Stop Codon Research Articles

Modulation of prion protein expression through cryptic splice site manipulation

Lowering expression of prion protein (PrP) is a well-validated therapeutic strategy in prion disease, but additional modalities are urgently needed. In other diseases, small molecules have proven capable of modulating pre-mRNA splicing, sometimes by forcing inclusion of cryptic exons that reduce gene expression. Here, we characterize a cryptic exon located in human PRNP’s sole intron and evaluate its potential to reduce PrP expression through incorporation into the 5’ untranslated region (5’UTR). This exon is homologous to exon 2 in non-primate species, but contains a start codon that would yield an upstream open reading frame (uORF) with a stop codon prior to a splice site if included in PRNP mRNA, potentially downregulating PrP expression through translational repression or nonsense-mediated decay. We establish a minigene transfection system and test a panel of splice site alterations, identifying mutants that reduce PrP expression by as much as 78%. Our findings nominate a new therapeutic target for lowering PrP.

Characterization of Caulimovirid-like Sequences from Upland Cotton (Gossypium hirsutum L.) Exhibiting Terminal Abortion in Georgia, USA.

In this study, we investigated the potential involvement of endogenous viral elements (EVEs) in the development of apical tissue necrosis, resulting in the terminal abortion of upland cotton (Gossypium hirsutum L.) in Georgia. The high-throughput sequence analysis of symptomatic and asymptomatic plant tissue samples revealed near-complete EVE-Georgia (EVE-GA) sequences closely related to caulimoviruses. The analysis of EVE-GA's putative open reading frames (ORFs) compared to cotton virus A and endogenous cotton pararetroviral elements (eCPRVE) revealed their similarity in putative ORFs 1-4. However, in the ORF 5 and ORF 6 encoding putative coat protein and reverse transcriptase, respectively, the sequences from EVE-GA have stop codons similar to eCPRVE sequences from Mississippi. In silico mining of the cotton genome database using EVE-GA as a query uncovered near-complete viral sequence insertions in the genomes of G. hirsutum species (~7 kb) but partial in G. tomentosum (~5.3 kb) and G. mustelinum (~5.1 kb) species. Furthermore, cotton EVEs' episomal forms and messenger RNA (mRNA) transcripts were detected in both symptomatic and asymptomatic plants collected from cotton fields. No significant yield difference was observed between symptomatic and asymptomatic plants of the two varieties evaluated in the experimental plot. Additionally, EVEs were also detected in cotton seeds and seedlings. This study emphasizes the need for future research on EVE sequences, their coding capacity, and any potential role in host immunity or pathogenicity.

Regulatory mechanism of cold-inducible diapause in Caenorhabditis elegans

Temperature is a critical environmental cue that controls the development and lifespan of many animal species; however, mechanisms underlying low-temperature adaptation are poorly understood. Here, we describe cold-inducible diapause (CID), another type of diapause induced by low temperatures in Caenorhabditis elegans. A premature stop codon in heat shock factor 1 (hsf-1) triggers entry into CID at 9 °C, whereas wild-type animals enter CID at 4 °C. Furthermore, both wild-type and hsf-1(sy441) mutant animals undergoing CID can survive for weeks, and resume growth at 20 °C. Using epistasis analysis, we demonstrate that neural signalling pathways, namely tyraminergic and neuromedin U signalling, regulate entry into CID of the hsf-1 mutant. Overexpression of anti-ageing genes, such as hsf-1, XBP1/xbp-1, FOXO/daf-16, Nrf2/skn-1, and TFEB/hlh-30, also inhibits CID entry of the hsf-1 mutant. Based on these findings, we hypothesise that regulators of the hsf-1 mutant CID may impact longevity, and successfully isolate 16 long-lived mutants among 49 non-CID mutants via genetic screening. Furthermore, we demonstrate that the nonsense mutation of MED23/sur-2 prevents CID entry of the hsf-1(sy441) mutant and extends lifespan. Thus, CID is a powerful model to investigate neural networks involving cold acclimation and to explore new ageing mechanisms.

