Biology subjects

Broughton, J.

Publications and source records attributed to Broughton, J..

4 recordsLinked to original sources

bioRxiv2025

Single-AAV CRISPR editing of skeletal muscle in non-human primates with NanoCas, an ultracompact nuclease

CRISPR gene editing is a transformative technology for addressing genetic diseases, but delivery constraints have largely limited its therapeutic applications to liver-targeted and ex vivo therapies. Here, we present the discovery and engineering of NanoCas, an ultracompact CRISPR nuclease capable of extending CRISPRs reach in vivo beyond liver targets. We experimentally screened 176 ultracompact CRISPR systems found in metagenomic data and applied protein engineering approaches to enhance the editing efficiency of NanoCas. The optimized NanoCas exhibits potent editing capabilities across various cell systems and tissues in vivo when administered via adeno-associated viral (AAV) vectors. This is accomplished despite NanoCas being approximately one-third the size of conventional CRISPR nucleases. In proof-of-concept experiments, we observed robust editing with our optimized NanoCas in mouse models targeting Pcsk9, a gene involved in cholesterol regulation, and targeting exon splice sites in dystrophin to address Duchenne muscular dystrophy (DMD) mutations. We further tested the efficacy of our NanoCas system in vivo in non-human primates (NHPs) resulting in editing levels above 30% in muscle tissues. The compact size of NanoCas, in combination with robust nuclease editing, opens the door for single-AAV editing of non-liver tissues in vivo, including the use of newer editing modalities such as reverse transcriptase (RT) editing, base editing, and epigenetic editing.

bioengineering↗

bioRxiv2024

Suppression of bacterial cell death underlies the antagonistic interaction between ciprofloxacin and tetracycline in Escherichia coli

Antibiotic combinations are an attractive strategy to maximise the efficiency of drug treatment and minimise resistance evolution, but we still lack a full understanding of their effect on bacterial cells. The interaction between DNA-targeting antibiotics, such as ciprofloxacin, and translation inhibitors, such as tetracycline, is antagonistic, resulting in a weaker effect on bacterial growth than expected from the effect of each single drug. This antagonism has been analysed in detail at the population level, but we lack a single-cell understanding of its effect and how it depends on nutrient availability. Here, we used a microfluidic device to quantify the antagonism between ciprofloxacin and tetracycline at the single-cell level in three nutrient conditions. We showed that improved growth is due to increased survival of cells under the drug combination compared to ciprofloxacin alone. This effect is growth-dependent, with better suppression in rich nutrient conditions. Quantification of the DNA damage response (SOS response) revealed two sub-populations among the cells that die upon ciprofloxacin treatment, with some cells reaching a very high level of SOS while others had a lower level of SOS, similar to surviving cells. The low-SOS cells were more frequent in fast growth conditions and showed increased survival under the drug combination but the high-SOS cells were hardly rescued by the drug combination. This result explains the stronger antagonistic effect of tetracycline on ciprofloxacin in fast growth compared to slow growth. Our results underscore the importance of single-cell quantification in understanding the bacterial response to antibiotic combinations and devising new treatment strategies.

cell biology↗

bioRxiv2021

Growth-dependent heterogeneity in the DNA damage response in Escherichia coli

In natural environments bacteria are frequently exposed to sub-lethal levels of DNA damage which leads to the induction of a stress response (the SOS response in Escherichia coli). Natural environments also vary in nutrient availability, resulting in distinct physiological changes in bacteria which may have direct implications on their capacity to repair their chromosomes. Here, we evaluated the impact of varying the nutrient availability on the expression of the SOS response induced by chronic sub-lethal DNA damage in E. coli. The expression of the SOS regulon was found to be highly heterogeneous at the single-cell level in all growth conditions. Surprisingly, we observed a larger fraction of high SOS-induced cells in slow growth as compared with fast growth, despite a higher rate of SOS induction in fast growth. This counter-intuitive result can be explained by the dynamic balance between the rate of SOS induction and the division rates of cells exposed to DNA damage. Taken together, our data illustrates how cell division and physiology come together to produce growth-dependent heterogeneity in the DNA damage response.

cell biology↗

bioRxiv2018

Dynamic Regulation of RNA Structure in Mammalian Cells

RNA structure is intimately connected to each step of gene expression. Recent advances have enabled transcriptome-wide maps of RNA secondary structure, termed RNA structuromes. However, previous whole-cell analyses lacked the resolution to unravel the dynamic regulation of RNA structure across subcellular states. Here we reveal the RNA structuromes in three compartments -- chromatin, nucleoplasm and cytoplasm. The cytotopic structuromes substantially expand RNA structural information, and enable detailed investigation of the central role of RNA structure in linking transcription, translation, and RNA decay. Through comparative structure analysis, we develop a resource to visualize the interplay of RNA-protein interactions, RNA chemical modifications, and RNA structure, and predict both direct and indirect reader proteins of RNA modifications. We validate the novel role of the RNA binding protein LIN28A as an N6-methyladenosine (m6A) modification \"anti-reader\". Our results highlight the dynamic nature of RNA structures and its functional significance in gene regulation.

molecular biology↗