Biology subjects

Chen, C.

Publications and source records attributed to Chen, C..

At least 307 records · Page 17Linked to original sources

bioRxiv2024

Extracellular plant subtilases dampen cold shock peptide elicitor levels

Recognizing pathogen-associated molecular patterns (PAMPs) on the cell surface is crucial for plant immunity. The proteinaceous nature of many of these patterns suggests that secreted proteases play important roles in their formation and stability. Here, we demonstrate that the apoplastic subtilase SBT5.2a inactivates the immunogenicity of cold-shock proteins of the bacterial plant pathogen Pseudomonas syringae by cleaving within the immunogenic csp22 epitope. Consequently, mutant plants lacking SBT5.2a activity retain higher levels of csp22, leading to enhanced immune responses and reduced pathogen growth. SBT5.2 sensitivity is influenced by sequence variation surrounding the cleavage site and likely extends to CSPs from other bacterial species. These findings suggest that variations in csp22 stability among bacterial pathogens are a crucial factor in plant-bacteria interactions and that pathogens might exploit plant proteases to avoid pattern recognition.

plant biology↗

bioRxiv2024

A nanobody-enzyme fusion protein targeting PD-L1 and sialic acid exerts anti-tumor effects by affecting tumor associated macrophages

Cancer cells employ various mechanisms to evade immune surveillance. Their surface features, including a protective "sugar coat" and immune checkpoints like PD-L1 (programmed death ligand 1), can impede immune cell recognition. Sialic acids, which carry negative charges, may hinder cell contact through electrostatic repulsion, while PD-L1 transmits immunosuppressive signals to T cells. Furthermore, cancer cells manipulate macrophages within the tumor microenvironment to facilitate immune escape. Prior research has demonstrated the effectiveness of separately blocking the PD-L1 and sialic acid pathways in eliciting anti-tumor effects. In this study, we investigated the relationship between PD-L1 expression and genes associated with sialic acid in clinical databases. Subsequently, we developed a novel nanobody enzyme fusion protein termed Nb16-Sia to simultaneously target both PD-L1 and sialic acid pathways. In vivo experiments confirmed the anti-tumor activity of Nb16-Sia and highlighted its dependence on macrophages. Further investigations revealed that Nb16-Sia could polarize macrophages towards the M1 phenotype through the C-type lectin pathway in vitro and eliminate tumor-associated macrophages in vivo. In conclusion, our findings demonstrate that the fusion of PD-L1 nanobody with sialidase effectively targets tumor-associated macrophages, resulting in significant anti-tumor effects. This approach holds promise for drug development aimed at enhancing immune responses against cancer.

pharmacology and toxicology↗

bioRxiv2024

LRRK2 mediates haloperidol-induced changes in indirect pathway striatal projection neurons

Haloperidol is used to manage psychotic symptoms in several neurological disorders through mechanisms that involve antagonism of dopamine D2 receptors that are highly expressed in the striatum. Significant side effects of haloperidol, known as extrapyramidal symptoms, lead to motor deficits similar to those seen in Parkinsons disease and present a major challenge in clinical settings. The underlying molecular mechanisms responsible for these side effects remain poorly understood. Parkinsons disease-associated LRRK2 kinase has an important role in striatal physiology and a known link to dopamine D2 receptor signaling. Here, we systematically explore convergent signaling of haloperidol and LRRK2 through pharmacological or genetic inhibition of LRRK2 kinase, as well as knock-in mouse models expressing pathogenic mutant LRRK2 with increased kinase activity. Behavioral assays show that LRRK2 kinase inhibition ameliorates haloperidol-induced motor changes in mice. A combination of electrophysiological and anatomical approaches reveals that LRRK2 kinase inhibition interferes with haloperidol-induced changes, specifically in striatal neurons of the indirect pathway. Proteomic studies and targeted intracellular pathway analyses demonstrate that haloperidol induces a similar pattern of intracellular signaling as increased LRRK2 kinase activity. Our study suggests that LRRK2 kinase plays a key role in striatal dopamine D2 receptor signaling underlying the undesirable motor side effects of haloperidol. This work opens up new therapeutic avenues for dopamine-related disorders, such as psychosis, also furthering our understanding of Parkinsons disease pathophysiology. SummaryChen et al. demonstrate that haloperidol mediated changes in the striatal indirect pathway neurons and circuits are linked to Parkinsons disease associated LRRK2. Inhibiting LRRK2 kinase activity ameliorates the motoric side effects of haloperidol, suggesting a potential approach to alleviating the unwanted side effects of antipsychotics.

