Biology subjects

Abreu Torres, A.

Publications and source records attributed to Abreu Torres, A..

2 recordsLinked to original sources

bioRxiv2025

Minimal activation of the p53 DNA damage response by a modular cytosine base editor enables effective multiplexed gene knockout in induced pluripotent stem cells

Precise genome editing of induced pluripotent stem cells (iPSC) holds great promise for engineering advanced cell therapies. CRISPR-Cas systems have been widely adopted in genome engineering applications, however their dependence on genotoxic DNA double strand breaks (DSBs) presents challenges in hypersensitive iPSCs. Base editors are capable of both modifying and ablating gene function without generating DSBs making them an attractive solution for iPSC engineering. Here we report efficient and durable multiplexed target knockout with minimal impact on cell viability and expansion with a cytosine base editor composed of nCas9-UGI and Rat APOBEC1 assembled using the Pin-point platform (nCas9-UGI:rAPO). Minimal p53-mediated DNA damage signalling occurred independently of the number of simultaneous edits installed, and this could be further reduced by modulating the assembly of the base editor complex. Whereas non-homologous end-joining-mediated DNA damage repair led to p53-mediated selection against imprecise editing outcomes and an associated reduction in the on-target efficiency of multiplexed SpCas9 nuclease editing, p53 activity was dispensable for maintaining genome integrity during the base editing process. The Pin-point platform therefore enables the assembly of base editors optimised for high editing efficiency with substantially reduced risk of selecting for defective DNA damage responses inherent to DSB-dependent editing systems.

molecular biology↗

bioRxiv2020

Viral mimicry of p65/RelA transactivation domain to inhibit NF-κB activation

Sensing of virus infection activates NF-{kappa}B to induce the expression of interferons, cytokines and chemokines to initiate the antiviral response. Viruses antagonise these antiviral defences by interfering with immune sensing and blocking the actions of antiviral and inflammatory molecules. Here, we show that a viral protein mimics the transactivation domain of the p65 subunit of NF-{kappa}B. The C terminus of vaccinia virus (VACV) protein F14 (residues 51-73) activates transcription when fused to a DNA-binding domain-containing protein and F14 associates with NF-{kappa}B co-activator CBP, disrupting p65-CBP interaction. Consequently, F14 diminishes CBP-mediated acetylation of p65 and the downstream recruitment of the transcriptional regulator BRD4 to the promoter of the NF-{kappa}B-responsive genes CXCL10 and CCL2, hence inhibiting their expression. Conversely, the recruitment of BRD4 to the promoters of NFKBIA, which encodes the inhibitor of NF-{kappa}B (I{kappa}B), and CXCL8 remains unaffected in the presence of either F14 or JQ1, a competitive inhibitor of BRD4 bromodomains, indicating its recruitment is acetylation-independent. Therefore, unlike other viral NF-{kappa}B antagonists, F14 is a selective inhibitor of NF-{kappa}B-dependent gene expression. A VACV strain lacking F14 showed that it contributes to virulence in an intradermal model of infection. Our results uncover a mechanism by which viruses disarm the antiviral defences through molecular mimicry of a conserved host protein and provide insight into the regulation of NF-{kappa}B-dependent gene expression by BRD4.

microbiology↗