CRISPR/Cas9-mediated generation of biallelic F0 anemonefish (Amphiprion ocellaris) mutants

Genomic manipulation is a useful approach for elucidating the molecular pathways underlying aspects of development, physiology, and behaviour. However, a lack of gene-editing tools appropriated for use in reef fishes has meant the genetic underpinnings for many of their unique traits remain to be investigated. One iconic group of reef fishes ideal for applying this technique are anemonefishes (Amphiprioninae) as they are widely studied for their symbiosis with anemones, sequential hermaphroditism, complex social hierarchies, skin pattern development, and vision, and are raised relatively easily in aquaria. In this study, we developed a gene-editing protocol for applying the CRISPR/Cas9 system in the false clown anemonefish, Amphiprion ocellaris. Microinjection of zygotes was used to demonstrate the successful use of our CRISPR/Cas9 approach at two separate target sites: the rhodopsin-like 2B opsin encoding gene (RH2B) involved in vision, and Tyrosinase-producing gene (tyr) involved in the production of melanin. Analysis of the sequenced target gene regions in A. ocellaris embryos showed that uptake was as high as 73.3% of injected embryos. Further analysis of the subcloned mutant gene sequences combined with amplicon shotgun sequencing revealed that our approach had a 75% to 100% efficiency in producing biallelic mutations in F0 A. ocellaris embryos. Moreover, we clearly show a loss-of-function in tyr mutant embryos which exhibited typical hypomelanistic phenotypes. This protocol is intended as a useful starting point to further explore the potential application of CRISPR/Cas9 in A. ocellaris, as a platform for studying gene function in anemonefishes and other reef fishes.

Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice

Background This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53em2Hwl/Korl and C57BL/6-Trp53em1Hwl/Korl knockout (KO) mice over 16 weeks. Results Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53em2Hwl/Korl KO mice, but were not detected in C57BL/6-Trp53em1Hwl/Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53em2Hwl/Korl KO mice. Conclusions Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice over 16 weeks.

Rice Brittle Culm19 Encoding Cellulose Synthase Subunit CESA4 Causes Dominant Brittle Phenotype But has No Distinct Influence on Growth and Grain Yield

Background Mechanical strength is a crucial agronomic trait in rice (Oryza sativa), and brittle mutants are thought suitable materials to investigate the mechanism of cell wall formation. So far, almost all brittle mutants are recessive, and most of them are defected in multiple morphologies and/or grain yield, limiting their application in hybrid breeding and in rice straw recycling. Results We identified a semi-dominant brittle mutant Brittle culm19 (Bc19) isolated from the japonica variety Nipponbare through chemical mutagenesis. The mutant showed the same apparent morphologies and grain yield to the wild type plant except for its weak mechanical strength. Its development of secondary cell wall in sclerenchyma cells was affected, along with reduced contents of cellulose, hemicellulose, lignin and sugars in culms and leaves. Positional cloning suggested that the Bc19 gene was allelic to OsCESA4, encoding one of the cellulose synthase A (CESA) catalytic subunits. In this mutant, a C-to-T substitution occurred in the coding sequence of BC19, causing the P507S missense mutation in its encoded product, which was located in the second cytoplasmic region of the OsCESA4 protein. Furthermore, introducing mutant gene Bc19 into the wild-type plant resulted in brittle plants, confirming that the P507S point mutation in OsCESA4 protein was responsible for the semi-dominant brittle phenotype of Bc19 mutant. Reverse correlation was revealed between cellulose contents and expression levels of mutant gene Bc19 among the homozygous mutant, the hybrid F1 plant, and the Bc19 overexpression transgenic plants, implying that gene Bc19 might affect cellulose synthesis in a dosage-dependent manner. Conclusions Bc19, a semi-dominant brittle mutant allele of gene OsCESA4, was identified using map-based cloning approach. The mutated protein of Bc19 possessing the P507S missense mutation behaved in a dosage-dependent semi-dominant manner. Unique brittle effect on phenotype and semi-dominant genetic quality of gene Bc19 indicated its potential application in grain-straw dual-purpose hybrid rice breeding.

