Biology subjects

Zahra, H.

Publications and source records attributed to Zahra, H..

3 recordsLinked to original sources

bioRxiv2026

Prime Editing Corrects the HBB Codon 8/9 (+G) Mutation in Patient-Derived Induced Pluripotent Stem Cells and Restores β-Globin Expression in iPSC-Derived Erythroid Cells

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

cell biology↗

bioRxiv2024

A Rapid, Cost-Effective Method for High-Yield DNA Purification from PCR Products and Agarose Gels.

Nucleic acid purification is essential for molecular biology workflows, enabling successful downstream applications like cloning, sequencing, and PCR amplification. While commercial kits are widely used for DNA extraction from PCR products and agarose gels, their high costs can burden resource-limited laboratories. To provide a viable alternative, we developed an optimized, cost-effective in-house protocol for high-yield DNA purification. This study evaluates the performance of our protocol for plasmid DNA and PCR products, comparing it with commercial kits from Qiagen, Thermo Fisher, and WizBio in terms of cost, time, DNA concentration, and purity. Results from gel electrophoresis demonstrated that the in-house protocol significantly enhances PCR product clarity and reduces background smearing, yielding high-purity DNA compatible with sensitive applications. Restriction-digested plasmid samples showed successful ligation and transformation in E. coli (DH5), with Sanger sequencing chromatogram further confirming the sequence integrity of the purified DNA. Our findings highlight the in-house protocol as a cost-effective, efficient, and reliable alternative to commercial kits, delivering high-quality DNA suitable for various molecular applications. This method offers an accessible and practical solution for laboratories seeking to optimize DNA purification under budget constraints.

molecular biology↗

bioRxiv2024

Evidence that Dmrta2 acts as a transcriptional repressor of Pax6 in murine cortical progenitors and identification of a mutation crucial for DNA recognition associated with microcephaly in human

Dmrta2 (also designated Dmrt5) is a transcriptional regulator expressed in cortical progenitors in a caudomedialhigh/rostrolaterallow gradient with important roles at different steps of cortical development. Dmrta2 has been suggested to act in cortex development mainly by differential suppression of Pax6 and other homeobox transcription factors such as the ventral telencephalic regulator Gsx2, which remains to be fully demonstrated. Here we have addressed the epistatic relation between Pax6 and Dmrta2 by comparing phenotypes in mutant embryos or embryos overexpressing both genes in various allelic combinations. We showed that Dmrta2 cooperates with Pax6 in the maintenance of cortical identity in dorsal telencephalic progenitors and that it acts as a transcriptional repressor of Pax6 to control cortical patterning. Mechanistically, we show that in P19 cells, Dmrta2 can act as a DNA-binding dependent repressor on the Pax6 E60 enhancer and that a point mutation that affects its DNA binding properties leads to agenesis of the corpus callosum, pachygyria, and the absence of the cingulate gyrus. Finally, we provide evidence that Dmrta2 binds to the Zfp423 zinc finger protein and that it enhances its ability to recruit the NurD repressor complex. Together, our results highlight the importance and conserved function of Dmrta2 in cortical development and provide novel insights into its mechanism of action. SIGNIFICANCE STATEMENTCorticogenesis is controlled by an array of transcription factors that coordinate neural progenitor self-renewal and differentiation to generate correct cortical cell number and diversity. However, how this complex array of transcription factors works in concert to regulate this delicate process remains largely unknown. Here we provide important insights into the mechanism of action of Dmrta2 by demonstrating that it cooperates with the transcription factor Pax6 to define the pallium-subpallium boundary and that it acts by repressing it, likely via the recruitment of Zfp423 and the NurD repressor complex, to control cortical patterning. Our data also reveal that a point mutation that affects its DNA binding causes cortical abnormalities in human, further highlighting its importance in cortex development.

neuroscience↗