Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 775 records · Page 43Linked to original sources

bioRxiv2020

Non-coding germline GATA3 variants alter chromatin topology and contribute to pathogenesis of acute lymphoblastic leukemia

Inherited non-coding genetic variants confer significant disease susceptibility in many cancers. However, the molecular processes of by which germline variants contribute to somatic lesions are poorly understood. We performed targeted sequencing in 5,008 patients and identified a key regulatory germline variant in GATA3 strongly associated with Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL). By creating an isogenic cellular model with CRISPR-Cas9 system, we showed that this variant activated a strong enhancer that significantly upregulated GATA3 transcription, which in turn reshaped the global chromatin accessibility and 3D genome organization. Remarkably, this genotype switch induced a chromatin loop between the CRLF2 oncogene and a distal enhancer, similar to the somatically acquired super-enhancer hijacking event in patients. GATA3 genotype-related alterations in transcriptional control and 3D chromatin organization were further validated in Ph-like ALL patients. Finally, we showed that GATA3 directly regulates CRLF2 and potentiates the oncogenic effects of JAK-STAT signaling in leukemogenesis. Altogether, our results provide evidence for a novel mechanism by which a germline non-coding variant contributes to oncogene activation epigenetic regulation and 3D genome reprogramming.

cancer biology↗

bioRxiv2020

Cellular heterogeneity-adjusted clonal methylation (CHALM) provides better prediction of gene expression

Promoter DNA methylation is a well-established mechanism of transcription repression, but its global correlation with gene expression is surprisingly weak. This weak correlation is due in part to the failure of current methylation quantification methods to consider the heterogeneity among sequenced bulk cells. Here, we introduce Cell Heterogeneity-Adjusted cLonal Methylation (CHALM) as a novel methylation quantification method. CHALM better explains the functional consequences of DNA methylation, such as its correlations with gene expression and H3K4me3. When applied to different methylation datasets, the CHALM method has uniquely detected differentially methylated genes that exhibit distinct biological functions supporting underlying mechanisms.

bioinformatics↗

bioRxiv2020

Adhesivity and motility of a zoonotic spirochete: Implications in host-dependent pathogenicity

Bacterial motility is crucial for many pathogenic species in the process of invasion and/or dissemination. The spirochete bacteria Leptospira spp. cause symptoms, such as hemorrhage, jaundice, and nephritis, in diverse mammals including humans. Although loss-of-motility attenuate the spirochete, the mechanism of the motility-dependent pathogenicity is unknown. Here, focusing on that Leptospira spp. swim in liquid and crawl on solid surfaces, we investigated the spirochetal dynamics on the host tissues by infecting cultured kidney cells from various species with pathogenic and nonpathogenic leptospires. We found that, in the case of the pathogenic leptospires, a larger fraction of bacteria attached to the host cells and persistently traveled long distances using the crawling mechanism. Our results associate the kinetics and kinematic features of the spirochetal pathogens with their virulence. One Sentence SummaryAdhesivity and crawling motility over host tissue surfaces are closely related to the pathogenicity of a zoonotic spirochete.

microbiology↗

bioRxiv2020

Profiling of Human Gut Virome with Oxford Nanopore Technology

Human gut virome play critical roles in maintaining gut microbial composition and functionality, as well as host physiology and immunology. Yet, there are insufficient amount of studies on this topic mainly due to methodological limitations, including enrichment of viruses (phages and host viruses) as well as short read-length from current sequencing technology. Here we developed a full working protocol for analyzing human gut virome using physical enrichment, reverse transcription and random amplification, and eventually the state-of-art single-molecule real-time sequencing (SMRT) platform of Oxford Nanopore Technology (ONT). We demonstrate that sequencing viral DNA directly, or viral DNA/cDNA after amplification using ONT achieves much longer reads and provides more information regarding virome diversity, many of the virome sequences do not have match in current databases. Moreover, direct DNA sequencing of virome provides first insights into the epigenetic modifications on phages, where signals of methylations can be directly detected. Our study demonstrates that progressing sequencing technology and bioinformatic improvements will bring more knowledge into virome composition, diversity and potentially their important functions. HighlightsO_LIVirus-like particles were enriched from human stool samples; C_LIO_LIViral nucleotides were sequenced with Oxford Nanopore Technology with and without amplification; C_LIO_LIGut virome in humans showed highly individualized composition; C_LIO_LINovel sequences and contigs were found to be the majority in the resulted sequences; C_LIO_LIEpigenetic modifications were detected directly on virus genomes. C_LI

bioinformatics↗

bioRxiv2020

Regulation of olfactory-based sex behaviors in the silkworm by genes in the sex-determination cascade