neuroscience↗

bioRxiv2024

Molecular and spatial transcriptomic classification of midbrain dopamine neurons and their alterations in a LRRK2G2019S model of Parkinson's disease

Several studies have revealed that midbrain dopamine (DA) neurons, even within a single neuroanatomical area, display heterogeneous properties. In parallel, studies using single cell profiling techniques have begun to cluster DA neurons into subtypes based on their molecular signatures. Recent work has shown that molecularly defined DA subtypes within the substantia nigra (SNc) display distinctive anatomic and functional properties, and differential vulnerability in Parkinsons disease (PD). Based on these provocative results, a granular understanding of these putative subtypes and their alterations in PD models, is imperative. We developed an optimized pipeline for single-nuclear RNA sequencing (snRNA-seq) and generated a high-resolution hierarchically organized map revealing 20 molecularly distinct DA neuron subtypes belonging to three main families. We integrated this data with spatial MERFISH technology to map, with high definition, the location of these subtypes in the mouse midbrain, revealing heterogeneity even within neuroanatomical sub-structures. Finally, we demonstrate that in the preclinical LRRK2G2019S knock-in mouse model of PD, subtype organization and proportions are preserved. Transcriptional alterations occur in many subtypes including those localized to the ventral tier SNc, where differential expression is observed in synaptic pathways, which might account for previously described DA release deficits in this model. Our work provides an advancement of current taxonomic schemes of the mouse midbrain DA neuron subtypes, a high-resolution view of their spatial locations, and their alterations in a prodromal mouse model of PD. Teaser: Using snRNASeq and MERFISH we identified midbrain DA subtypes, mapped their spatial location, and identified alterations in a LRRK2 model

neuroscience↗

bioRxiv2024

Genome-wide mapping of native co-localized G4s and R-loops in living cells

The non-B DNA structures can act as dynamic functional genomic elements regulating gene expression. Among them, G4s and R-loops are two of the best studied. The interplay between R-loops and G4s are emerging in regulating DNA repair, replication and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops - associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease.

genomics↗

bioRxiv2024

SpaNorm: spatially-aware normalisation for spatial transcriptomics data

Library size normalisation is necessary to enable comparisons between observations in transcriptomic datasets. Numerous methods have been developed to normalise these effects with sample and gene specific adjustments. However, in spatial transcriptomics data, normalisation is complicated by the fact that spatial region-specific library size confounds biology. The most popular approach of adapting methods developed for single-cell RNA-seq data has been shown to excessively remove biological signals associated with spatial domains and thus results in poorer downstream domain identification. To this end, we propose the first spatially-aware normalisation method, SpaNorm. SpaNorm concurrently models spatial library size effects and the underlying smooth biology, to tease apart these effects, and thereby remove library size effects without removing biology. This is achieved through optimal decomposition of spatially smooth variation into those related and unrelated to library size and the use of location-specific scaling factors. Using 27 tissue samples from 6 datasets spanning 4 spatial platforms, we show that SpaNorm outperforms current state of the art methods at retaining biological information in the form of spatial domains and spatially variable genes (SVGs) better than 4 commonly used single-cell normalisation approaches. SpaNorm is versatile and it can be used for both spot-based and subcellular spatial transcriptomics data. Notably, the benefit of using SpaNorm is more pronounced for the latter data such as those from Xenium, STOmics and CosMx platforms for which the proportion of genes exhibiting region-specific library size effect is higher. SpaNorm works equally well with segmented cell-level data and spot-based data where each spot contains multiple cells.