Complete mitochondrial genome of Melia azedarach L., reveals two conformations generated by the repeat sequence mediated recombination

Melia azedarach is a species of enormous value of pharmaceutical industries. Although the chloroplast genome of M. azedarach has been explored, the information of mitochondrial genome (Mt genome) remains surprisingly limited. In this study, we used a hybrid assembly strategy of BGI short-reads and Nanopore long-reads to assemble the Mt genome of M. azedarach. The Mt genome of M. azedarach is characterized by two circular chromosomes with 350,142 bp and 290,387 bp in length, respectively, which encodes 35 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes. A pair of direct repeats (R1 and R2) were associated with genome recombination, resulting in two conformations based on the Sanger sequencing and Oxford Nanopore sequencing. Comparative analysis identified 19 homologous fragments between Mt and chloroplast genome, with the longest fragment of 12,142 bp. The phylogenetic analysis based on PCGs were consist with the latest classification of the Angiosperm Phylogeny Group. Notably, a total of 356 potential RNA editing sites were predicted based on 35 PCGs, and the editing events lead to the formation of the stop codon in the rps10 gene and the start codons in the nad4L and atp9 genes, which were verified by PCR amplification and Sanger sequencing. Taken together, the exploration of M. azedarach gap-free Mt genome provides a new insight into the evolution research and complex mitogenome architecture.

Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces

Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.

Genetic diversity of astroviruses detected in wild aquatic birds in Hong Kong

Wild waterfowl serve as a reservoir of some astroviruses. Fecal samples from wild waterfowl collected at Hong Kong's Marshes were tested using pan-astrovirus reverse transcription-PCR. Positive samples underwent subsequent host identification using DNA barcoding. Based on deduced partial sequences, noteworthy samples from three astrovirus groups (mammalian, avian and unclassified astroviruses) were further analyzed by next-generation sequencing. One sample of Avastrovirus 4 clade, MP22-196, had a nearly complete genome identified. The results of ORF2 phylogenetic analysis and genetic distance analysis indicate that Avastrovirus 4 is classified as a distinct subclade within Avastrovirus. MP22-196 has typical astrovirus genome characteristics. The unique characteristics and potential differences of this genome, compared to other avian astrovirus sequences, involve the identification of a modified sgRNA sequence situated near the ORF2 start codon, which precedes the ORF1b stop codon. Additionally, the 3' UTR of MP22-196 is shorter than other avian astroviruses. This study expands our understanding of the Avastrovirus 4 clade.

SARS-CoV-2 genomic surveillance of migrants arriving to Europe through the Mediterranean routes.

The implementation genomic-based surveillance on emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in low-income countries, which have inadequate molecular and sequencing capabilities and limited vaccine storage, represents a challenge for public health. To date, there is little evidence on molecular investigations of SARS-CoV-2 variants in areas where they might emerge. We report the findings of an experimental SARS-CoV-2 molecular surveillance programme for migrants, refugees, and asylum seekers arriving to Europe via Italy through the Mediterranean Sea. We descriptively analysed data on migrants collected at entry points in Sicily from February 2021 to May 2022. These entry points are integrated with a network of laboratories fully equipped for molecular analyses, which performed next-generation sequencing and used Nextclade and the Pangolin coronavirus disease 2019 (COVID-19) tools for clade/lineage assignment. We obtained 472 full-length SARS-CoV-2 sequences and identified 12 unique clades belonging to 31 different lineages. The delta variant accounted for 43.6% of all genomes, followed by clades 21D (Eta) and 20A (25.4% and 11.4%, respectively). Notably, some of the identified lineages (A.23.1, A.27, and A.29) predicted their introduction into the migration area. The mutation analysis allowed us to identify 617 different amino acid substitutions, 156 amino acid deletions, 7 stop codons, and 6 amino acid insertions. Lastly, we highlighted the geographical distribution patterns of some mutational profiles occurring in the migrants' countries of origin. Genome-based molecular surveillance dedicated to migrant populations from low-resource areas may be useful for forecasting new epidemiological scenarios related to SARS-CoV-2 variants or other emerging pathogens, as well as for informing the updating of vaccination strategies.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

N6-Methyladenosine (m6A) Sequencing Reveals Heterodera glycines-Induced Dynamic Methylation Promoting Soybean Defense.

