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Stem Cell Research May 2021When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point...
When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-Puro cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-Puro cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.
Topics: CRISPR-Cas Systems; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Homologous Recombination; Humans; Induced Pluripotent Stem Cells; Mutation; Point Mutation
PubMed: 33857832
DOI: 10.1016/j.scr.2021.102332 -
Journal of Clinical Neuromuscular... Dec 2022Dystrophinopathies result from mutations to the DMD gene. We report 5 boys in 3 families with heterogenous phenotypes due to a point mutation in the DMD gene: a... (Review)
Review
Dystrophinopathies result from mutations to the DMD gene. We report 5 boys in 3 families with heterogenous phenotypes due to a point mutation in the DMD gene: a hemizygous tyrosine-to-cysteine change in exon 15 (c.1724T>C) resulting in an amino acid substitution of leucine to proline at codon 575. This mutation has been reported before, with at least 3 prior patients presenting with similar clinical findings of myalgia, myoglobinuria, and occasional muscle cramping. The mutation on DMD c.1724T>C (p.Leu575Pro) is listed in the Clinvar database as a variant of unknown significance. Our report provides contributing evidence that this alteration should be classified as pathogenic.
Topics: Humans; Muscular Dystrophy, Duchenne; Dystrophin; Point Mutation; Myalgia; Exons
PubMed: 36409343
DOI: 10.1097/CND.0000000000000413 -
Scientific Reports Feb 2023An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6...
An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6 interacts with even higher affinity with ceramide phosphoethanolamine (CPE), a sphingolipid that dominates in invertebrate cell membranes. In the presence of pleurotolysin B, a protein bearing the membrane-attack complex/perforin domain, OlyA6 forms pores in insect midgut cell membranes and acts as a potent bioinsecticide. It has been shown that a point mutation of glutamate 69 to alanine (E69A) allows OlyA6 to bind to cholesterol-free sphingomyelin. Using artificial lipid membranes and mammalian MDCK cells, we show that this mutation significantly enhances the interaction of OlyA6 with sphingomyelin and CPE, and allows recognition of these sphingolipids even in the absence of cholesterol. Our results suggest that OlyA6 mutant E69A could serve as complementary tool to detect and study cholesterol-associated and free sphingomyelin or CPE in membranes. However, the mutation does not improve the membrane-permeabilizing activity after addition of pleurotolysin B, which was confirmed in toxicity tests on insect and mammalian cell lines, and on Colorado potato beetle larvae.
Topics: Animals; Sphingomyelins; Point Mutation; Cell Membrane; Cholesterol; Insecta; Membranes, Artificial; Mammals
PubMed: 36750638
DOI: 10.1038/s41598-023-28949-7 -
Nature Biomedical Engineering Jan 2020
Topics: Adenine; Animals; Mice; Point Mutation; Tyrosinemias
PubMed: 31937937
DOI: 10.1038/s41551-019-0489-x -
Gastrointestinal Endoscopy Mar 2021
Topics: Adenocarcinoma; Biomarkers; High-Throughput Nucleotide Sequencing; Humans; Pancreatic Neoplasms; Point Mutation
PubMed: 33583519
DOI: 10.1016/j.gie.2020.07.024 -
Scientific Reports Jun 2023Protein-protein interactions (PPIs) play a critical role in all biological processes. Menin is tumor suppressor protein, mutated in multiple endocrine neoplasia type 1...
