic architectures, each with unique SATR satellite composition, segmental duplication organization, and epigenetic signatures, distinct from the subtelomeric architectures observed in hominid genomes. Notably, in contrast to the largely gene-poor subtelomeric regions in African hominids, the SATR architectures in macaques harbor 58 actively transcribed genes, supported by open chromatin and expression data, suggesting gene innovation within these repetitive regions. Functionally, T2T-MMU8v2.0 improves read mappability and accuracy across sequencing platforms, and results in a 19% improvement of transcription start site enrichment scores and 5,821 additional chromatin accessibility peaks on average, thereby enhancing variant detection, regulatory annotation, and transcriptomic resolution in population genetics or single-nucleus studies. Together, this work establishes a new benchmark for genomics, offers a roadmap for resolving complex repetitive regions, and reveals previously unrecognized features of subtelomeric genome structure and evolution.

genomics↗

bioRxiv2025

A Paracrine-to-Autocrine Shunt of GREM1 Fuels Colorectal Cancer Metastasis via ACVR1C

Tumor cells typically rely on paracrine stromal signals to guide malignant behaviors, but whether they acquire signaling autonomy to support metastasis remains unclear. We elucidate this in colorectal cancer (CRC) by uncovering a paracrine-to-autocrine shunt of Gremlin1 (GREM1), a canonical stromal-secreted antagonist of bone morphogenetic proteins (BMPs). We demonstrate that while GREM1 remains restricted to stromal cells in earlier-stage (I-III) CRC, its ectopic expression in tumor epithelium increases markedly in stage IV. Mechanistically, we identify ACVR1C as a novel, high-affinity epithelial receptor for GREM1. Their interaction activates SMAD2/3 signaling, which upregulates SNAI1 and GREM1, establishing a feedback loop that amplifies epithelial-mesenchymal transition (EMT). Disrupting this loop impairs CRC metastasis in vivo. Clinically, epithelial GREM1 or ACVR1C expression predicts metastasis and poor survival. These findings define a paradigm in which tumor cells hijack stromal GREM1 to establish a GREM1-ACVR1C autocrine loop that sustains EMT and metastasis, marking a shift toward signaling autonomy and revealing a targetable vulnerability in advanced CRC.

cancer biology↗

bioRxiv2025

Separable Spatial Single-cell Transcriptome Representation Learning via Graph Transformer and Hyperspherical Prototype Clustering

Spatial transcriptomics enables the exploration of cell types, spatial domain organization, and cell-cell communication at tissue scale, serving as a powerful link between tissue morphology and molecular function. However, existing methods still struggle with limited spatial resolution, poor robustness in multi-slice alignment, and suboptimal ligand-receptor (L-R) detection in complex tissues. To address these challenges, we propose S3RL (Separable Spatial Single-cell transcriptome Representation Learning via Graph Transformer and Hyperspherical Prototype Clustering), a unified framework that integrates gene expression, spatial coordinates, and histological image features via a graph neural network and hyperspherical prototype-based separable representation learning. In spatial clustering tasks, S3RL improves the average Adjusted Rand Index (ARI) by nearly 120% on the Nanostring lung cancer dataset and over 26% on the 10X DLPFC dataset compared to state-of-the-art methods. For multi-slice spatial alignment, S3RL achieves an average ARI improvement of over 65.4% in partial brain slices and over 48.3% in complete slices relative to GraphST, highlighting its superior robustness and alignment consistency across heterogeneous spatial inputs. Moreover, S3RL reveals more biologically meaningful ligand-receptor signaling relationships, enhancing the interpretability of cell-cell communication patterns. Together, these results demonstrate S3RLs effectiveness in enhancing spatial resolution, cell type identification, and biological insight across diverse spatial transcriptomics datasets.

bioinformatics↗

bioRxiv2025

Tissue-wide profiling of human lungs reveals spatial sequestration of macrophages in tuberculosis

The immune response to human tuberculosis (TB), particularly in the context of complex lung pathology, remains incompletely understood. Here, we employed whole-slide spatial proteomics to map immune cell organization in TB-affected human lung tissues. Our analysis revealed pronounced spatial segregation of major immune cell populations in non-necrotizing TB lesions. At the tissue level, macrophages and lymphocytes formed distinct cellular communities associated with specific pathological features. At the lesion level, macrophages and B cells showed an inverse relationship in both abundance and spatial distribution. Proinflammatory T cells preferentially accumulated in macrophage-rich lesions but remained largely separated from macrophages. Interestingly, lesions exhibiting clear segregation between T cells and macrophages were more common in subclinical TB than in active disease. These findings suggest that spatial isolation of macrophages from effector lymphocytes may help temper inflammation and potentially prevent lesion progression to necrosis, while also enabling immune evasion by Mycobacterium tuberculosis. One Sentence SummaryXiao et al. reveal spatial segregation of immune cells in TB-lung tissue and link the microenvironmental dynamics to disease states of tuberculosis.