Insect courtship and mating depend on integration of olfactory, visual, and tactile cues. Compared to other insects, Bombyx mori, the domesticated silkworm, has relatively simple sexual behaviors as it cannot fly. Here by using CRISPR/Cas and electrophysiological techniques we found that courtship and mating behaviors are regulated in male silk moths by mutating genes in the sex determination cascade belonging to two conserved pathways. Loss of Bmdsx gene expression significantly reduced the peripheral perception of the major pheromone component bombykol by reducing expression of the product of the BmOR1 gene which completely blocked courtship in adult males. Interestingly, we found that mating behavior was regulated independently by another sexual differentiation gene, Bmfru. Loss of Bmfru completely blocked mating, but males displayed normal courtship behavior. Lack of Bmfru expression significantly reduced the perception of the minor pheromone component bombykal due to the down regulation of BmOR3 expression; further, functional analysis revealed that loss of the product of BmOR3 played a key role in terminating male mating behavior. Our results suggest that Bmdsx and Bmfru are at the base of the two primary pathways that regulate olfactory-based sexual behavior. Author SummaryThe fundamental insect sexual behaviors, courtship and mating, result from successful integration of olfactory, vision, tactile and other complex innate behaviors. In the widely used insect model, Drosophila melanogaster, the sex determination cascade genes fruitless and doublesex are involved in the regulation of courtship and mating behaviors; however, little is known about the function of these sexual differentiation genes in regulating sex behaviors of Lepidoptera. Here we combine genetics and electrophysiology to investigate regulation pathway of sexual behaviors in the model lepidopteran insect, the domesticated silk moth, Bombyx mori. Our results support the presence of two genetic pathways in B. mori, named Bmdsx-BmOR1-bombykol and Bmfru-BmOR3-bombykal, which control distinct aspects of male sexual behavior that are modulated by olfaction. This is the first comprehensive report about the role of sex differentiation genes in the male sexual behavior in the silk moth.

genetics↗

bioRxiv2020

Ultra-deep sequencing differentiates patterns of skin clonogenic mutations associated with sun-exposure status

Non-melanoma skin cancer is the most common human malignancy and is primarily caused by exposure to ultraviolet (UV) radiation. The earliest detectable precursor of UV-mediated skin cancer is the growth of cell groups harboring clonal mutation (CM) in clinically normal appearing skin. Systematic evaluation of CMs is crucial to understand early photo-carcinogenesis. Previous studies confirmed the presence of CMs in sun-exposed skin. However, the relationship between UV-exposure and the accumulation of CMs, and the correlation of CMs with skin cancer risk remain poorly understood. To elucidate the exact molecular and clinical effects of long-term UV-exposure on skin, we performed targeted ultra-deep sequencing in 450 individual-matched sun-exposed (SE) and non-sun-exposed (NE) epidermal punch biopsies obtained from clinically normal skin from 13 donors. A total of 638 CMs were identified, including 298 UV-signature mutations (USMs). The numbers of USMs per sample were three times higher in the SE samples and were associated with significantly higher variant allele frequencies (VAFs), compared with the NE samples. We identified genomic regions in TP53, NOTCH1 and GRM3 where mutation burden was significantly associated with UV-exposure. Six mutations were almost exclusively present in SE epidermis and accounted for 42% of the overall difference between SE and NE mutation burden. We defined Cumulative Relative Clonal Area (CRCA), a single metric of UV-damage calculated by the overall relative percentage of the sampled skin area affected by CMs. The CRCA was dramatically elevated by a median of 11.2 fold in SE compared to NE samples. In an extended cohort of SE normal skin samples from patients with a high- or low-burden of cutaneous squamous cell carcinoma (cSCC), the SE samples in high-cSCC patients contained significantly more USMs than SE samples in low-cSCC patients, with the difference mostly conferred by mutations from low-frequency clones (defined by VAF≤1%) but not expanded clones (VAF>1%). Our studies of differential mutational features in normal skin between paired SE/NE body sites and high/low-cSCC patients provide novel insights into the carcinogenic effect of UV exposure, and suggest CMs might be used to develop novel biomarkers for predicting cancer risk.Significance statement In UV radiation exposed skin, mutations fuel clonal cell growth. We established a sequencing-based method to objectively assess the mutational differences between sun-exposed (SE) and non-sun-exposed (NE) areas of normal human skin. Striking differences, in both the numbers of mutations and variant allele frequencies, were found between SE and NE areas. Furthermore, we identified specific genomic regions where mutation burden is significantly associated with UV-exposure status. These findings revealed previously unknown mutational patterns associated with UV-exposure, providing important insights into UV radiation’s early carcinogenic effects. Additionally, in an extended cohort, we identified preliminary association between normal skin mutation burden and cancer risk. These findings pave the road for future development of quantitative measurement of subclinical UV damage and skin cancer risk.Competing Interest StatementThe authors have declared no competing interest.Common AbbreviationsUVUltravioletCMClonogenic mutationNMSCNonmelanoma skin cancerSESun-exposedNENon-sun-exposedUSMUV-signature mutationNUSMNon-UV-signature mutationCRCACumulative Relative Clonal AreacSCCCutaneous squamous cell carcinomaAKActinic keratosisSNVSingle nucleotide variant Indels – Insertions/deletionsDNVDinucleotide variantCSNVCluster of single nucleotide variantMACMulti-Nucleotide Variant Annotation CorrectorVAFVariant allele frequencyView Full Text