Protein-protein interactions (PPIs) play a critical role in all biological processes. Menin is tumor suppressor protein, mutated in multiple endocrine neoplasia type 1 syndrome and has been shown to interact with multiple transcription factors including (RPA2) subunit of replication protein A (RPA). RPA2, heterotrimeric protein required for DNA repair, recombination and replication. However, it's still remains unclear the specific amino acid residues that have been involved in Menin-RPA2 interaction. Thus, accurately predicting the specific amino acid involved in interaction and effects of MEN1 mutations on biological systems is of great interests. The experimental approaches for identifying amino acids in menin-RPA2 interactions are expensive, time-consuming, and challenging. This study leverages computational tools, free energy decomposition and configurational entropy scheme to annotate the menin-RPA2 interaction and effect on menin point mutation, thereby proposing a viable model of menin-RPA2 interaction. The menin-RPA2 interaction pattern was calculated on the basis of different 3D structures of menin and RPA2 complexes, constructed using homology modeling and docking strategy, generating three best-fit models: Model 8 (- 74.89 kJ/mol), Model 28 (- 92.04 kJ/mol) and Model 9 (- 100.4 kJ/mol). The molecular dynamic (MD) was performed for 200 ns and binding free energies and energy decomposition analysis were calculated using Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) in GROMACS. From binding free energy change, model 8 of Menin-RPA2 exhibited most negative binding energy of - 205.624 kJ/mol, followed by model 28 of Menin-RPA2 with - 177.382 kJ/mol. After S606F point mutation in Menin, increase of BFE (ΔG) by - 34.09 kJ/mol in Model 8 of mutant Menin-RPA2 occurs. Interestingly, we found a significant reduction of BFE (ΔG) and configurational entropy by - 97.54 kJ/mol and - 2618 kJ/mol in mutant model 28 as compared the o wild type. Collectively, this is the first study to highlight the configurational entropy of protein-protein interactions thereby strengthening the prediction of two significant important interaction sites in menin for the binding of RPA2. These predicted sites could be vulnerable for structural alternation in terms of binding free energy and configurational entropy after missense mutation in menin.
Topics: Humans; Point Mutation; Mutation; Transcription Factors; Binding Sites; Multiple Endocrine Neoplasia Type 1; Amino Acids; Replication Protein A
PubMed: 37291166
DOI: 10.1038/s41598-023-35599-2 -
STAR Protocols Sep 2023Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been...
Chimeric mouse models have recently been developed to study human microglia in vivo. However, widespread engraftment of donor microglia within the adult brain has been challenging. Here, we present a protocol to introduce the G795A point mutation using CRISPR-Cas9 into the CSF1R locus of human pluripotent stem cells. We also describe an optimized microglial differentiation technique for transplantation into newborn or adult recipients. We then detail pharmacological paradigms to achieve widespread and near-complete engraftment of human microglia. For complete details on the use and execution of this protocol, please refer to Chadarevian et al. (2023)..
Topics: Adult; Animals; Mice; Infant, Newborn; Humans; Microglia; Brain; Disease Models, Animal; Pluripotent Stem Cells; Point Mutation
PubMed: 37516973
DOI: 10.1016/j.xpro.2023.102490 -
The Journal of Biological Chemistry Feb 2022The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR mice with reduced dimerization propensity due...
The glucocorticoid (GC) receptor (GR) is essential for normal development and in the initiation of inflammation. Healthy GR mice with reduced dimerization propensity due to a point mutation (A465T) at the dimer interface of the GR DNA-binding domain (DBD) (here GR) have previously helped to define the functions of GR monomers and dimers. Since GR retains residual dimerization capacity, here we generated the dimer-nullifying double mutant GR mice, featuring an additional mutation (I634A) in the ligand-binding domain (LBD) of GR. These mice are perinatally lethal, as are GR mice (these mice have the I634A mutation but not the A465T mutation), displaying improper lung and skin formation. Using embryonic fibroblasts, high and low doses of dexamethasone (Dex), nuclear translocation assays, RNAseq, dimerization assays, and ligand-binding assays (and K values), we found that the lethal phenotype in these mice is due to insufficient ligand binding. These data suggest there is some correlation between GR dimerization potential and ligand affinity. We conclude that even a mutation as subtle as I634A, at a position not directly involved in ligand interactions sensu stricto, can still influence ligand binding and have a lethal outcome.