Unraveling the intricacies of soybean cyst nematode (Heterodera glycines) race 4 resistance and susceptibility in soybean breeding lines-11-452 (highly resistant) and Dongsheng1 (DS1, highly susceptible)-was the focal point of this study. Employing cutting-edge N6-methyladenosine (m6A) and RNA sequencing techniques, we delved into the impact of m6A modification on gene expression and plant defense responses. Through the evaluation of nematode development in both resistant and susceptible roots, a pivotal time point (3 days postinoculation) for m6A methylation sequencing was identified. Our sequencing data exhibited robust statistics, successful soybean genome mapping, and prevalent m6A peak distributions, primarily in the 3' untranslated region and stop codon regions. Analysis of differential methylation peaks and differentially expressed genes revealed distinctive patterns between resistant and susceptible genotypes. In the highly resistant line (11-452), key resistance and defense-associated genes displayed increased expression coupled with inhibited methylation, encompassing crucial players such as R genes, receptor kinases, and transcription factors. Conversely, the highly susceptible DS1 line exhibited heightened expression correlated with decreased methylation in genes linked to susceptibility pathways, including Mildew Locus O-like proteins and regulatory elements affecting defense mechanisms. Genome-wide assessments, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses, and differential methylation peak/differentially expressed gene overlap emphasized the intricate interplay of m6A modifications, alternative splicing, microRNA, and gene regulation in plant defense. Protein-protein interaction networks illuminated defense-pivotal genes, delineating divergent mechanisms in resistant and susceptible responses. This study sheds light on the dynamic correlation between methylation, splicing, and gene expression, providing profound insights into plant responses to nematode infection.

Fluorescence labeling strategies for the study of ion channel and receptor cell surface expression: A comprehensive toolkit for extracellular labeling of TRPV1.

Regulation of ion channel expression on the plasma membrane is a major determinant of neuronal excitability, and identifying the underlying mechanisms of this expression is critical to our understanding of neurons. A critical aspect of measuring changes in ion channel expression is uniquely identifying ion channels located on the cell surface. To accomplish this goal we demonstrate two orthogonal strategies to label extracellular sites of the ion channel TRPV1 that minimally perturb the function of the channel: 1) We use the amber codon suppression technique to introduce a non-canonical amino acid (ncAA) with tetrazine click chemistry compatible with a trans-cyclooctene coupled fluorescent dye. 2) By inserting the circularly permutated HaloTag (cpHaloTag) in an extracellular loop of TRPV1, we incorporate a click-chemistry site for a chloroalkane-linked fluorescent dye of our choosing. Optimization of ncAA insertion sites was accomplished by screening residue positions between the S1 and S2 transmembrane domains with elevated missense variants in the human population, and we identified T468 as a rapid labeling site (~5 minutes) based on functional as well as biochemical assays in HEK293T/17 cells. After several rounds of adapting the linker lengths and backbone placement of cpHaloTag on the extracellular side of TRPV1, our efforts led to a channel construct that robustly expressed as a fully functional TRPV1exCellHalo fusion with intact wild-type gating properties. The TRPV1exCellHalo construct was used in a single molecule experiment to track TRPV1 on the cell surface and validate studies that show decreased mobility of the channel upon activation. The success of these extracellular label TRPV1 (exCellTRPV1) constructs as tools to track surface expression of the channel will shed significant light on the mechanisms regulating expression and provide a general scheme to introduce similar modifications to other cell surface receptors.

