Search Results (6)

Background: Fabry disease (FD) results from pathogenic GLA variants, causing lysosomal α-galactosidase A (α-GalA) deficiency and sphingolipid ceramide trihexoside (Gb3 or THC) accumulation. Disease phenotype varies widely but cardiomyopathy is commonly life-limiting. As a multisystemic disorder, FD initiates at the cellular level; however, the mechanism/s underlying Gb3-induced cell dysfunction remains largely unknown. This study established an in vitro mutation-specific model of Fabry cardiomyopathy using human-induced pluripotent stem cell (iPSC)-derived cardiomyocytes to explore underlying cell pathology. Methods: Skin biopsies from consenting Fabry patients and normal control subjects were reprogrammed to iPSCs then differentiated into cardiomyocytes. The GLA mutations in Fabry cell lines were corrected using CRISP-Cas9. Phenotypic characteristics, α-Gal A activity, Gb3 accumulation, functional status, and lipid analysis were assessed. Cardiomyocytes derived from two patients with severe clinical phenotype and genotypes, GLA^c.851T>C, GLA^{c.1193_1196del}, and their respective corrected lines, GLA^{corr c.851T>C}, GLA^{corr c.1193_1196del}, were selected for further studies. Results: Cardiomyocytes derived from individuals with FD iPSCs exhibited stable expression of cardiomyocyte markers and spontaneous contraction, morphological features of FD, reduced α-Gal A activity, and accumulation of Gb3. Lipidomic profiling revealed differences in the Gb3 isoform profile between the control and FD patient iPSC-derived cardiomyocytes. Contraction strength was unchanged but relaxation after contraction was delayed, mimicking the diastolic dysfunction typical of Fabry cardiomyopathy. Conclusions: iPSC-derived cardiomyocytes provide a useful model to explore aspects of Fabry cardiomyopathy, including disruptions in sphingolipid pathways, proteomics, and multigene expression that together link genotype to phenotype. The platform potentially offers broad applicability across many genetic diseases and offers the prospect of testing and implementation of individualised therapies. Full article

The demand for high-precision CRISPR/Cas9 systems in biomedicine is experiencing a notable upsurge. The editing system fdCas9 employs a dual-sgRNA strategy to enhance editing accuracy. However, the application of fdCas9 is constrained by the stringent requirement for two protospacer adjacent motifs (PAMs) of Cas9. Here, we devised an optimized editor, fRYdCas9, by merging FokI with the nearly PAM-less RYdCas9 variant, and two fRYdCas9 systems formed a dimer in a proper spacer length to accomplish DNA cleavage. In comparison to fdCas9, fRYdCas9 demonstrates a substantial increase in the number of editable genomic sites, approximately 330-fold, while maintaining a comparable level of editing efficiency. Through meticulous experimental validation, we determined that the optimal spacer length between two FokI guided by RYdCas9 is 16 base pairs. Moreover, fRYdCas9 exhibits a near PAM-less feature, along with no on-target motif preference via the library screening. Meanwhile, fRYdCas9 effectively addresses the potential risks of off-targets, as analyzed through whole genome sequencing (WGS). Mouse embryonic editing shows fRYdCas9 has robust editing capabilities. This study introduces a potentially beneficial alternative for accurate gene editing in therapeutic applications and fundamental research. Full article

2,5-Furandicarboxylic acid (FDCA) is an important monomer for manufacturing biobased plastics. Biocatalysis has been recognized as a sustainable tool in organic synthesis. To date, the efficiencies of most biocatalytic processes toward FDCA remain low. So, it is highly desired to develop efficient processes. In this work, a biocatalytic route toward FDCA was developed by integrating a cell-free extract of galactose oxidase variant M_3–5 with a whole-cell biocatalyst harboring NAD⁺-dependent vanillin dehydrogenases and NADH oxidase, starting from 5-hydroxymethylfurfural. FDCA was produced in a concurrent mode with >90% yields within 36 h at 20 mM substrate concentration. In addition, biocatalytic synthesis of FDCA was performed on a preparative scale, with 78% isolated yield. The present work may lay the foundation for sustainable production of FDCA. Full article