cancer biology↗

bioRxiv2020

MAnorm2 for quantitatively comparing groups of ChIP-seq samples

Eukaryotic gene transcription is regulated by a large cohort of chromatin associated proteins, and inferring their differential binding sites between cellular contexts requires a rigorous comparison of the corresponding ChIP-seq data. We present MAnorm2, a new computational tool for quantitatively comparing groups of ChIP-seq samples. MAnorm2 uses a hierarchical strategy to normalize ChIP-seq data and then performs differential analysis by assessing within-group variability of ChIP-seq signals under an empirical Bayes framework. In this framework, MAnorm2 considers the abundance of differential ChIP-seq signals between groups of samples and the possibility of different within-group variability between groups. When samples in each group are biological replicates, MAnorm2 can reliably identify differential binding events even between highly similar cellular contexts. Using a number of real ChIP-seq data sets, we observed that MAnorm2 clearly outperformed existing tools for differential ChIP-seq analysis, with the improvement in performance being most dramatic when the groups of samples being compared had distinct global within-group variability.

bioinformatics↗

bioRxiv2020

Biophysical basis of cellular multi-specificity encoded in a model molecular switch

Molecular switches are central to signal transduction in protein interaction networks. One switch protein can independently regulate distinct cellular processes, but the molecular mechanisms enabling this functional multi-specificity remain unclear. Here we integrate system-scale cellular and biophysical measurements to study how a paradigm switch, the small GTPase Ran/Gsp1, achieves its functional multi-specificity. We make 55 targeted point mutations to individual interactions of Ran/Gsp1 and show through quantitative, systematic genetic and physical interaction mapping that Ran/Gsp1 interface perturbations have widespread cellular consequences that cluster by biological processes but, unexpectedly, not by the targeted interactions. Instead, the cellular consequences of the interface mutations group by their biophysical effects on kinetic parameters of the GTPase switch cycle, and cycle kinetics are allosterically tuned by distal interface mutations. We propose that the functional multi-specificity of Ran/Gsp1 is encoded by a differential sensitivity of biological processes to different kinetic parameters of the Gsp1 switch cycle, and that Gsp1 partners binding to the sites of distal mutations act as allosteric regulators of the switch. Similar mechanisms may underlie biological regulation by other GTPases and biological switches. Finally, our integrative platform to determine the quantitative consequences of cellular perturbations may help explain the effects of disease mutations targeting central switches.

systems biology↗

bioRxiv2019

Spen links RNA-mediated endogenous retrovirus silencing and X chromosome inactivation

