Silent Mutations Research Articles

Thermosensitive pilus production by FCT type 3 Streptococcus pyogenes controlled by Nra regulator translational efficiency.

Summary Streptococcus pyogenes produces a diverse variety of pili in a serotype‐dependent manner and thermosensitive expression of pilus biogenesis genes was previously observed in a serotype M49 strain. However, the precise mechanism and biological significance remain unclear. Herein, the pilus expression analysis revealed the thermosensitive pilus production only in strains possessing the transcriptional regulator Nra. Experimental data obtained for nra deletion and conditional nra‐expressing strains in the background of an M49 strain and the Lactococcus heterologous expression system, indicated that Nra is a positive regulator of pilus genes and also highlighted the importance of the level of intracellular Nra for the thermoregulation of pilus expression. While the nra mRNA level was not significantly influenced by a temperature shift, the Nra protein level was concomitantly increased when the culture temperature was decreased. Intriguingly, a putative stem‐loop structure within the coding region of nra mRNA was a factor related to the post‐transcriptional efficiency of nra mRNA translation. Either deletion of the stem‐loop structure or introduction of silent chromosomal mutations designed to melt the structure attenuated Nra levels, resulting in decreased pilus production. Consequently, the temperature‐dependent translational efficacy of nra mRNA influenced pilus thermoregulation, thereby potentially contributing to the fitness of nra‐positive S. pyogenes in human tissues.

Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects

BackgroundGenetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes.ResultsHere, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.ConclusionsA single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.

Regulatory Role of ERG3 and Efg1 in Azoles-Resistant Strains of Candida albicans Isolated from Patients Diagnosed with Vulvovaginal Candidiasis.

Vulvovaginal candidiasis (VVC), caused by Candida albicans, affects women's health and life. We aimed to explore the correlation between ERG3 as well as Efg1 mutation/overexpression and azoles-resistance, and the correlation between ERG3 and Efg1 mRNA expression in C. albicans. First, C. albicans was isolated from clinical VVC patients. ERG3 and Efg1 mutations were detected by polymerase chain reaction (PCR) and sequencing, and the expression levels of these two genes were also identified by qRT-PCR. Correlations between mutation/overexpression of ERG3/Efg1 and azoles-resistance as well as ERG3 and Efg1 mRNA expression were analyzed. Based on the ERG3 sequencing, the results showed that there were 2 missense mutation sites, 1 nonsense mutation site, and 4 silent mutation sites, while 1 missense mutation sites, 1 nonsense mutation site, and 12 silent mutation sites were found in Efg1. Furthermore, the mRNA levels of ERG3 gene in the strains sensitive to FCA, ITR or VRC were higher than those in the strains resistant to FCA, ITR, VRC (P < 0.05). While for the mRNA levels of Efg1, susceptible strains were lower than resistant strains. Besides, there was a significant linear negative correlation between ERG3 and Efg1 mRNA expression (r = - 0.614, P < 0.001).

Effectiveness of integrated interpretation of exome and corresponding transcriptome data for detecting splicing variants of genes associated with autosomal recessive disorders

PurposePart of the weakness of exome analysis lies in the inability to detect aberrant splicing. An evaluation of the post-splicing mRNA sequence concurrently with genomic variants could improve the diagnostic rate. We aimed to investigate publicly available exome sequencing data and its matching transcriptomics data of phenotypically normal individuals to identify alternatively spliced variants from known genes associated with autosomal recessive disorders under the premise that some of the subjects could be carriers of such disorders. MethodsAberrant splicing events and their triggering genomic variants were detected with the aid of Bayesian network method “SAVNet” which was originally developed for cancer genomics. ResultsForty aberrant splicing events including exon skipping, the creation of a new splice site, and the use of a cryptic splice site in response to the disruption of the authentic site were detected in 1916 genes among 31 of the 179 subjects from the 1000 Genomes Project. The predicted effects on proteins were either frameshift mutations (30) or large in-frame insertions or deletions (10). Five missense mutations and 2 silent mutations were reinterpreted as triggering major changes in transcript sequences. The detection rate of provisionally truncating pathogenic variants increased by 19%, compared with a conventional exome analysis. ConclusionThe coupling interpretation of exome and transcriptome data enhances the performance of conventional exome analyses through the proper interpretation of intronic variants that are outside of the GT/AG splicing consensus sequences and also allows the reinterpretation of “missense” or “silent” substitutions that can indeed have drastic effects on splicing.