bioinformatics↗

bioRxiv2024

Development of Optimization of Expected Cross Value for Mate Selection Problems

In this study, we address the mate selection problem in the hybridization stage of a breeding pipeline, which constitutes the multi-objective breeding goal key to the performance of a variety development program. The solution framework we formulate seeks to ensure that individuals with the most desirable genomic characteristics are selected to cross in order to maximize the likelihood of the inheritance of desirable genetic materials to the progeny. Unlike approaches that use phenotypic values for parental selection and evaluate individuals separately, we use a criterion that relies on the genetic architecture of traits and evaluates combinations of genomic information of the pairs of individuals. We introduce the expected cross value (ECV) criterion that measures the expected number of desirable alleles for gametes produced by pairs of individuals sampled from a population of potential parents. We use the ECV criterion to develop an integer linear programming formulation for the parental selection problem. The formulation is capable of controlling the inbreeding level between selected mates. We evaluate the approach for two applications: (i) improving multiple target traits simultaneously, and (ii) finding a multi-parental solution to design crossing blocks. We evaluate the performance of the ECV criterion using a simulation study. Finally, we discuss how the ECV criterion and the proposed integer linear programming techniques can be applied to improve breeding efficiency while maintaining genetic diversity in a breeding program.

bioinformatics↗

bioRxiv2024

An intra-family conserved high-order RNA structure within the M ORF is important for arterivirus subgenomic RNA accumulation and infectious virus production

Synthesis of subgenomic RNAs is a strategy commonly used by polycistronic positive sense single-stranded RNA viruses to express 3'-proximal genes. Members of the order of Nidovirales, including coronaviruses and arteriviruses, use a unique discontinuous transcription strategy to synthesize subgenomic RNAs. In this study, in silico synonymous site conservation analysis and RNA structure folding predicted the existence of intra-family conserved high-order RNA structure within the M ORF of arteriviral genomes, which was further determined to be important for the transcription/accumulation of subgenomic RNAs and production of infectious viral particles. Mutations disrupting the stability of the RNA structures significantly decreased the accumulation of multiple subgenomic RNAs. In contrast, the impact of mutagenesis on full-length genomic RNA accumulation was limited. The degree to which wild-type levels of subgenomic RNA accumulation were maintained was found to correlate with the efficiency of infectious virus production. Moreover, the thermo-stability of stems within the high-order RNA structure is also well correlated with viral replication capacity and the maintenance of subgenomic RNA accumulation. This study is the first to report an intra-Arteriviridae conserved high-order RNA structure that is located in a protein-coding region and functions as an important cis-acting element to control the accumulation/transcription of arteriviral subgenomic RNAs. This work suggests a complex regulation mechanism between genome replication and discontinuous transcription in nidoviruses. IMPORTANCEArteriviruses are a group of RNA viruses that infect different animal species. They can cause diseases associated with respiratory/reproductive syndromes, abortion, or haemorrhagic fever. Among arteriviruses, porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) are economically important veterinary pathogens. The challenge in control of arterivirus infection reflects our limited knowledge of viral biology. In this study, we conducted a comprehensive bioinformatical analysis of arteriviral genomes and discovered intra-family conserved regions in the M ORF with a high-order RNA structure. The thermo-stability of the RNA structure influences sgRNA transcription/accumulation and correlates with the level of infectious virus production. Our studies provide a new insight on arterivirus replication mechanism, which may have implications in developing disease control and prevention strategies.

molecular biology↗

bioRxiv2024

Dual nuclease single-cell lineage tracing by Cas9 and Cas12a

Single-cell lineage tracing based on CRISPR/Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool, which utilizes two orthogonal gene editing systems to record deep cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability of enhanced lineage recording with minimized target dropouts and deepened tree depth. Application of DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers. Moreover, we have developed an entropy-based approach to quantify the lineage recording ability of DuTracer in cell differentiation models. Together, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

developmental biology↗

bioRxiv2024

Brain-wide functional connectome analysis of 40,000 individuals reveals brain networks that show aging effects in older adults

The functional connectome changes with aging. We systematically evaluated aging related alterations in the functional connectome using a whole-brain connectome network analysis in 39,675 participants in UK Biobank project. We used adaptive dense network discovery tools to identify networks directly associated with aging from resting-state fMRI data. We replicated our findings in 499 participants from the Lifespan Human Connectome Project in Aging study. The results consistently revealed two motor-related subnetworks (both permutation test p-values <0.001) that showed a decline in resting-state functional connectivity (rsFC) with increasing age. The first network primarily comprises sensorimotor and dorsal/ventral attention regions from precentral gyrus, postcentral gyrus, superior temporal gyrus, and insular gyrus, while the second network is exclusively composed of basal ganglia regions, namely the caudate, putamen, and globus pallidus. Path analysis indicates that white matter fractional anisotropy mediates 19.6% (p<0.001, 95% CI [7.6% 36.0%]) and 11.5% (p<0.001, 95% CI [6.3% 17.0%]) of the age-related decrease in both networks, respectively. The total volume of white matter hyperintensity mediates 32.1% (p<0.001, 95% CI [16.8% 53.0%]) of the aging-related effect on rsFC in the first subnetwork.