Dosage compensation between the sexes has emerged independently multiple times during evolution, often harnessing long noncoding RNAs (lncRNAs) to alter gene expression on the sex chromosomes. In eutherian mammals, X chromosome inactivation (XCI) in females proceeds via the lncRNA Xist, which coats one of the two X chromosomes and recruits repressive proteins to epigenetically silence gene expression in cis1,2. How Xist evolved new functional RNA domains to recruit ancient, pleiotropic protein partners is of great interest. Here we show that Spen, an Xist-binding repressor protein essential for XCI3-7, binds to ancient retroviral RNA, performing a surveillance role to recruit chromatin silencing machinery to these parasitic loci. Spen inactivation leads to de-repression of a subset of endogenous retroviral (ERV) elements in embryonic stem cells, with gain of chromatin accessibility, active histone modifications, and ERV RNA transcription. Spen binds directly to ERV RNAs that show structural similarity to the A-repeat of Xist, a region critical for Xist-mediated gene silencing8-9. ERV RNA and Xist A-repeat bind the RRM3 domain of Spen in a competitive manner. Insertion of an ERV into an A-repeat deficient Xist rescues binding of Xist RNA to Spen and results in local gene silencing in cis. These results suggest that insertion of an ERV element into proto-Xist may have been a critical evolutionary event, which allowed Xist to coopt transposable element RNA-protein interactions to repurpose powerful antiviral chromatin silencing machinery for sex chromosome dosage compensation.

genomics↗

bioRxiv2019

To mock or not: a comprehensive comparison of mock IP and DNA input for ChIP-seq

Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to detect genome-wide DNA-protein binding. The binding sites of transcription factors facilitate many biological studies. Of emerging concern is the abundance of spurious sites in ChIP-seq, which are mainly caused by uneven genomic sonication and nonspecific interactions between chromatin and antibody. A "mock" IP is designed to correct for both factors, whereas a DNA input control corrects only for uneven sonication. However, a mock IP is more susceptible to technical noise than a DNA input, and empirically, these two controls perform similarly for ChIP-seq. Therefore, DNA input is currently being used almost exclusively. With a large dataset, we demonstrate that using a DNA input control results in a definable set of spurious sites, and their abundance is tightly associated with the intrinsic properties of the ChIP-seq sample. For example, compared to human cell lines, samples such as human tissues and whole worm and fly have more accessible genomes, and thus have more spurious sites. The large and varying abundance of spurious sites may impede comparative studies across multiple samples. In contrast, using a mock IP as control substantially removes these spurious sites, resulting in high-quality binding sites and facilitating their comparability across samples. Although outperformed by mock IP, DNA input is still informative and has unique advantages. Therefore, we have developed a method to use both controls in combination to further improve binding site detection.

genomics↗

bioRxiv2019

Regulation of heterotopic ossification through local inflammatory monocytes in a mouse model of aberrant wound healing

Heterotopic ossification (HO) is an aberrant regenerative process with ectopic bone induction in response to musculoskeletal trauma, in which mesenchymal stem cells (MSC) differentiate into osteochondrogenic cells instead of myocytes or tenocytes. Despite frequent cases of hospitalized musculoskeletal trauma, the inflammatory responses and cell population dynamics that regulate subsequent wound healing and tissue regeneration are still unclear. Here we examine, using a mouse model of trauma-induced HO, the local microenvironment of the initial post-injury inflammatory response. Single cell transcriptome analyses identify distinct monocyte/macrophage populations at the injury site, with their dynamic changes over time elucidated using trajectory analyses. Mechanistically, transforming growth factor beta-1 (TGF{beta}1)-producing monocytes/macrophages are associated with HO and aberrant chondrogenic progenitor cell differentiation, while CD47-activating peptides that reduce systemic TGF{beta} levels help ameliorate HO. Our data thus implicate CD47 activation as a novel therapeutic approach for modulating monocyte/macrophage phenotypes, MSC differentiation and HO formation during wound healing.

immunology↗

bioRxiv2019

Male accessory gland factors increased sex pheromone titres in the glands of Spodoptera litura female moths by inhibiting calling behavior

Moths are the most widely studied example of pheromones in animals. However, little is known about the mechanism of intrasexual and mating-related intersexual regulation of pheromone production and release in female moths. Our previous studies in Spodoptera litura found that mating induced a higher sex pheromone titre in the pheromone gland (PG) and mating or male accessory gland (MAG) extract suppressed female calling behavior. We therefore hypothesize that the inhibition of female calling behavior by mating or MAG factors likely suppresses the release of sex pheromones and thus results in a higher pheromone titre in the PG. To test this hypothesis, in the present study, we introduced an artificial calling behavior suppression treatment by gently knocking on and shaking the testing boxes contained moths once every 10 minutes. Results show that this treatment significantly increased pheromone titres in virgin or saline injected virgin females, and the increase rates are similar to those of mating and MAG extract treated ones. These results have suggested that the increase of sex pheromone titer in the female PG after mating in S. litura is due to the inhibition of female calling behavior by MAG factors. Moreover, results of this study also suggest that female calling behavior is positively correlated to pheromone release and likewise, the calling behavior and sex pheromone release in S. litura females are directly under the neural control, and modulated by molecular and environmental factors.