The predictive capability of immunohistochemistry and DNA sequencing for determining TP53 functional mutation status: a comparative study of 41 glioblastoma patients.

Tumor protein 53 (p53) regulates fundamental pathways of cellular growth and differentiation. Aberrant p53 expression in glioblastoma multiforme, a terminal brain cancer, has been associated with worse patient outcomes and decreased chemosensitivity. Therefore, correctly identifying p53 status in glioblastoma is of great clinical significance. p53 immunohistochemistry is used to detect pathological presence of the TP53 gene product. Here, we examined the relationship between p53 immunoreactivity and TP53 mutation status by DNA Sanger sequencing in adult glioblastoma. Of 41 histologically confirmed samples, 27 (66%) were immunopositive for a p53 mutation via immunohistochemistry. Utilizing gene sequencing, we identified only eight samples (20%) with TP53 functional mutations and one sample with a silent mutation. Therefore, a ≥10% p53 immunohistochemistry threshold for predicting TP53 functional mutation status in glioma is insufficient. Implementing this ≥10% threshold, we demonstrated a remarkably low positive-predictive value (30%). Furthermore, the sensitivity and specificity with ≥10% p53 immunohistochemistry to predict TP53 functional mutation status were 100% and 42%, respectively. Our data suggests that unless reliable sequencing methodology is available for confirming TP53 status, raising the immunoreactivity threshold would increase positive and negative predictive values as well as the specificity without changing the sensitivity of the immunohistochemistry assay.

Molecular Characterization of Leishmania RNA virus 2 in Leishmaniamajor from Uzbekistan.

Here we report sequence and phylogenetic analysis of two new isolates of Leishmania RNA virus 2 (LRV2) found in Leishmania major isolated from human patients with cutaneous leishmaniasis in south Uzbekistan. These new virus-infected flagellates were isolated in the same region of Uzbekistan and the viral sequences differed by only nineteen SNPs, all except one being silent mutations. Therefore, we concluded that they belong to a single LRV2 species. New viruses are closely related to the LRV2-Lmj-ASKH documented in Turkmenistan in 1995, which is congruent with their shared host (L. major) and common geographical origin.

Molecular characterization of high stearic acid soybean mutants and post-transcriptional control of GmSACPD genes in the mutant with a single nucleotide deletion

Soybean (Glycine max) oil normally contains 2%–4% stearic acid. Increased saturated fatty acid levels elevate the melting point of soybean oil and improve oxidative stability. Using X-ray irradiation, we isolated six high stearic acid mutants and investigated mutations in the gene encoding Δ9-stearoyl-ACP-desaturase (SACPD). Our analyses of the KK2 mutant revealed an approximately 155-kbp deletion in the GmSACPD-B region. We also identified a frameshift mutation in the KK24 mutant, corresponding with a base-pair deletion in the GmSACPD-C coding region. The other four mutants M2, M18, MM106, and MS827 also had 2.2–21.8-kbp deletions in the GmSACPD-C region. Thus, we designed co-dominant marker sets for KK2 and KK24 mutant alleles using multiplex polymerase chain reactions and the CEL I mismatch detection method, respectively. In addition, we quantified the degradation of GmSACPD-C mRNA with a single base-pair deletion and related it to nonsense-mediated mRNA decay and simultaneous reductions in GmSACPD-A and -B mRNA levels. This molecular mechanism may be exploited to introduce silent mutations in duplicate genes of soybean plants and other organisms.

A silent mutation in codon 228 results in a novel allele HLA-C*04:06:03 in an Ao-Naga individual from North-East India.

One nucleotide substitution (C>T) in codon 228 of HLA-C*04:06:01 results in the novel allele, HLA-C*04:06:03.

Unraveling the Role of Silent Mutation in the ω-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription.