neuroscience↗

bioRxiv2024

Androgen receptor activity inversely correlates with immune cell infiltration and immunotherapy response across multiple cancer lineages

There is now increasing recognition of the important role of androgen receptor (AR) in modulating immune function. To gain a comprehensive understanding of the effects of AR activity on cancer immunity, we employed a computational approach to profile AR activity in 33 human tumor types using RNA-Seq datasets from The Cancer Genome Atlas. Our pan-cancer analysis revealed that the genes most negatively correlated with AR activity across cancers are involved in active immune system processes. Importantly, we observed a significant negative correlation between AR activity and IFN{gamma} pathway activity at the pan-cancer level. Indeed, using a matched biopsy dataset from subjects with prostate cancer before and after AR-targeted treatment, we verified that inhibiting AR enriches immune cell abundances and is associated with higher IFN{gamma} pathway activity. Furthermore, by analyzing immunotherapy datasets in multiple cancers, our results demonstrate that low AR activity was significantly associated with a favorable response to immunotherapy. Together, our data provide a comprehensive assessment of the relationship between AR signaling and tumor immunity.

cancer biology↗

bioRxiv2024

Exosomal miRNA-124 is involved in the immune regulation of cervical cancer by regulating CD45 alternative splicing

ObjectiveTo investigate the regulatory mechanism of miRNA-124 in tumor immune regulation of cervical cancer. MethodsPeripheral blood samples of cervical cancer patients and transient infection controls were collected to extract exosomal miR-124 and Stem-loop Q-PCR to detect the expression level of miR-124. QPCR, WB, and flow cytometry were used to detect the mRNA and protein expression of Th cell differentiation and function-related genes. Transfection, QPCR, and flow cytometry were used to detect the effects of miR-124 overexpression and silencing on the differentiation of Th immune memory cells. In vitro, cell-killing experiments were performed to detect the tumor-killing activity of TH immune memory cells co-cultured with the Hela cell line, and the downstream target genes of miR-124 were predicted and verified by luciferase. Multiplex PCR was used to detect the genotype of miR-124 SNP in peripheral blood DNA. QPCR and WB were used to detect the mRNA and protein expression and phosphorylation of miR-124 target gene GSK3-PSF-CD45 pathway, and GSK3 inhibitor was used to block the pathway. ResultsWe found that exosomal miRNA-124 was significantly downregulated in cervical cancer tissues and affected Th cell immune function and memory cell generation. The overexpression of miRNA-124 can directly target GSK3, phosphorylate the target gene, and inhibit the expression of GSK3, thereby increasing the expression of CD45, increasing the ability of Th immune cells to recognize and kill the HPV virus, and significantly inhibiting the proliferation and migration of cervical cancer cells.

molecular biology↗

bioRxiv2024

The Brain Computes Dynamic Facial Movements for Emotion Categorization Using a Third Pathway

Recent theories suggest a new brain pathway dedicated to processing social movement is involved in understanding emotions from biological motion, beyond the well-known ventral and dorsal pathways. However, how this social pathway functions as a network that computes dynamic biological motion signals for perceptual behavior is unchartered. Here, we used a generative model of important facial movements that participants (N = 10) categorized as "happy," "surprise," "fear," "anger," "disgust," "sad" while we recorded their MEG brain responses. Using new representational interaction measures (between facial features, MEGt source, and behavioral responses), we reveal per participant a functional social pathway extending from occipital cortex to superior temporal gyrus. Its MEG sources selectively represent, communicate and compose facial movements to disambiguate emotion categorization behavior, while occipital cortex swiftly filters out task-irrelevant identity-defining face shape features. Our findings reveal how social pathway selectively computes complex dynamic social signals to categorize emotions in individual participants.

