Biology subjects

Blassberg, R.

Publications and source records attributed to Blassberg, R..

4 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↗

bioRxiv2023

An aptamer-mediated base editing platform for simultaneous knock-in and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply the novel modular RNA aptamer-mediated Pin-point base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

molecular biology↗

bioRxiv2021

A quantitative landscape of cell fate transitions identifies principles of cellular decision-making

Fate decisions in developing tissues involve cells transitioning between a set of discrete cell states, each defined by a distinct gene expression profile. Geometric models, often referred to as Waddington landscapes, in which developmental paths are given by the gradient and cell states by the minima of the model, are an appealing way to describe differentiation dynamics and developmental decisions. To construct and validate accurate dynamical landscapes, quantitative methods based on experimental data are necessary. To this end we took advantage of the differentiation of neural and mesodermal cells from pluripotent mouse embryonic stem cells exposed to different combinations and durations of signalling factors. We developed a principled statistical approach using flow cytometry data to quantify differentiating cell states. Then, using a framework based on Catastrophe Theory and approximate Bayesian computation, we constructed the corresponding dynamical landscape. The result was a quantitative model that accurately predicted the proportions of neural and mesodermal cells differentiating in response to specific signalling regimes. Analysis of the geometry of the landscape revealed two distinct ways in which cells make a binary choice between one of two fates. We discuss the biological relevance of these mechanisms and suggest that they represent general archetypal designs for developmental decisions. Taken together, the approach we describe is broadly applicable for the quantitative analysis of differentiation dynamics and for determining the logic of developmental cell fate decisions.

developmental biology↗

bioRxiv2020

Sox2 levels configure the WNT response of epiblast progenitors responsible for vertebrate body formation

WNT signalling has multiple roles. It maintains pluripotency of embryonic stem cells, assigns posterior identity in the epiblast and induces mesodermal tissue. We provide evidence that these distinct functions are conducted by the transcription factor SOX2, which adopts different modes of chromatin interaction and regulatory element selection depending on its level of expression. At high levels, SOX2 acts as a pioneer factor, displacing nucleosomes from regulatory elements with high affinity SOX2 binding sites and recruiting the WNT effector, TCF/{beta}-catenin, to maintain pluripotent gene expression. Reducing SOX2 levels destabilises pluripotency and reconfigures SOX2/TCF/{beta}-catenin occupancy to caudal epiblast expressed genes. These contain low-affinity SOX2 sites and are co-occupied by T/Bra and CDX. The loss of SOX2 allows WNT induced mesodermal differentiation. These findings define a role for Sox2 levels in dictating the chromatin occupancy of TCF/{beta}-catenin and reveal how context specific responses to a signal are configured by the level of a transcription factor.

developmental biology↗