The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, α2ββ’σω. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ω-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the mutant was shown to render the RNA polymerase defective in transcription initiation (ω6, N60D) and gave an insight on the function of ω in RNA polymerase. Serendipitously, we also obtained a silent mutant, and the mutant was found to be lethal during the isolation of toxic mutants. The primary focus of this study is to understand the mechanistic details of this lethality. Isolated ω shows a predominantly unstructured circular dichroism profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for this lack of function. Subsequently, we generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The CodonPlus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

Developmental Toxicity Assessment of Piperonyl Butoxide Exposure Targeting Sonic Hedgehog Signaling and Forebrain and Face Morphogenesis in the Mouse: An in Vitro and in Vivo Study.

Background:Piperonyl butoxide (PBO) is a pesticide synergist used in residential, commercial, and agricultural settings. PBO was recently found to inhibit Sonic hedgehog (Shh) signaling, a key developmental regulatory pathway. Disruption of Shh signaling is linked to birth defects, including holoprosencephaly (HPE), a malformation of the forebrain and face thought to result from complex gene–environment interactions.Objectives:The impact of PBO on Shh signaling in vitro and forebrain and face development in vivo was examined.Methods:The influence of PBO on Shh pathway transduction was assayed in mouse and human cell lines. To examine its teratogenic potential, a single dose of PBO () was administered by oral gavage to mice at gestational day 7.75, targeting the critical period for HPE. Gene–environment interactions were investigated using mice, which model human HPE-associated genetic mutations.Results:PBO attenuated Shh signaling in vitro through a mechanism similar to that of the known teratogen cyclopamine. In utero PBO exposure caused characteristic HPE facial dysmorphology including dose-dependent midface hypoplasia and hypotelorism, with a lowest observable effect level of . Median forebrain deficiency characteristic of HPE was observed in severely affected animals, whereas all effective doses disrupted development of Shh-dependent transient forebrain structures that generate cortical interneurons. Normally silent heterozygous Shh null mutations exacerbated PBO teratogenicity at all doses tested, including .Discussion:These findings demonstrate that prenatal PBO exposure can cause overt forebrain and face malformations or neurodevelopmental disruptions with subtle or no craniofacial dysmorphology in mice. By targeting Shh signaling as a sensitive mechanism of action and examining gene–environment interactions, this study defined a lowest observable effect level for PBO developmental toxicity in mice more than 30-fold lower than previously recognized. Human exposure to PBO and its potential contribution to etiologically complex birth defects should be rigorously examined. https://doi.org/10.1289/EHP5260

GENE EDITING AND MOLECULAR THERAPY: P.285Effective gene editing of the human DYSF founder mutation c.1624delG in patient-derived cells and generation of analogous "humanized" mouse models for in vivo treatment

Dysferlinopathy, also known as limb-girdle muscular dystrophy type 2B (LGMD2B), is caused by mutations in DYSF. It is one of the most frequent of > 40 untreatable muscular dystrophies leading to progressive muscle degeneration and wheel-chair-dependency. We generated hiPSC from two patients carrying a founder frameshift mutation (c.1624delG) in DYSF exon 44 that results in a complete absence of dysferlin protein and developed various gene editing approaches to repair this mutation using CRISPR/Cas9. First, using mutation-specific sgRNAs in combination with the enhanced specificity Cas9 variant eSpCas9(1.1), we were able to restore the mutant sequence to its wild-type form via homology directed repair without introducing any silent mutations in the repair template. Second, we used Cas9-induced NHEJ, in particular the indel signature bias of specific sgRNAs, to restore the DYSF reading frame in patient-derived cells with a high efficiency. We could readily demonstrate rescue of the re-framed dysferlin protein by western blot after editing. In parallel, we generated two related transgenic mouse lines, one with the murine Dysf exon 44 exchanged for the human wild-type DYSF exon 44 and the other containing the human c.1624delG founder mutation within the human DYSF exon 44. The latter lacks dysferlin protein and recapitulates the progressive muscular dystrophy phenotype seen in LGMD2B patients. Using these models, we are assessing ways to re-create in vivo our DYSF editing approaches directly on the human sequence, utilizing genetic tools that can be applied to patient cells.

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells.