Sequence diversity of apidaecin-like peptides arresting the terminating ribosome.

The Proline-rich Antimicrobial Peptide (PrAMP) apidaecin (Api) inhibits translation by binding in the ribosomal nascent peptide exit tunnel, trapping release factors RF1 or RF2, and arresting ribosomes at stop codons. To explore the extent of sequence variations of the native 18-amino acid Api that allows it to preserve its activity, we screened a library of synthetic mutant Api genes expressed in bacterial cells, resulting in nearly 350000 peptide variants with multiple substitutions. By applying orthogonal negative and positive selection strategies, we identified a number of multi-substituted Api variants capable of arresting ribosomes at stop codons. Our findings underscore the critical contribution of specific amino acid residues of the peptide for its on-target function while significantly expanding the variety of PrAMPs acting on the terminating ribosome. Additionally, some of the tested synthesized multi-substituted Api variants exhibit improved antibacterial activity compared to that of the wild type PrAMP and may constitute the starting point to develop clinically useful antimicrobials.

JunctionCounts: comprehensive alternative splicing analysis and prediction of isoform-level impacts to the coding sequence.

Alternative splicing (AS) is emerging as an important regulatory process for complex biological processes. Transcriptomic studies therefore commonly involve the identification and quantification of alternative processing events, but the need for predicting the functional consequences of changes to the relative inclusion of alternative events remains largely unaddressed. Many tools exist for the former task, albeit each constrained to its own event type definitions. Few tools exist for the latter task; each with significant limitations. To address these issues we developed junctionCounts, which captures both simple and complex pairwise AS events and quantifies them with straightforward exon-exon and exon-intron junction reads in RNA-seq data, performing competitively among similar tools in terms of sensitivity, false discovery rate and quantification accuracy. Its partner utility, cdsInsertion, identifies transcript coding sequence (CDS) information via in silico translation from annotated start codons, including the presence of premature termination codons. Finally, findSwitchEvents connects AS events with CDS information to predict the impact of individual events to the isoform-level CDS. We used junctionCounts to characterize splicing dynamics and NMD regulation during neuronal differentiation across four primates, demonstrating junctionCounts' capacity to robustly characterize AS in a variety of organisms and to predict its effect on mRNA isoform fate.

The complete mitochondrial genome of Exoristobia philippinensis (Hymenoptera: Chalcidoidea: Encyrtidae) and phylogenetic analysis

Exoristobia philippinensis (Hymenoptera: Encyrtidae) is a worldwide parasitic wasp. This work presents the mitochondrial genome (mitogenome) of E. philippinensis for the first time. The complete mitochondrial genome of E. philippinensis was sequenced and annotated, which was 15,751 bp in length, and encoded 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and two ribosomal RNA genes (rRNAs). All 13 PCGs were initiated by the ATN (ATG, ATT, and ATA) codon, terminated with the stop codon TAA except for ND1 which ends with TAG. Phylogenetic analysis showed that E. philippinensis has a sister relationship with the genus Lamennaisia.

Complete mitochondrial genome and phylogenetic analysis of Cirriformia tentaculata (Annelida, Polychaeta, Cirratulidae) from Weihai, Shandong, China

Cirriformia species usually inhabit intertidal zones and deep-sea sediments. Their accurate identification has proven to be challenging. Here, we present the complete mitochondrial genome of one Cirriformia tentaculata_Montagu 1808 specimen collected from China. The total length of the complete mitochondrial sequence of C. tentaculata is 15,516 bp and consists of 13 protein-coding genes (PCGs), 23 tRNA genes, two rRNA genes, and an A + T rich region (64.20%). All PCGs begin with the typical ATN start codon, except for cox1, which uses TTG. TAA or TAG serve as termination codons for twelve PCGs, while nad5 terminates with an incomplete codon, T. The phylogenetic tree revealed a close relationship between C. tentaculata in this study, and Cirriformia cf. tentaculata and Timarete posteria from Korea. The information will assist in the future identification and understanding of this species and offers a novel point of reference for identifying Cirriformia species, and phylogenetic studies.