Topics: Animals; Dexamethasone; Glucocorticoids; Ligands; Mice; Point Mutation; Receptors, Glucocorticoid
PubMed: 35007536
DOI: 10.1016/j.jbc.2022.101574 -
Genes Oct 2021The OTUD6B and ZMIZ1 genes were recently identified as causes of syndromic intellectual disability (ID) with shared phenotypes of facial dysmorphism, distal limb...
The OTUD6B and ZMIZ1 genes were recently identified as causes of syndromic intellectual disability (ID) with shared phenotypes of facial dysmorphism, distal limb anomalies, and seizure disorders. OTUD6B- and ZMIZ1-related ID are inherited in autosomal recessive and autosomal dominant patterns, respectively. We report a 5-year-old girl with developmental delay, facial phenotypes resembling Williams syndrome, and cardiac defects. The patient also had terminal broadening of the fingers and polydactyly. Cytogenomic microarray (CMA), whole exome sequencing (WES), and mRNA analysis were performed. The CMA showed a paternally inherited 0.118 Mb deletion of 8q21.3, chr8:92084087-92202189, with OTUD6B involved. The WES identified a hemizygous OTUD6B variant, c.873delA (p.Lys291AsnfsTer3). The mother was heterozygous for this allele. The WES also demonstrated a heterozygous ZMIZ1 variant, c.1491 + 2T > C, in the patient and her father. This ZMIZ1 variant yielded exon 14 skipping, as evidenced by mRNA study. We suggest that Williams syndrome-like phenotypes, namely, periorbital edema, hanging cheek, and long and smooth philtrum represent expanded phenotypes of OTUD6B-related ID. Our data expand the genotypic spectrum of OTUD6B- and ZMIZ1-related disorders. This is the first reported case of a compound heterozygote featuring point mutation, chromosomal microdeletion of OTUD6B, and the unique event of OTUD6B, coupled with ZMIZ1 variants.
Topics: Alleles; Child, Preschool; Chromosome Deletion; Endopeptidases; Exome; Female; Genetic Predisposition to Disease; Heterozygote; Humans; Infant; Intellectual Disability; Male; Pedigree; Phenotype; Point Mutation; Transcription Factors; Exome Sequencing
PubMed: 34680978
DOI: 10.3390/genes12101583 -
Reproduction in Domestic Animals =... Aug 2022Cytosine base editors (CBEs) and CRISPR/Cas9-mediated HDR method both have the ability to introduce nucleotide substitution into genomes, which exhibit great potential...
Cytosine base editors (CBEs) and CRISPR/Cas9-mediated HDR method both have the ability to introduce nucleotide substitution into genomes, which exhibit great potential for improving economically important traits in livestock species. The FecG mutation (g. C1184T, p. S395F) of growth differentiation factor 9 (GDF9) gene increases prolificacy in Cambridge sheep and Belclare sheep. In the present study, we aimed to compare the efficiency and precision of BE4-Gam and CRISPR/Cas9 systems on generating FecG mutation in ovine genome. First, the microinjection of BE4-Gam mRNA had no adverse effects on development rate after cleavage, and the efficiencies of total mutants and targeted mutants were 8.9% and 7.1%, respectively. Then, the total mutation and targeted mutation rates were improved from 8.5% to 22.5% (p < .01), and 6.4% to 16.3%, respectively, by adjusting the injection time of BE4-Gam mRNA from 14 to 12 hr post-insemination (hpi). Furthermore, CRISPR/Cas9-mediated HDR method introduced the FecG mutation at the efficiency of 16.1%, which was comparable to BE4-Gam system (16.3%). There was no bystander editing event happened in edited embryos caused by CRISPR/Cas9, but the bystander editing efficiency was as high as 15.0% in BE4-Gam-edited embryos. In summary, our findings demonstrated that CRISPR/Cas9-mediated HDR method was more accurate than BE4-Gam system in introducing FecG into ovine genome, and highlight the potential of the former strategy to modify economically important trait-associated SNPs.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Microinjections; Mutation; Point Mutation; RNA, Messenger; Sheep
PubMed: 35426186
DOI: 10.1111/rda.14124