neuroscience↗

bioRxiv2024

Down-regulation of AKT proteins slows the growth of mutant-KRAS pancreatic tumors

Serine/threonine kinase AKT isoforms play a well-established role in cell metabolism and growth. Most pancreatic adenocarcinoma (PDAC) harbors activation mutations of KRAS, which activates the PI3K/AKT signaling pathway. However, AKT inhibitors are not effective in the treatment of pancreatic cancer. To better understand the role of AKT signaling in mutant-KRAS pancreatic tumors, this study utilizes proteolysis-targeting chimeras (PROTACs) and CRISPR-Cas9-genome editing to investigate AKT proteins. PROTAC down-regulation of AKT proteins markedly slowed the growth of three pancreatic tumor cell lines harboring mutant KRAS. In contrast, inhibition of AKT kinase activity alone had very little effect on the growth of these cell lines. Concurrent genetic deletion of all AKT isoforms (AKT1, AKT2, and AKT3) in the KPC (KrasG12D; Trp53R172H; Pdx1-Cre) pancreatic cancer cell line also dramatically slowed its growth in vitro and when orthotopically implanted in syngeneic mice. Surprisingly, insulin-like growth factor-1 (IGF-1), but not epidermal growth factor (EGF), restored KPC cell growth in serum-deprived conditions and the IGF-1 growth stimulation effect was AKT dependent. RNA-seq analysis of AKT1/2/3-deficient KPC cells suggested that reduced cholesterol synthesis may be responsible for the decreased response to IGF-1 stimulation. These results indicate that the presence of all three AKT isoforms supports pancreatic tumor cell growth and pharmacological degradation of AKT proteins may be more effective than AKT catalytic inhibitors for treating pancreatic cancer.

cancer biology↗

bioRxiv2024

High glucose treatment induced nuclei aggregation of microvascular endothelial cells via foxo1a-klf2a pathway

BACKGROUNDHyperglycemia is a major contributor to endothelial dysfunction and blood vessel damage, leading to severe diabetic microvascular complications. Despite the growing body of research on the underlying mechanisms of endothelial cell dysfunction, the available drugs based on current knowledge fall short of effectively alleviating these complications. Therefore, our endeavor to explore novel insights into the cellular and molecular mechanisms of endothelial dysfunction is crucial for the field. METHODSIn this study, we carried out a high-resolution imaging and time-lapse imaging analysis of the behavior of endothelial cells in Tg(kdrl:ras-mCherry::fli1a:nEGFP) zebrafish embryos upon high glucose treatment. Genetic manipulation and chemical biology approaches were utilized to analyze the underlying mechanism of high-glucose-induced nuclei aggregation and aberrant migration of zebrafish endothelial cells and cultured human endothelial cells. Bioinformatical analysis of single-cell RNA sequencing data and molecular biological techniques to identify the target genes of Foxo1a. RESULTSIn this study, we observed that the high glucose treatment resulted in nuclei aggregation of endothelial cells in zebrafish intersegmental vessels (ISVs). Additionally, the aberrant migration of microvascular endothelial cells in high glucose-treated embryos, which might be a cause of nuclei aggregation, was discovered. High glucose-induced aggregation of vascular endothelial nuclei via foxo1a downregulation in zebrafish embryos. Then, we revealed that high glucose resulted in the downregulation of foxo1a expression and increased the expression of its direct downstream effector, klf2a, through which the aberrant migration and aggregation of vascular endothelial nuclei were caused. CONCLUSIONSHigh glucose treatment caused the nuclei of endothelial cells to aggregate in vivo, which resembles the crowded nuclei of endothelial cells in microaneurysms. High glucose suppresses foxo1a expression and increases the expression of its downstream effector, klf2a, thereby causing the aberrant migration and aggregation of vascular endothelial nuclei. Our findings provide a novel insight into the mechanism of microvascular complications in hyperglycemia.

cell biology↗

bioRxiv2024

A Libra in the Brain: Neural Correlates of Error Awareness Predict Moral Wrongness and Guilt Proneness