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

Detection of novel mitochondrial mutations in cytochrome C oxidase subunit 1 (COX1) in patients with familial adenomatous polyposis (FAP).

Familial adenomatous polyposis (FAP) is an Autosomal dominant inherited disorder and a rare form‌ of colorectal cancer (CRC) that is characterized by the development of hundreds to thousands of adenomas in the rectum and colon. Mostly, cancers develop after the advent of the polyps. It appears in both sexes evenly, and the occurrence of the disease is in the second decade of life. Mitochondrial genome mutations have been reported with a variety of Tumors, but the precise role of these mutations in the pathogenicity and tumor progression is not exactly clear. Cytochrome c oxidase subunit I (COX1) is the terminal enzyme of the mitochondrial respiratory chain. The present study aims at assessing the occurrence of mtDNA mutations in COX1 gene in FAP patients and attempts to find out the cause and effect relationship between mitochondrial mutations and tumor progression. In this study, 56 FAP patients were investigated for the presence of the mutations in mitochondrial COX1 coding gene by PCR and sequencing analysis. All sequences that differed from the revised Cambridge Reference Sequence (rCRS) were classified as missense/ nonsense or silent mutations. Functional genomic studies using Bio-informatics tools were performed on the founded mutations to understand the downstream alterations in structure and function of protein. We identified 38 changes in the COX1 gene in patients with FAP symptoms. Most of them were heteroplasmic changes of missense type (25/38). Tree of the changes (G6145A, C6988A, and T7306G) were nonsense mutations and had not been reported in the literature before. Our results of bioinformatics predictions showed that the identified mutations can affect mitochondrial functions, especially if the conservative domain of the protein is concerned. Our findings indicate a high frequency of mtDNA mutations in all of the FAP cases compared to matched controls. These data significantly enhance our understanding of how such mutations contribute to cancer pathologies and develop the cancer treatment methods by new diagnostic biomarkers, and new drugs for gene therapy.

A novel method to rescue and culture duck Astrovirus type 1 in vitro

BackgroundReverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1.MethodsIn this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined.ResultsThe infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 μg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings.ConclusionsAn improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.

Genetic Diversity in FUB Genes of Fusarium oxysporum f. sp. cubense Suggests Horizontal Gene Transfer.

Fusaric acid (FA) is an important secondary metabolite of many Fusarium species and involved in the wilt symptoms caused in banana by Fusarium oxysporum f. sp. cubense (Foc). To investigate the evolution characteristics of the 12 Foc FA biosynthetic genes (FUB), coding sequences of the 12 FUB genes and three housekeeping genes, EF-1α/RPB1/RPB2 (translation elongation factor-1α/RNA polymerase II subunit I/RNA polymerase II subunit II), were subjected to genetic diversity analysis, phylogenetic analysis, recombination detection, and selective pressure analysis. The results of selective pressure analysis showed that the 15 genes were mainly subjected to negative selection. However, a significantly higher number of silent mutations, which could not be simply explained by selective pressure difference, were observed in the 12 FUB genes in Foc than in the three housekeeping genes. Infraspecies phylogeny and recombination detection analysis showed that significantly more horizontal gene transfer (HGT) events (normalized) had occurred in the FUB genes than in the three housekeeping genes. In addition, many of these events involved outgroup isolates and significantly increased the genetic diversity of FUB genes in Foc. The infraspecies phylogenetic analysis suggested that the polyphyletic phylogeny proposed for Foc requires further discussion, and the divergence of race 1, race 4, and the common ancestor of several F. oxysporum (Fo) isolates pathogenic to nonbanana plants should have diverged over a short period. Finally, our results suggest that the FUB genes in Fo should have benefited from HGT to gain a relatively high genetic diversity to respond to different host plants and environments despite mainly being subject to negative selection.

Phenotypically Silent Bone Morphogenetic Protein Receptor 2 Mutations Predispose Rats to Inflammation-Induced Pulmonary Arterial Hypertension by Enhancing the Risk for Neointimal Transformation.

