Biology subjects

Tavassoli, M.

Publications and source records attributed to Tavassoli, M..

4 recordsLinked to original sources

bioRxiv2024

Harnessing RNA-based DNA repair pathways for targeted gene editing

Recent studies have revealed a role for RNA in the repair of DNA double-strand breaks. Here, we show that the asymmetric DNA overhangs generated by the small TevSaCas9 dual nuclease informs a simple and robust editing strategy in human cells whereby Pol{theta} and Rad52 are recruited to repair the double-strand break. The 2-nt, 3 DNA overhang generated by the I-TevI nuclease domain of TevSaCas9 hybridizes with the 3 end of a co-localized repair template guide RNA to specifically license repair. Substitutions that destabilize the repair duplex reduce editing efficiency. Targeted RNA-templated repair (rep-editing) harnesses cellular RNA-based DNA repair pathways to introduce precise nucleotide edits, deletions and insertions in human cells with high efficiency and fidelity independent of co-delivered repair functions. The small size of TevSaCas9 and RNA repair template offers delivery advantages over size-constrained or multi-component editing systems.

molecular biology↗

bioRxiv2022

Semisynthesis reveals Apoptin as a tumour-selective protein prodrug that causes cytoskeletal collapse

Apoptin is a small viral protein capable of inducing cell death selectively in cancer cells. Despite its potential as an anticancer agent, relatively little is known about its mechanism of toxicity and cancer-selectivity. Previous experiments suggest that cancer-selective phosphorylation modulates Apoptin toxicity, although a lack of chemical tools has hampered the dissection of underlying mechanisms. Here, we describe structure-function studies with site-specifically phosphorylated Apoptin (Apoptin-T108ph) in living cells which revealed that Thr108 phosphorylation is the selectivity switch for Apoptin toxicity. Mechanistic investigations link T108ph to actin binding, cytoskeletal disruption and downstream inhibition of anoikis-resistance as well as cancer cell invasion. These results establish Apoptin as a protein pro-drug, selectively activated in cancer cells by phosphorylation, which disrupts the cytoskeleton and promotes cell death. We anticipate that this mechanism provides a framework for the design of next generation anticancer proteins with enhanced selectivity and potency.

cancer biology↗

bioRxiv2021

An Efficient and Cost-effective Purification Methodology for Cas9 Nuclease

With an ever-increasing demand for laboratory-grade Cas9 proteins by many groups advancing the use of CRISPR technology, a more efficient and scalable process for generating the proteins, coupled with rapid purification methods is in urgent demand. Here, we introduce a modified methodology for rapid purification of active SaCas9 protein within 24 hours. The product has over 90% protein purity. The simplicity and cost-effectiveness of such methodology will enable general labs to produce a sizable amount of Cas9 proteins, further accelerating the advancement of CRISPR/Cas9-based research.

biochemistry↗

bioRxiv2020

ImmunoCluster: A computational framework for the non-specialist to immune profile cellular heterogeneity using liquid and imaging mass, and flow cytometry datasets

High dimensional cytometry is an innovative tool for immune monitoring in health and disease, it has provided novel insight into the underlying biology as well as biomarkers for a variety of diseases. However, the analysis of multiparametric "big data" usually requires specialist computational knowledge. Here we describe ImmunoCluster (https://github.com/kordastilab/ImmunoCluster) an R package for immune profiling cellular heterogeneity in high dimensional liquid and imaging mass cytometry, and flow cytometry data, designed to facilitate computational analysis by a non-specialist. The analysis framework implemented within ImmunoCluster is readily scalable to millions of cells and provides a variety of visualization and analytical approaches, as well as a rich array of plotting tools that can be tailored to users needs. The protocol consists of three core computational stages: 1, data import and quality control, 2, dimensionality reduction and unsupervised clustering; and 3, annotation and differential testing, all contained within an R-based open-source framework.

systems biology↗