Biology subjects

Giovannangeli, C.

Publications and source records attributed to Giovannangeli, C..

4 recordsLinked to original sources

bioRxiv2026

Error-free and efficient prime editing in absence of maternal Polθ

Prime editing is a versatile genome-editing technology developed to make small edits while minimising the unwanted mutations associated with approaches based on double-strand breaks. In zebrafish embryos, however, prime editing is highly error-prone, typically yielding more unwanted mutations than precise edits. Here, we show that most unwanted mutations are deletions flanked by microhomologies and insertions templated from neighbouring sequences, both hallmarks of microhomology-mediated end-joining of double-strand breaks. These breaks are unlikely to result from direct cleavage of both genomic strands by the prime editor. Instead, we propose that the rapid cell divisions of early zebrafish embryos promote the conversion of nicks into double-strand breaks by replication forks. Loss of maternally deposited DNA polymerase {theta} (Pol{theta}), the core factor in microhomology-mediated end-joining, abolishes unwanted mutations and can increase edit rates above 50%, resulting in error-free and efficient prime editing. Our work identifies the source of unwanted prime-editing mutations in zebrafish and establishes a practical strategy to achieve high edit rates and near-perfect precision.

genetics↗

bioRxiv2025

A prime editing strategy to rewrite the γ-globin promoters and reactivate fetal hemoglobin for sickle cell disease

Fetal hemoglobin (HbF) reactivation is a promising therapy for {beta}-hemoglobinopathies. We developed a prime editing strategy that introduces multiple mutations in the fetal {gamma}-globin promoters of patients hematopoietic stem/progenitor cells (HSPCs), boosting HbF expression and offering alternative therapeutic perspectives.

molecular biology↗

bioRxiv2023

Comparative transcriptomics reveal a novel tardigrade specific DNA binding protein induced in response to ionizing radiation

Tardigrades, microscopic animals found in virtually all ecosystems, are renowned for their remarkable ability to withstand extreme conditions. Recent studies have identified novel tardigrade specific protein families that aid in resistance to desiccation and ionizing radiation (IR). Notably, a tardigrade specific DNA binding protein called Dsup (for DNA damage suppressor) has been found to protect from X-ray damage in human cells and from hydroxyl radicals in vitro. However, Dsup has only been found in two species within the Hypsibioidea superfamily. To better understand mechanisms underlying radio-resistance in the Tardigrada phylum, we first characterized DNA damage and repair in response to IR in the model species Hypsibius exemplaris. By analysis of phosphorylated H2AX, we demonstrated the induction and repair of DNA double-strand breaks after IR exposure. Importantly, the rate of single-strand breaks induced was roughly equivalent to that in human cells, suggesting that DNA repair plays a predominant role in the remarkable radio-resistance of tardigrades. In order to identify novel tardigrade specific genes involved, we next conducted a comparative transcriptomics across three species, H. exemplaris, Acutuncus antarcticus and Paramacrobiotus fairbanksi, the latter belonging to the Macrobiotoidea superfamily known to lack Dsup homologs. In all three species, many genes of DNA repair were among the most strongly overexpressed genes alongside a novel tardigrade specific gene, named Tardigrade DNA damage Response protein 1 (TDR1). We found that TDR1 protein interacts with DNA and forms aggregates at high concentration suggesting it may condensate DNA and act by preserving chromosome organization until DNA repair is accomplished. Remarkably, when expressed in human cells, TDR1 improved resistance to Bleomycin, a radiomimetic drug. Based on these findings, we propose that TDR1 is a novel tardigrade specific gene responsible for conferring resistance to IR. Our study sheds light on mechanisms of DNA repair helping to cope with high levels of DNA damage. Furthermore, it suggests that at least two tardigrade specific genes, respectively for Dsup and TDR1, have independently evolved DNA-binding functions that contribute to radio-resistance in the Tardigrada phylum.

molecular biology↗

bioRxiv2019

Differential Requirements for the RAD51 Paralogs in Genome Repair and Maintenance in Human Cells

Deficiency in several of the classical human RAD51 paralogs [RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3] is associated with cancer predisposition and Fanconi anemia. To investigate their functions, isogenic disruption mutants for each were generated in non-transformed MCF10A mammary epithelial cells and in transformed U2OS and HEK293 cells. In U2OS and HEK293 cells, viable ablated clones were readily isolated for each RAD51 paralog; in contrast, with the exception of RAD51B, RAD51 paralogs are cell-essential in MCF10A cells. Underlining their importance for genomic stability, mutant cell lines display variable growth defects, impaired sister chromatid recombination, reduced levels of stable RAD51 nuclear foci, and hypersensitivity to mitomycin C and olaparib. Altogether these observations underscore the contributions of RAD51 paralogs in diverse DNA repair processes, and demonstrate essential differences in different cell types. Finally, this study will provide useful reagents to analyze patient-derived mutations and to investigate mechanisms of chemotherapeutic resistance deployed by cancers.

cell biology↗