immunology↗

bioRxiv2025

Nanosecond pulsed electric field precisely manipulates cytoplasmic Ca2+ oscillations during oocyte activation

Oocyte activation deficiency (OAD) is a primary cause of fertilization failure following intracytoplasmic sperm injection (ICSI), a problem that can potentially be overcome through artificial oocyte activation (AOA). However, concerns persist regarding the safety and efficacy of AOA techniques in clinical practice. In this study, nanosecond pulsed electric field (nsPEF) is proposed as a safe and controllable method for oocyte activation in vitro by precisely manipulating Ca2+ signaling in the ooplasm. Mouse oocytes collected from oviducts were exposed to nsPEF stimulation at varying intensities to induce distinct Ca2+ signaling patterns. Subsequently, these oocytes underwent parthenogenetic activation and were cultured to assess developmental potential up to the blastocyst stage. The sperm-initiated physiological Ca2+ oscillations are successfully mimicked by one series of Ca2+ signals induced by nsPEF at low or medium intensities, and improve activation efficiency and developmental potential compared to calcium ionophore A23187. Low-intensity nsPEF pulses mediated repetitive extracellular Ca2+ influx in a non-invasive, electro-permeable manner. Medium-intensity nsPEF stimulation triggered periodic Ca2+ release from the endoplasmic reticulum (ER) via the PIP2-IP3-IP3R pathway, producing physiological-like Ca2+ oscillations. The non-invasive nsPEF method ensures safe oocyte activation by preserving cellular integrity and minimizing stress responses. The efficacy of nsPEF in precisely manipulating Ca2+ signaling patterns was further validated in human unfertilized oocytes. Our findings present a novel AOA approach with enhanced safety and efficacy, especially for patients experiencing repeated fertilization failures. It is anticipated that nsPEF is emerging as a promising technology for addressing male infertility issues, and may offer a potential alternative fertility treatment, bypassing sperm for initiation of oocyte activation.

developmental biology↗

bioRxiv2025

Governed by surface amino acid composition: HIV capsid passage through the NPC barrier

Nuclear transport receptors (NTRs) carry cargo across the permeability barrier of nuclear pore complexes (NPCs) - an FG phase condensed from disordered but cohesive FG repeats. This phase repels normal macromolecules but allows NTR passage. When HIV infects non-dividing cells, its capsid is transported into nuclei not like cargo but crosses NPCs like NTRs. We now uncovered the molecular determinants of the capsids NTR behavior: The FG-binding pocket is insufficient. Hexameric and pentameric capsomers contribute. The highly exposed outer capsid surface is key. It lacks FG-repulsive charged residues (K,D,E) that are very abundant on normal protein surfaces. FG-attractive residues dominate the capsid surface. Introducing FG-repulsive ones impedes FG phase-partitioning, NPC-targeting and NPC-passage of assembled capsids. Capsids are thus made FG phase-soluble by myriads of transient FG-attractive interactions originating from individual surface sidechains. We discuss that CPSF6 releases the capsid from NPCs by switching its surface from FG-attractive to FG-repulsive.

cell biology↗

bioRxiv2025

Achievement-Based Differences in Cognitive-Emotional Interplay During Classroom Learning: A Multimodal Analysis with Experience Sampling Method

BackgroundThe relationship between cognition and emotion during learning is crucial for educational theories and has been under debate for a long time, from the ancient philosophy to current scientific analyses. However, the relationship may not be uniform and may vary across diverse individual groups. AimsTherefore, this study aims to explore whether the relationship between cognition and emotion varies across different student groups. Furthermore, it examines whether neurophysiological evidence can provide insights into such group differences. SampleThe study involved 22 high school students (11 high-performing and 11 low-performing) over 13 math lessons. Surveys were administered three times during each lesson, yielding a multilevel dataset with 777 samples. MethodsThe experience sampling method was employed to gather data on cognitive and emotional states periodically, as well as their regulations. Simultaneously, multimodal neurophysiological data, including electroencephalograph (EEG), electrodermal activity (EDA), and photoplethysmography (PPG), were collected during classroom learning to analyze the relationship further. ResultsCognitive state, cognitive regulation, emotional state, and emotional regulation were highly correlated overall. In high-performing students, the association was higher than that in low-performing students, especially at the level of within-subject repeated-measures. Multimodal data revealed the common neurophysiological basis of the correlated variation between cognition and emotion, and highlighted different neurophysiological patterns between high and low achievers. ConclusionsThe joint evidence from the experience sampling survey and neurophysiological measures reveals that the cognitive-emotional relationship differs by academic achievement levels, with high achievers revealing a more consistent variation between cognition and emotion. Such co-variation shares a common neurophysiological pattern with features associated with high student-teacher interaction and high concentration, further explaining the group differences.