Conserved omicron RNA (COR) is a 40 base long 99.9% conserved sequence in SARS-CoV-2 Omicron variant, predicted to form a stable stem loop, the targeted cleavage of which can be an ideal next step in controlling the spread of variants. The Cas9 enzyme has been traditionally utilized for gene editing and DNA cleavage. Previously Cas9 has been shown to be capable of RNA editing under certain conditions. Here we investigated the ability of Cas9 to bind to single-stranded conserved omicron RNA (COR) and examined the effect of copper nanoparticles (Cu NPs) and/or polyinosinic-polycytidilic acid (poly I:C) on the RNA cleavage ability of Cas9. The interaction of the Cas9 enzyme and COR with Cu NPs was shown by dynamic light scattering (DLS) and zeta potential measurements and was confirmed by two-dimensional fluorescence difference spectroscopy (2-D FDS). The interaction with and enhanced cleavage of COR by Cas9 in the presence of Cu NPs and poly I:C was shown by agarose gel electrophoresis. These data suggest that Cas9-mediated RNA cleavage may be potentiated at the nanoscale level in the presence of nanoparticles and a secondary RNA component. Further explorations in vitro and in vivo may contribute to the development of a better cellular delivery platform for Cas9. Full article

What is known as Furan-2,5-dicarboxylic acid (FDCA) is an attractive compound since it has similar properties to terephthalic acid. Further, 5-hydroxymethylfurfural oxidase (HMFO) is an enzyme, which could convert HMF to FDCA directly. Most wild types of HMFO have low activity on the oxidation of HMF to FDCA. The variant of 8BxHFMO from Methylovorus sp. MP688 was the only reported enzyme that was able to perform FDCA production. However, the stabilization of 8BxHMFO is still not that satisfactory, and further improvement is necessary for the industrial application of the enzyme. In this work, stability-enhanced HMFO from 8BxHFMO was engineered through employing B-factor analysis. The mutation libraries were created based on the NNK degeneracy of residues with the top ten highest B-factor value, and two of the effective mutants were screened out through the high throughput selection with the horseradish peroxidase (HRP)-Tyr assay. The mutants Q319K and N44G show a significantly increased yield of FDCA in the reaction temperature range of 30 to 40 °C. The mutant Q319K shows the best performance at 35 °C with a FDCA yield of 98% (the original 8BxHMFO was only 85%), and a half-life exceeding 72 h. Moreover, molecular dynamic simulation indicates that more hydrogen bonds are formed in the mutants, which improves the stability of the protein structure. The method could enhance the design of more stable biocatalysts; and provides potential for the further optimization and utilization of HMFO in biotechnological processes. Full article

The identification of the robust clustered regularly interspersed short palindromic repeats (CRISPR) associated endonuclease (Cas9) system gene-editing tool has opened up a wide range of potential therapeutic applications that were restricted by more complex tools, including zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Nevertheless, the high frequency of CRISPR system off-target activity still limits its applications, and, thus, advanced strategies for highly specific CRISPR/Cas9-mediated genome editing are continuously under development including CRISPR–FokI dead Cas9 (fdCas9). fdCas9 system is derived from linking a FokI endonuclease catalytic domain to an inactive Cas9 protein and requires a pair of guide sgRNAs that bind to the sense and antisense strands of the DNA in a protospacer adjacent motif (PAM)-out orientation, with a defined spacer sequence range around the target site. The dimerization of FokI domains generates DNA double-strand breaks, which activates the DNA repair machinery and results in genomic edit. So far, all the engineered fdCas9 variants have shown promising gene-editing activities in human cells when compared to other platforms. Herein, we review the advantages of all published variants of fdCas9 and their current applications in genome engineering. Full article