Bmpr2 (bone morphogenetic protein receptor 2) mutations are critical risk factors for hereditary pulmonary arterial hypertension (PAH) with approximately 20% of carriers developing disease. There is an unmet medical need to understand how environmental factors, such as inflammation, render Bmpr2 mutants susceptible to PAH. Overexpressing 5-LO (5-lipoxygenase) provokes lung inflammation and transient PAH in Bmpr2+/- mice. Accordingly, 5-LO and its metabolite, leukotriene B4, are candidates for the second hit. The purpose of this study was to determine how 5-LO-mediated pulmonary inflammation synergized with phenotypically silent Bmpr2 defects to elicit significant pulmonary vascular disease in rats. Monoallelic Bmpr2 mutant rats were generated and found phenotypically normal for up to 1 year of observation. To evaluate whether a second hit would elicit disease, animals were exposed to 5-LO-expressing adenovirus, monocrotaline, SU5416, SU5416 with chronic hypoxia, or chronic hypoxia alone. Bmpr2-mutant hereditary PAH patient samples were assessed for neointimal 5-LO expression. Pulmonary artery endothelial cells with impaired BMPR2 signaling were exposed to increased 5-LO-mediated inflammation and were assessed for phenotypic and transcriptomic changes. Lung inflammation, induced by intratracheal delivery of 5-LO-expressing adenovirus, elicited severe PAH with intimal remodeling in Bmpr2+/- rats but not in their wild-type littermates. Neointimal lesions in the diseased Bmpr2+/- rats gained endogenous 5-LO expression associated with elevated leukotriene B4 biosynthesis. Bmpr2-mutant hereditary PAH patients similarly expressed 5-LO in the neointimal cells. In vitro, BMPR2 deficiency, compounded by 5-LO-mediated inflammation, generated apoptosis-resistant and proliferative pulmonary artery endothelial cells with mesenchymal characteristics. These transformed cells expressed nuclear envelope-localized 5-LO consistent with induced leukotriene B4 production, as well as a transcriptomic signature similar to clinical disease, including upregulated nuclear factor Kappa B subunit (NF-κB), interleukin-6, and transforming growth factor beta (TGF-β) signaling pathways. The reversal of PAH and vasculopathy in Bmpr2 mutants by TGF-β antagonism suggests that TGF-β is critical for neointimal transformation. In a new 2-hit model of disease, lung inflammation induced severe PAH pathology in Bmpr2+/- rats. Endothelial transformation required the activation of canonical and noncanonical TGF-β signaling pathways and was characterized by 5-LO nuclear envelope translocation with enhanced leukotriene B4 production. This study offers an explanation of how an environmental injury unleashes the destructive potential of an otherwise silent genetic mutation.

Little evidence of adaptation potential to ocean acidification in sea urchins living in "Future Ocean" conditions at a CO2 vent.

Ocean acidification (OA) can be detrimental to calcifying marine organisms, with stunting of invertebrate larval development one of the most consistent responses. Effects are usually measured by short‐term, within‐generation exposure, an approach that does not consider the potential for adaptation. We examined the genetic response to OA of larvae of the tropical sea urchin Echinometra sp. C. raised on coral reefs that were either influenced by CO2 vents (pH ~ 7.9, future OA condition) or nonvent control reefs (pH 8.2). We assembled a high quality de novo transcriptome of Echinometra embryos (8 hr) and pluteus larvae (48 hr) and identified 68,056 SNPs. We tested for outlier SNPs and functional enrichment in embryos and larvae raised from adults from the control or vent sites. Generally, highest F ST values in embryos were observed between sites (intrinsic adaptation, most representative of the gene pool in the spawned populations). This comparison also had the highest number of outlier loci (40). In the other comparisons, classical adaptation (comparing larvae with adults from the control transplanted to either the control or vent conditions) and reverse adaptation (larvae from the vent site returned to the vent or explanted at the control), we only observed modest numbers of outlier SNPs (6–19) and only enrichment in two functional pathways. Most of the outliers detected were silent substitutions without adaptive potential. We conclude that there is little evidence of realized adaptation potential during early development, while some potential (albeit relatively low) exists in the intrinsic gene pool after more than one generation of exposure.