zoology↗

bioRxiv2019

Lateral mechanical impedance rather than frontal, promotes cortical expansion of roots

It has long been considered that mechanical impedance on root will restrict root elongation and consequently promote radial growth. However, we did observe radial expansion but not elongation restriction in maize seedlings after short growth in sands. Mechanical impedance of soil can be classified into frontal- and lateral-type based on the interaction site of root. Therefore, we suspected that radial expansion might be mainly stimulated by lateral rather than frontal impedance. To verify our speculation, frontal and lateral impedance was provided separately. Small plastic caps were used to provide unique frontal impedance on root tip and cylindrical plastic containers were used to provide lateral impedance. Plastic caps could reduce root length remarkably. However, the radial expansion of plastic-cap-fitted roots was significantly inferior to that of the sand-cultured roots. Microstructural analysis revealed that sand-condition thickened root largely dependents on cortical expansion, whereas plastic cap did it mainly by thickening stele. In cylindrical plastic containers, mechanical impedance came only from the lateral direction and promoted the expansion of cortex just as sand-condition. Thus, we proposed that the expansion of the cortex and the consequent radial growth is mainly due to lateral impedance when growing in sands.

plant biology↗

bioRxiv2019

Single-cell RNA-seq data reveals TNBC tumor heterogeneity through characterizing subclone compositions and proportions

Understanding subclonal architecture and their biological functions poses one of the key challenges to deeply portray and investigative the cause of triple-negative breast cancer (TNBC). Here we combine single-cell and bulk sequencing data to analyze tumor heterogeneity through characterizing subclone compositions and proportions. Based on sing-cell RNA-seq data (GSE118389) we identified five distinct cell subpopulations and characterized their biological functions based on their gene markers. According to the results of functional annotation, we found that C1 and C2 are related to immune functions, while C5 is related to programmed cell death. Then based on subclonal basis gene expression matrix, we applied deconvolution algorithm on TCGA tissue RNA-seq data and observed that microenvironment is diverse among TNBC subclones, especially C1 is closely related to T cells. Whats more, we also found that high C5 proportions would led to poor survival outcome, log-rank test p-value and HR [95%CI] for five years overall survival in GSE96058 dataset were 0.0158 and 2.557 [1.160-5.636]. Collectively, our analysis reveals both intra-tumor and inter-tumor heterogeneity and their association with subclonal microenvironment in TNBC (subclone compositions and proportions), and uncovers the organic combination of subclones dictating poor outcomes in this disease. HighlightsWe applied deconvolution algorithm on subclonal basis gene expression matrix to link single cells and bulk tissue together.

cancer biology↗

bioRxiv2019

Protective functions of ZO-2/Tjp2 expressed in hepatocytes and cholangiocytes against liver injury and cholestasis

BACKGROUND & AIMSTight junctions (TJs) establish tissue barriers that maintain osmotic homeostasis and, in the liver, isolate bile flow from the blood circulation. ZO-2/Tjp2 is a scaffold protein that tethers TJ transmembrane proteins to the actin cytoskeleton. Missense mutations in Tjp2 have recently been shown to cause progressive cholestatic liver disease in humans. However, the underlying mechanisms still remain elusive. To study the role of Tjp2 in cholestatic liver disease, we generated and characterized mice lacking Tjp2 in hepatocytes, cholangiocytes, or both. METHODSTjp2 was inactivated in the mouse liver (both in hepatocytes and cholangiocytes) or hepatocytes or cholangiocytes only. Liver function tests were carried out by biochemical analysis of plasma and liver samples and liver tissue was evaluated by immunohistochemistry and histology. The mice were also subjected to cholic acid (CA) diet to assess their susceptibility to liver insults. RESULTSDeletion of Tjp2 in the mouse liver did not result in apparent changes in TJ structure and composition, but lead to progressive cholestasis with lower expression levels of the bile acid (BA) transporter ABCB11/Bsep and the detoxification enzyme Cyp2b10. Feeding a CA diet that is well tolerated by control mice caused severe cholestasis and necrotic liver injury in mice lacking hepatic Tjp2. Administration of a CAR agonist, TCPOBOP, protected these mice from CA induced injury by enhancing the expression of the detoxifying enzyme Cyp2b10 in hepatocytes. Mice lacking Tjp2 in only hepatocytes or in only cholangiocytes showed less severe CA diet induced liver injury. CONCLUSIONLoss of Tjp2 from hepatocytes and cholangiocytes both contribute to progressive cholestatic liver disease and higher susceptibility to liver injury. In hepatocytes, Tjp2 exerts a protective role by regulating expression levels of BA transporters and detoxification enzymes. The mice may provide a new animal model for cholestatic liver disease linked to Tjp2 mutations in humans.