Error awareness is a fundamental mechanism in humans. Through traditional psychological tasks, certain neural activities that represent errors in objective manners have been identified. However, there is limited knowledge on how humans subjectively determine right from wrong in moral contexts. In this study, participants (N=39) mentally simulated themselves as the agents of moral and immoral behaviors, while viewing a series of actions with EEG recording and MRI scanning, respectively. A significant difference in error-related negativity (ERN) was observed among morally wrong scenarios, accompanied by higher wrongfulness ratings. Additionally, individual differences in guilt-proneness could predict the subjects ERN amplitude. The ERN amplitude was correlated with the BOLD activity in the anterior mid-cingulate cortex and anterior insula to immoral scenarios, reflecting error awareness toward moral wrongfulness. The late potential component displayed greater negativity to immoral scenarios and was correlated with BOLD activities in the amygdala, ventromedial prefrontal cortex, and temporoparietal junction, indicating cognitive and affective evaluation in moral judgment. In line with the moral dynamic framework, our results demonstrated individual variability in moral judgments, as indicated by dispersed and overlapping cognitive neural networks. This suggests that subjective evaluations of wrongfulness are underpinned by neural mechanisms, associated with those involved in objective error awareness.

neuroscience↗

bioRxiv2024

Precision and Accuracy of Single-Cell/Nuclei RNA Sequencing Data

Single-cell and single-nucleus RNA sequencing (sc/snRNA-Seq) have become essential tools for profiling gene expression across different cell types in biomedical research. While factors like RNA integrity, cell count, and sequencing depth are known to influence data quality, quantitative benchmarks and actionable guidelines are lacking. This gap contributes to variability in study designs and inconsistencies in downstream analyses. In this study, we systematically evaluated quantitative precision and accuracy in expression measures across 23 sc/snRNA-Seq datasets comprising 3,682,576 cells from 339 samples. Precision was assessed using technical replicates based on pseudo-bulks created from subsampling. Accuracy was evaluated using sample-matched scRNA-Seq and pooled-cell RNA-Seq data of mononuclear phagocytes from four species. Our results show that precision and accuracy are generally low at the single-cell level, with reproducibility being strongly influenced by cell count and RNA quality. We establish data-driven thresholds for optimizing study design, recommending at least 500 cells per cell type per individual to achieve reliable quantification. Furthermore, we show that signal-to-noise ratio is a key metric for identifying reproducible differentially expressed genes. To support future research, we developed VICE (Variability In single-Cell gene Expressions), a tool that evaluates sc/snRNA-seq data quality and estimates the true positive rate of differential expression results based on sample size, observed noise levels, and expected effect size. These findings provide practical, evidence-based guidelines to enhance the reliability and reproducibility of sc/snRNA-seq studies.

genomics↗

bioRxiv2024

Efficient Gene Editing and Overexpression of Gametophyte Transformation in a Model Fern

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-related nuclease (Cas) system allows precise and easy editing of genes in many plant species. However, this system has not yet been applied to any fern species due to the complex characteristics of fern genomes, genetics and physiology. Here, we established, for the first time, a protocol for gametophyte-based screening single-guide RNAs (sgRNAs) with high efficiency for CRISPR/Cas-mediated gene editing in a model fern species, Ceratopteris richardii. We utilized the C. richardii Actin promoter to drive sgRNA expression and enhanced CaMV 35S promoter to drive the expression of Streptococcus pyogenes Cas9 in this CRISPR-mediated editing system, which was employed to successfully edit a few genes (e.g., nucleotidase/phosphatase 1, CrSAL1; Cryptochrome 4, CRY4) and CrPDS, encoding a phytoene desaturase protein that resulted in an albino phenotype in C. richardii. Knockout of CrSAL1 resulted in significantly reduced stomatal conductance (gs), leaf transpiration rate (E), stomatal/pore length, and abscisic acid (ABA)-induced reactive oxygen species (ROS) accumulation in guard cells. Moreover, CrSAL1 overexpressing plants showed significantly increased net photosynthetic rate (A), gs, E and intrinsic water use efficiency (iWUE) as well as most of the stomatal traits and ROS production in guard cells compared to those in the wild-type (WT) plants. Taken together, the optimized CRISPR/Cas9 system provides a useful tool for functional genomics in a model fern species, allowing the exploration of fern gene functions for evolutionary biology, herbal medicine discovery and agricultural applications.

plant biology↗