A case study of Duchenne muscular dystrophy caused by Alu element insertion in DMD gene and analysis of its gray-hair symptoms.

Duchenne muscular dystrophy (DMD) is a severe X-linked recessive genetic disorder caused by mutations in the DMD gene, which leads to a deficiency of the dystrophin protein. The main mutation types of this gene include exon deletions and duplications, point mutations, and insertions. These mutations disrupt the normal expression of dystrophin, ultimately leading to the disease. In this study, we reported a case of DMD caused by an insertion mutation in exon 59 (E59) of the DMD gene. The affected child exhibited significant abnormalities in related biochemical markers, early symptoms of DMD, and multiple gray hair. His mother and sister were carriers with slightly abnormal biochemical markers. The mother had mild clinical symptoms, while the sister had no clinical symptoms. Other family members were genetically and physically normal. Sequencing and sequence alignment revealed that the inserted fragment was an Alu element from the AluYa5 subfamily. This insertion produced two stop codons and a polyadenylate (polyA) tail. To understand the impact of this insertion on the DMD gene and its association with clinical symptoms, exonic splicing enhancer (ESE) prediction indicated that the insertion did not affect the splicing of E59. Therefore, we speculated that the insertion sequence would be present in the mRNA sequence of the DMD gene. The two stop codons and polyA tail likely terminate translation, preventing the production of functional dystrophin protein, which may be the mechanism leading to DMD. In addition to typical DMD symptoms, the child also exhibited premature graying of hair. This study reports, for the first time, a case of DMD caused by the insertion of an Alu element into the coding region of the DMD gene. This finding provides clues for studying gene mutations induced by Alu sequence insertion and expands the understanding of DMD gene mutations.

Comparative genomics reveals the evolutionary history of the unicellular eukaryote class Litostomatea and its adaptive evolution based on biochemical metabolic capacity

Abstract Ciliated protists are unicellular eukaryotic organisms characterized by their morphological diversity, ubiquitous distribution, and the important roles they play in a wide range of biological studies. The class Litostomatea is a morphologically diverse ciliate group that comprises hundreds of free-living and endosymbiotic species. Here, we sequenced 14 predatory litostomateans, i.e. 12 haptorians and two rhynchostomatians. A comparative analysis was performed with other published omics’ data on litostomateans. Our first phylogenomic analysis of litostomateans showed the monophyly of the subclasses Trichostomatia and Rhynchostomatia, the non-monophyly of the subclass Haptoria, and the monophyly of all orders and families that were analysed. Evolutionary history analysis suggested that Litostomatea diverged during the Late Neoproterozoic, the family Chaeneidae was the earliest diverging haptorian lineage, and the Rhynchostomatia probably separated from the order Lacrymariida (subclass Haptoria) during the Early Palaeozoic. Stop codon usage analysis of 28 litostomateans showed that they use TAA as the biased stop codon and reassign the other two stop codons (TAG and TGA) to code for amino acids. In addition, the preferred codons in the 14 newly sequenced litostomateans are strongly biased towards A/U bases in the third position, very probably due to the comparatively low GC content. Genes encoding carbohydrate-active enzymes (CAZymes) are more diversified in the endosymbiotic Trichostomatia than in the free-living predatory Rhynchostomatia and Haptoria, suggesting that trichostomes have the strongest capability of carbohydrate utilization. Notably, we found that three free-living litostomateans (Didinium sp.1, Myriokaryon sp., and Apodileptus visscheri) exhibit substantial differences from other free-living ciliates in terms of their number of CAZymes. Considering the potency and versatility of CAZymes in the degradation and biotransformation of carbohydates, we propose that the multifarious CAZymes in these three ciliates could be a survival strategy for nutrient acquisition and niche adaptation. Finally, the functional annotation of significantly expanded gene families in these three ciliates revealed their vigorous potency in biochemical metabolism. These findings will facilitate wider omic-scale phylogenetic analyses of Litostomatea and deepen our understanding of this group from an evolutionary standpoint.