physiology↗

bioRxiv2019

Lfc/Arhgef2 regulates mitotic spindle orientation in hematopoietic stem and progenitor cells and is essential for productive hematopoiesis

How hematopoietic stem cells (HSCs) coordinate their divisional axis relative to supportive niche cells and whether or not their divisional orientation is important for stem cell-driven hematopoiesis is poorly understood. Single cell RNA sequencing data from patients with the inherited bone marrow failure Shwachman-Diamond syndrome (SDS) show that ARHGEF2, a RhoA-specific guanine nucleotide exchange factor (GEF) and determinant of mitotic spindle orientation, is one of a restricted group of genes specifically downregulated in SDS HSCs and multipotent progenitors. Here, we describe Lfc/Arhgef2 as an important regulator of hematopoiesis in vivo. Transplanted Lfc/Arhgef2-/- bone marrow shows impaired hematopoietic recovery and a production deficit of long-term HSCs. These phenotypes cannot be explained by differences in numbers of transplanted HSCs, their cell cycle status, level of apoptosis, progenitor output or homing ability. Using live imaging of dividing hematopoietic stem and progenitor cells (HSPCs), we show an increased frequency of misoriented divisions in the absence of Lfc/Arhgef2. Functional ARHGEF2 knockdown in human HSCs also impairs their ability to regenerate hematopoiesis, culminating in significantly smaller hematopoietic xenografts. Together, these data provide evidence demonstrating a conserved role for Lfc/Arhgef2 in orienting HSPC division and suggest that HSCs divide in certain orientations to establish hematopoiesis, the loss of which leads to their exhaustion in a mechanism that may underlie certain bone marrow failure syndromes.

cell biology↗

bioRxiv2019

qKW9 encodes a pentatricopeptide repeat protein affecting photosynthesis and grain filling in maize

Kernel weight is an important yield component in maize that was selected during domestication. Many kernel weight genes have been identified through mutant analysis, and are mostly involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, only a limited number of loci underlying quantitative variation in kernel weight have been cloned. Here we characterize a maize kernel weight QTL, qKW9 and find that it encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of NdhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qKW9 background, C-to-U editing of NdhB was abolished, and photosynthesis was reduced, suggesting that qKW9 regulates kernel weight by controling the maternal source of photosynthate for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis on maize grain yield production.

plant biology↗

bioRxiv2019

Programmable CRISPR interference for gene silencing using Cas13a in mosquitoes

In the CRISPR-Cas systems, Cas13a is an RNA-guided RNA nuclease specifically targeting single strand RNA. We developed a Cas13a mediated CRISPR interference tool to target mRNA for gene silencing in mosquitoes. The machinery was tested in two mosquito species. A Cas13a expressing plasmid was delivered to mosquitoes by intrathoracic injection, and Cas13a transcripts were detectable at least10 days post-delivery. In Anopheles gambiae, vitellogenin gene was silenced by Vg-crRNA injection two hours post-blood meal, which was accompanied by a significant reduction in egg production. In Aedes aegypti, the - and {delta}-subunits of COPI genes were silenced by a post-blood meal crRNA injection, which resulted in mortality and fragile midguts, reproducing a phenotype reported previously. Co-silencing genes simultaneously is achievable when a cocktail of target crRNAs is given. No detectable collateral cleavages of non-target transcripts were observed in the study. This study adds a programmable CRISPR tool to manipulate RNA in mosquitoes.

molecular biology↗