Successful establishment of a reverse genetic system for QX-type infectious bronchitis virus and technical improvement of the rescue procedure

In this study, a pathogenic avian infectious bronchitis virus (IBV) QX-type strain YN was successfully rescued by vaccinia virus based reverse genetic technology. Ten fragments contiguously spanning the complete IBV genome were amplified and cloned into the vaccinia virus genome by homologous recombination. The full-length genomic cDNA was transcribed in vitro, and its transcript was transfected into BHK-21/N cells that could stably express IBV N protein. At 48 h post transfection, the culture medium was harvested and inoculated into 10-day-old specific-pathogen-free embryonated chicken eggs to replicate the rescued virus. This strategy was chosen to facilitate the rescue procedure and to ensure that the recombinant rYN virus will not require any cell culture adaptations. After only one in ovo passage, the recombinant YN virus (rYN) was successfully recovered and confirmed to possess the introduced silent marker mutation in its genome. Biological characteristics of rYN such as the EID50, TCID50, replication in ovo, and replication kinetcs in vitro were tested and all were similar to its parental strain YN. Our findings demonstrate the successful construction of highly-pathogenic QX-type IBV using a modified rescue procedure, allowing for future studies of the molecular biology and pathogenicity of IBV field strains.

A CRISPR/Cas9 method to generate heterozygous alleles in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a genetically facile organism, yet multiple CRISPR/Cas9 techniques are widely used to edit its genome more efficiently and cost effectively than conventional methods. The absence of selective markers makes CRISPR/Cas9 editing particularly useful when making mutations within genes or regulatory sequences. Heterozygous mutations within genes frequently arise in the winners of evolution experiments. The genetic dissection of heterozygous alleles can be important to understanding gene structure and function. Unfortunately, the high efficiency of genome cutting and repair makes the introduction of heterozygous alleles by standard CRISPR/Cas9 technique impossible. To be able to quickly and reliably determine the individual phenotypes of the thousands of heterozygous mutations that can occur during directed evolutions is of particular interest to industrial strain improvement research. In this report, we describe a CRISPR/Cas9 method that introduces specific heterozygous mutations into the S.cerevisiae genome. This method relies upon creating silent point mutations in the protospacer adjacent motif site or removing the protospacer adjacent motif site entirely to stop the multiple rounds of genome editing that prevent heterozygous alleles from being generated. This technique should be able to create heterozygous alleles in other diploid yeasts and different allelic copy numbers in polyploid cells.

A Specific Macula-Predominant Retinal Phenotype Is Associated With the CDHR1 Variant c.783G>A, a Silent Mutation Leading to In-Frame Exon Skipping.

To report the clinical and molecular findings in patients with retinal dystrophy associated with the c.783G>A variant in CDHR1. The retinal phenotype of 10 patients with CDHR1-related retinopathy was characterized by multimodal imaging including color fundus photography, optical coherence tomography (OCT), and blue- and near-infrared fundus autofluorescence imaging. Functional testing included electroretinography, visual acuity, and visual field testing. Six patients homozygous for the c.783G>A variant in CDHR1 showed a retinal phenotype resembling central areolar choroidal dystrophy (CACD) on multimodal imaging. Retinal function outside an area of slowly progressive macular atrophy remained relatively preserved. In contrast, biallelic severe/truncating CDHR1 mutations result in retina-wide retinal degeneration in addition to macular atrophy, with overall severely reduced retinal function. Patients compound heterozygous for the c.783G>A mutation and a truncating mutation in CDHR1 showed an intermediate phenotype. All patients except one with biallelic severe CDHR1 mutations were asymptomatic in the first four decades of life, irrespective of their individual CDHR1 mutations. Analysis of blood RNA from patients with the c.783G>A variant revealed in-frame skipping of exon 8 in vivo, predicting a partial deletion of CDHR1 ectodomains 2 and 3. Patients with biallelic c.783G>A CDHR1 mutations demonstrate a retinal phenotype consistent with autosomal recessive CACD. The apparently silent dbSNP-annotated c.783G>A CDHR1 variant (rs147346345) has a relatively high minor allele frequency (0.31%), with homozygous individuals annotated in the general population, and it may therefore have been disregarded in many next-generation sequencing (NGS)-based studies. The differential diagnosis includes PRPH2-associated CACD and age-related macular degeneration.