ATR2C ala2 from Arabidopsis-infecting downy mildew requires 4 TIR-NLR immune receptors for full recognition.

Arabidopsis Col-0 RPP2A and RPP2B confer recognition of Arabidopsis downy mildew (Hyaloperonospora arabidopsidis [Hpa]) isolate Cala2, but the identity of the recognized ATR2Cala2 effector was unknown. To reveal ATR2Cala2, an F2 population was generated from a cross between Hpa-Cala2 and Hpa-Noks1. We identified ATR2Cala2 as a non-canonical RxLR-type effector that carries a signal peptide, a dEER motif, and WY domains but no RxLR motif. Recognition of ATR2Cala2 and its effector function were verified by biolistic bombardment, ectopic expression and Hpa infection. ATR2Cala2 is recognized in accession Col-0 but not in Ler-0 in which RPP2A and RPP2B are absent. In ATR2Emoy2 and ATR2Noks1 alleles, a frameshift results in an early stop codon. RPP2A and RPP2B are essential for the recognition of ATR2Cala2. Stable and transient expression of ATR2Cala2 under 35S promoter in Arabidopsis and Nicotiana benthamiana enhances disease susceptibility. Two additional Col-0 TIR-NLR (TNL) genes (RPP2C and RPP2D) adjacent to RPP2A and RPP2B are quantitatively required for full resistance to Hpa-Cala2. We compared RPP2 haplotypes in multiple Arabidopsis accessions and showed that all four genes are present in all ATR2Cala2-recognizing accessions.

Exploitation of enhanced prime editing for blocking aberrant angiogenesis

IntroductionAberrant angiogenesis plays an important part in the development of a variety of human diseases including proliferative diabetic retinopathy, with which there are still numerous patients remaining a therapeutically challenging condition. Prime editing (PE) is a versatile gene editing approach, which offers a novel opportunity to genetically correct challenging disorders. ObjectivesThe goal of this study was to create a dominant-negative (DN) vascular endothelial growth factor receptor (VEGFR) 2 by editing genomic DNA with an advanced PE system to block aberrant retinal angiogenesis in a mouse model of oxygen-induced retinopathy. MethodsAn advanced PE system (referred to as PE6x) was established within two lentiviral vectors, with one carrying an enhanced PE guide RNA and a canonical Cas9 nickase fused with an optimized reversal transcriptase, and the other conveying a nicking guide RNA and a DN-MLH1 to improve PE efficiency. Dual non-integrating lentiviruses (NILVs) produced with the two lentiviral PE6x vectors were then employed to create a mutation of VEGFR2 T17967A by editing the Mus musculus VEGFR2 locus in vitro and in vivo, leading to generation of a premature stop codon (TAG, K796stop) to produce DN-VEGFR2, to interfere with the wild type VEGFR2 which is essential for angiogenesis. ResultsNILVs targeting VEGFR2 delivered into cultured murine vascular endothelial cells led to 51.06 % VEGFR2 T17967A in the genome analyzed by next generation sequencing and the production of DN-VEGFR2, which was found to hamper VEGF-induced VEGFR2 phosphorylation, as demonstrated by Western blot analysis. Intravitreally injection of the dual NILVs into postnatal day 12 mice in a model of oxygen-induced retinopathy, led to production of retinal DN-VEGFR2 in postnatal day 17 mice which blocked retinal VEGFR2 expression and activation as well as abnormal retinal angiogenesis without interfering with retinal structure and function, as assessed by electroretinography, optical coherence tomography, fundus fluorescein angiography and histology. ConclusionDN-VEGFR2 resulted from editing genomic VEGFR2 using the PE6x system can be harnessed to treat intraocular pathological angiogenesis.