Distribution of Pfmdr 1 and Kelch 13 Genes Among the Children 5 Years and Below Attending a Secondary Health Facility, South-West, Nigeria.

Background: Malaria is a major public health concern in some part of the world especially in the tropical Africa where children are more vulnerable. The occurrence of resistant gene in Plasmodium falciparum to some antimalarial drugs could increase the malaria morbidity and mortality among the children. The study evaluates the distribution of P. falciparum resistant kelch protein gene on chromosome 13 (PfKelch 13) and multidrug resistant (Pfmdr1) mutant genes among children aged five years and below who attended Mother and Child Hospital, Akure, Nigeria. Methods: Thin and thick smears were prepared from the blood collected aseptically through venepuncture from five hundred (500) children (age 5years and below). Two hundred (200) malaria positive samples were randomly selected from the 500 samples for PCR analysis to detect Pfmdr1 and Kelch 13 mutant genes from the positive samples. Discussion: The results showed that of the 500 respondents who gave their consent to participate in the study, 288 (57.6%) were males while 212 (42.4%) were females. The distribution of Pfmdr1 are; mixed group (mutant/wild) 38.5% (77/200), mutant gene 35.5% (71/200), wild gene 20.5% (41/200) and the resistant genes were absent in 5.5% (11/200) of the infected children. The mixed group of Pfmdr1 gene was higher among infants (51.9%), male (44.3%), children with birth order 4 (60.0%) and children that have blood group B (51.3%), however, there is no significant difference in the distribution of Pfmdr1 between gender (χ2 = 0.634, df = 1, p>0.05). There was a point mutation in the codon position 557 where the amino acid Alanine was replaced by Serine in the PfK13. The research revealed high prevalence of Pfmdr1 mutant genes and point mutation in the PfK13 gene of P. falciparum among children which may be as a result of treatment of malaria with different antimalarial drugs which the parasite has developed resistance against. It is therefore important to administer other malaria drugs apart from the drugs the parasite has developed resistance against.

PREDICTING THE RISK OF DEVELOPING ABDOMINAL ADHESIONS IN CHILDREN DEPENDING ON ACETYLATION GENOTYPE

Objective. To determine the predictive value of the genetic polymorphism of the N-arylacetyltransferase-2 (NAT-2) gene for assessing the risk of postoperative adhesive intestinal obstruction in children. Methods. In all children (36 children with adhesive intestinal obstruction (main group) and 35 planned patients (comparative group)) the acetylation genotype was studied by detecting point mutations of the NAT-2 gene using allele-specific amplification method with analysis of apolymerase chain reaction-restrictionfragmentlengthpolymorphism. Results. The study of the frequency of mutations at position 481 revealed the greatest diversity of the studied variants of genotypes: 33.3% of the children of the main group were homozygous for the wild-type gene, 44.4% were heterozygotes, 22.2% of patients had a homozygous mutant gene. According to the NAT-2 * 6A genotype (G 590 - A), the majority of patients (55.6%) were heterozygotes, 44.4% were homozygotes with the wild-type of the gene. Not a single case of mutation at position 857 has been identified. Among the children of the main group, the share of «fast» acetylators was 69.4%, in the comparison group - 40.0% (χ²=6.215; p=0.013). The development of postoperative adhesive intestinal obstruction in children with the “fast” acetylation genotype occurred in the absence of clinical and anamnestic risk factors and was characterized by a greater severity and prevalence of intra-abdominal adhesive process (PAI was (14.8±1.8) and (8.1±2.4 ), respectively). Conclusion. The risk of developing postoperative adhesive complications in children can be done preventively by determining the genetic polymorphism of the N-acetyltransferase-2 gene. The risk group for developing adhesive intestinal obstruction is made up of children who are the carriers of NAT-2 alleles and correspond to the genotype of «fast» and «moderate» acetylation. Children who are «fast» acetylators have a more pronounced intra-abdominal adhesion process and a higher risk of complications associated with excessive adhesion even in the absence of other risk factors. What this paper adds N-acetyltransferase 2 (NAT2) gene polymorphism as a prognostic risk factor for the development of adhesive intestinal obstruction in children has been studied. Children as the carriers of the «fast» acetylator genotype have a higher risk of developing intra-abdominal adhesions and therefore require more comprehensive preventive measures at all stages of possible influence.