neuroscience↗

bioRxiv2024

Incomplete lineage sorting of segmental duplications defines the human chromosome 2 fusion site early during African great ape speciation

All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Yet, the structure, function, and evolutionary history of the genomic regions associated with this fusion remain poorly understood. Here, we analyze finished telomere-to-telomere chromosomes in great apes and macaques to show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the rapid turnover of subterminal repetitive DNA. We characterized the fusion site at single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. Most conspicuously, one of these SDs shares homology to a hypomethylated SD spacer sequence present in hundreds of copies in the subterminal heterochromatin of chimpanzees and bonobos. The fusion in human was accompanied by a systematic degradation of the three divergent -satellite arrays representing the ancestral centromere creating five distinct structural haplotypes in humans. CRISPR/Cas9-mediated depletion of the fusion site in human cell lines significantly alters the expression of 108 genes, indicating a potential regulatory consequence to this human-specific karyotypic change.

evolutionary biology↗

bioRxiv2024

Integrating target capture with whole genome sequencing of recent and natural history collections to explain the phylogeography of wild-growing and cultivated Cannabis

O_LICannabis has provided important and versatile services to humans for millennia. Domestication and subsequent dispersal have resulted in various landraces and cultivars. Unravelling the phylogeography of this genus poses considerable challenges due to its complex history. C_LIO_LIWe relied on a Hyb-Seq approach (combining target capture with shotgun sequencing), with the universal Angiosperms353 enrichment panel, to explore the genetic structure of wild-growing accessions and cultivars by implementing phylogenomic and population genomic workflows on the same Hyb-Seq data. C_LIO_LIOur findings support the treatment of Cannabis as a monotypic genus (C. sativa L.), structured into three main genetic groups--E Asia, Paleotropis, and Boreal--with clear phylogeographic signal despite significant levels of admixture. The E Asia group was sister to the Paleotropis and the Boreal groups. Individuals within the Paleotropis group could be further structured into three subgroups: Iranian Plateau, C & S China and Himalayas, and Indoafrica. Individuals from the Boreal group split into two subgroups: Eurosiberia and W Mongolia and Caucasus and Mediterranean. Hemp and drug-type landraces and cultivars consistently matched their putative geographic origin. C_LIO_LIThese findings enhance our understanding of the genetic patterns in Cannabis and provide a framework for future research into its current and past genetic diversity. C_LI

genomics↗

bioRxiv2024

Expanding the CRISPR toolbox by engineering Cas12a orthologs of metagenomic discovery

Cas12a (Cpf1) is a CRISPR-associated nuclease with broad utility in genome editing and molecular diagnostic applications. However, the widespread adoption of CRISPR-Cas12a nucleases and their variants has been hindered by the requirement for a specific protospacer adjacent motif (PAM), relatively low CRISPR RNA (crRNA) activity and the inability to multiplex nucleic acid detection alone. To overcome these limitations, we employed a comprehensive framework combined with AlphaFold2 to de novo mine 1,261 previously unexploited Cas12a orthologs from the global microbiome. Following experimental analysis, we identified the most promising 21 Cas12a nuclease orthologs and designated them "Genie scissor 12" (Gs12). Our analysis uncovered two exceptional variants among these newly identified orthologs: Gs12-10, a first natural PAM-less Cas12a ortholog, which can recognize 52 distinct PAM types, representing a significant 1.8-fold expansion in recognition range compared to the relative LbCas12a PAM; and Gs12-7MAX, an engineered variant of Gs12-7 that exhibited 1.27-fold higher editing efficiency than enAsCas12a-HF. Furthermore, we harnessed Gs12-1, Gs12-4, Gs12-9, and Gs12-18, along with their corresponding engineered crRNAs, to develop a powerful four-channel multiplexed CRISPR-based nucleic acid detection system. The discovery of diverse functions in Cas12a offers a deeper understanding of the CRISPR/Cas12a family. Also, it holds great promise for expanding its applications and uncovering the untapped potential of other CRISPR/Cas systems. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/615316v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@bee75forg.highwire.dtl.DTLVardef@1480f61org.highwire.dtl.DTLVardef@1e9cbc9org.highwire.dtl.DTLVardef@4211b2_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