Clinical phenotypic and genetic analysis of syndrome families with EYA1 gene variants

Objective:To investigate the clinical phenotype of a family with branchio-oto syndrome （BOS） and to explore the genetic etiology of the syndrome in this family. Methods:Clinical data were collected from a child diagnosed with BOS and his family members. Genomic DNA was extracted from peripheral blood of the proband and his family members. Whole-exome sequencing was performed, and the mutation sites were verified and analyzed by Sanger sequencing. Results:The family consists of two generations with four members, three of whom exhibit the phenotype. Two members have hearing loss and bilateral preauricular fistulas and bilateral branchial cleft fistulas. One member has bilateral preauricular fistulas and bilateral branchial cleft fistulas. All of which were in line with the clinical diagnosis of gill ear syndrome, the inheritance mode of the family was autosomal dominant inheritance, genetic testing showed that all members of the family had c. 1744delC（p. L592Cfs*47） mutation in the EYA1 gene, while unaffected members have the wild-type allele at this locus. This mutation is a frameshift mutation, which results in the early appearance of the stop codon, and has not been reported so far. According to ACMG guidelines, the variant was preliminarily determined to be suspected pathogenic. Conclusion:The newly discovered EYA1c. 1744delC（p. L592Cfs*47） mutation in this family is the pathogenic mutant gene of the patients in this family, which further expands the mutation spectrum of EYA1 gene, gives us a new understanding of the disease, and provides an important reference for clinical diagnosis and genetic counseling.

Ash1 l loss-of-function results in structural birth defects and altered cortical development

Abstract The histone methyltransferase ASH1L plays a crucial role in regulating gene expression across various organ systems during development, yet its role in brain development remains largely unexplored. Over 130 individuals with autism harbour heterozygous loss-of-function ASH1L variants, and population studies confirm it as a high-risk autism gene. Previous studies on Ash1 l deficient mice have reported autistic-like behaviours and provided insights into the underlying neuropathophysiology. In this study, we used mice with a cre-inducible deletion of Ash1 l exon 4, which results in a frame shift and premature stop codon (p.V1693Afs*2). Our investigation evaluated the impact of Ash1 l loss-of-function on survival and craniofacial skeletal development. Using a tamoxifen-inducible cre strain, we targeted Ash1 l knockout early in cortical development (Emx1-Cre-ERT2; e10.5). Immunohistochemistry was utilized to assess cortical lamination, while EdU incorporation aided in birthdating cortical neurons. Additionally, single-cell RNA sequencing was employed to compare cortical cell populations and identify genes with differential expression. At e18.5, the proportion of homozygous Ash1 l germline knockout embryos appeared normal; however, no live Ash1 l null pups were present at birth (e18.5: n = 77, P = 0.90; p0: n = 41, P = 0.00095). Notably, Ash1l-/- exhibited shortened nasal bones (n = 31, P = 0.017). In the cortical-specific knockout model, SATB2 neurons showed increased numbers (n = 6/genotype, P = 0.0001) and were distributed through the cortical plate. Birthdating revealed generation of ectopically placed deep layer neurons that express SATB2 (e13.5 injection: n = 4/genotype, P = 0.0126). Single cell RNA sequencing revealed significant differences in gene expression between control and mutant upper layer neurons, leading to distinct clustering. Pseudotime analysis indicated that the mutant cluster followed an altered cell differentiation trajectory. This study underscores the essential role of Ash1 l in postnatal survival and normal craniofacial development. In the cortex, ASH1L exerts broad effects on gene expression and is indispensable for determining the fate of upper layer cortical neurons. These findings provide valuable insights into the potential mechanisms of ASH1L neuropathology, shedding light on its significance in neurodevelopmental disorders like autism.