An Exon Skipping in CRS1 Is Associated with Perturbed Chloroplast Development in Maize

Chloroplasts of higher plants are semi-autonomous organelles that perform photosynthesis and produce hormones and metabolites. They play crucial roles in plant growth and development. Although many seedling-lethal nuclear genes or regulators required for chloroplast development have been characterized, the understanding of chloroplast development is still limited. Using a genetic screen, we isolated a mutant named ell1, with etiolated leaves and a seedling-lethal phenotype. Analysis by BN-PAGE and transmission electron microscopy revealed drastic morphological defects of chloroplasts in ell1 mutants. Genetic mapping of the mutant gene revealed a single mutation (G-to-A) at the 5′ splice site of intron 5 in CRS1, resulting in an exon skipping in CRS1, indicating that this mutation in CRS1 is responsible for the observed phenotype, which was further confirmed by genetic analysis. The incorrectly spliced CRS1 failed to mediate the splicing of atpF intron. Moreover, the quantitative analysis suggested that ZmCRS1 may participate in chloroplast transcription to regulate the development of chloroplast. Taken together, these findings improve our understanding of the ZmCRS1 protein and shed new light on the regulation of chloroplast development in maize.

Characteristics of a Novel ATP2B3 K416_F418delinsN Mutation in a Classical Aldosterone-Producing Adenoma

In patients with primary aldosteronism (PA), the prevalence of ATP2B3 mutation is rare. The aim of this study is to report a novel ATP2B3 mutation in a PA patient. Based on our tissue bank of aldosterone-producing adenomas (APA), we identified a novel somatic ATP2B3 K416_F418delinsN mutation. The affected individual was a 53 year-old man with a 4 year history of hypertension. Computed tomography (CT) showed bilateral adrenal masses of 1.6 (left) and 0.5 cm (right) in size. An adrenal venous sampling (AVS) showed a lateralization index (LI) of 2.2 and a contralateral suppression index (CLS) of 0.12; indicating left functional predominance. After a left unilateral adrenalectomy, he achieved partial biochemical and hypertension–remission. This classical adenoma harbored a novel ATP2B3 K416_F418delinsN somatic mutation, which is a deletion from nucleotides 1248 to 1253. The translated amino acid sequence from 416 to 418, reading as lysine-phenylalanine-phenylalanine, was deleted; however, an asparagine was inserted due to merging of residual nucleotide sequences. The CYP11B2 immunohistochemistry staining demonstrated strong immunoreactivity in this classical adenoma. The ATP2B3 K416_F418delinsN mutation is a functional mutation in APA, since HAC15 cells, a human adrenal cell line, transfected with the mutant gene showed increased CYP11B2 expression and aldosterone production.

SYN1 Mutation Causes X-Linked Toothbrushing Epilepsy in a Chinese Family

Toothbrushing epilepsy is a rare form of reflex epilepsy (RE) with sporadic incidence. To characterize the genetic profile of reflex epilepsy patients with tooth brushing-induced seizures in a Chinese family. Solo clinical whole-exome sequencing (WES) of the proband, a 37-year-old Chinese man, was performed to characterize the genetic etiology of toothbrushing epilepsy. Mutations in the maternal X-linked synapsin 1 (SYN1) identified in the proband and his family members were confirmed by Sanger sequencing. The pathogenicity of these mutations was determined using in silico analysis. The proband had four episodes of toothbrushing-induced seizures. The semiology included nausea, twitching of the right side of the mouth and face, followed by a generalized tonic-clonic seizure (GTCS). The proband's elder maternal uncle had three toothbrushing-induced epileptic seizures at the age of 26. The proband's younger maternal uncle had no history of epileptic seizures but had a learning disability and aggressive tendencies. We identified a deleterious nonsense mutation, c.1807C>T (p.Q603Ter), in exon 12 of the SYN1 gene (NM_006950), which can result in a truncated SYN1 phosphoprotein with altered flexibility and hydropathicity. This novel mutation has not been reported in the 1000G, EVS, ExAC, gnomAD, or HGMD databases. We identified a novel X-linked SYN1 exon 12 mutant gene in a Chinese family with toothbrushing epilepsy. Our findings provide novel insights into the mechanism of this complex form of reflex epilepsy that could potentially be applied in disease diagnosis.

Rice Brittle Culm19 Encoding Cellulose Synthase Subunit CesA4 Causes Dominant Brittle Phenotype But Has No Distinct Influence on Growth and Grain Yield

Background: Mechanical strength is a crucial agronomic trait in rice (Oryza sativa), and brittle mutants are thought suitable materials to investigate the mechanism of cell wall formation. So far, almost all brittle mutants are recessive, and most of them are defected in multiple morphologies and/or grain yield, limiting their application in hybrid breeding and in rice straw recycling.Results: We identified a semi-dominant brittle mutant Brittle culm19 (Bc19) isolated from the japonica variety Nipponbare through chemical mutagenesis. The mutant showed the same apparent morphologies and grain yield to the wild type plant except for its weak mechanical strength. Its development of secondary cell wall in sclerenchyma cells was affected, along with reduced contents of cellulose, hemicellulose, lignin and sugars in culms and leaves. Positional cloning suggested that the Bc19 gene was allelic to OsCesA4, encoding one of the cellulose synthase A (CesA) catalytic subunits. In this mutant, a C-to-T substitution occurred in the coding sequence of BC19, causing the P507S missense mutation in its encoded product, which was located in the second cytoplasmic region of the OsCesA4 protein. Furthermore, introducing mutant gene Bc19 into the wild-type plant resulted in brittle plants, confirming that the P507S point mutation in OsCesA4 protein was responsible for the semi-dominant brittle phenotype of Bc19 mutant. Reverse correlation was revealed between cellulose contents and expression levels of mutant gene Bc19 among the homozygous mutant, the hybrid F1 plant, and the Bc19 overexpression transgenic plants, implying that gene Bc19 might affect cellulose synthesis in a dosage-dependent manner.Conclusions: Bc19, a semi-dominant brittle mutant allele of gene OsCesA4, was identified using map-based cloning approach. The mutated protein of Bc19 possessing the P507S missense mutation behaved in a dosage-dependent semi-dominant manner. Unique brittle effect on phenotype and semi-dominant genetic quality of gene Bc19 indicated its potential application in grain-straw dual-purpose hybrid rice breeding.

Huntington's disease gene therapy and nanomedicines may be available shortly

Although Huntington's disease-based gene mutation has long been established, pathophysiology from mutant gene to aberrant aggregation, neurotoxicity, metabolic dysfunction, and neuroimmunological dysfunction is convoluted and incomplete. We investigated innovative disease-modifying drugs that target various established pathogenic stages, from gene to RNA to protein pathways. Several of these medicines are being explored in human clinical trials, while others promise preclinical findings in vivo. The ultimate goal of these new treatments is to boost survival, function and quality of life and perhaps cure Huntington's disease. Gene therapies have tremendous potential to correct or alter the underlying defective trinucleotide expansion of DNA, which would prevent all erroneous downstream processes. In early pathogenic intervention, RNA-focused treatments also promise to avoid downstream HTT toxicity activation. For those targeting DNA and RNA levels, invasive administration (often necessitating direct intraparenychmal or intrathecal distribution) and potential off-target effects with accidental downregulation of non-HD-related genes or transcripts are typical treatment restrictions. Downstream therapies that are important include those that reduce MSN atrophy and neuroinflammation.Other novel preclinical development procedures include the use of endogenous, adult glial cells to repair striatal neurons and mutant HTT protein immunization. These drugs may be particularly relevant for usage in people with advanced disease if DNA or RNA-targeted therapy can not reverse previous neurotoxicity. These unique and new therapy approaches have generated hopes that Huntington's disease-modifying medicines may soon become reality.