Biology subjects

Benoit, C.

Publications and source records attributed to Benoit, C..

3 recordsLinked to original sources

bioRxiv2024

Functional benefit of CRISPR/Cas9-induced allele deletion for RYR1 dominant mutation

More than 700 pathogenic or probably pathogenic variations have been identified in the RYR1 gene causing various myopathies collectively known as "RYR1-related myopathies". Currently, there is no treatment for these myopathies, and gene therapy stands out as one of the most promising approaches. In the context of a dominant form of Central Core Disease due to a RYR1 mutation, we aimed at showing the functional benefit of inactivating specifically the mutated RYR1 allele by guiding CRISPR/Cas9 cleavages onto frequent single nucleotide polymorphisms (SNPs) segregating on the same chromosome. Whole-genome sequencing was used to pinpoint SNPs localized on the mutant RYR1 allele and identified specific CRISPR/Cas9 guide-RNAs. Lentiviruses encoding these guide-RNAs and the SpCas9 nuclease were used to transduce immortalized patient muscle cells, inducing the specific deletion of the mutant RYR1 allele. The efficiency of the deletion was assessed at both DNA and RNA levels and at the functional level after monitoring calcium release induced by the stimulation of the RyR1-channel. This study provides in-cellulo proof of concept regarding the benefits of mutant RYR1 allele deletion, in the case of a dominant RYR1 mutation, from both a molecular and functional perspective. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/576997v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@484a3borg.highwire.dtl.DTLVardef@1a842org.highwire.dtl.DTLVardef@cda3d5org.highwire.dtl.DTLVardef@befd91_HPS_FORMAT_FIGEXP M_FIG C_FIG eTOC synopsisMutations in the RYR1 gene, encoding a calcium channel required for muscle contraction, cause severe myopathies. In this study, Marty and colleagues demonstrate the functional benefit of suppression of a mutant RYR1 allele using CRISPR/Cas9, in the case of a dominant mutation, leaving the wild type allele alone.

neuroscience↗

bioRxiv2020

Dynamics of a neuronal pacemaker in the weakly electric fish Apteronotus

The precise timing of neuronal activity is critical for normal brain function. In weakly electric fish, the medullary pacemaker network (PN) sets the timing for an oscillating electric organ discharge (EOD) used for electric sensing. This network is the most precise biological oscillator known, with sub-microsecond variation in oscillator period. The PN consists of two principle sets of neurons, pacemaker and relay cells, that are connected by gap junctions and normally fire in synchrony, one-to-one with each EOD cycle. However, the degree of gap junctional connectivity between these cells appears insufficient to provide the population averaging required for the observed temporal precision of the EOD. This has led to the hypothesis that individual cells themselves fire with high precision, but little is known about the oscillatory dynamics of these pacemaker cells. To this end, we have developed a biophysical model of a pacemaker neuron action potential based on experimental recordings. We validated the model by comparing the changes in oscillatory dynamics produced by different experimental manipulations. Our results suggest that a relatively simple model captures the complex dynamics exhibited by pacemaker cells, and that these dynamics may enhance network synchrony and precision. Author summaryMany neural networks in the brain exhibit activity patterns which oscillate regularly in time. These oscillations, like a clock, can provide a precise sense of time, enabling drummers to maintain complex beat patterns and pets to anticipate "feeding time". The exact mechanisms by which brain networks give rise to these biological clocks are not clear. The pacemaker network of weakly electric fish has the highest precision of all known biological clocks. In this study, we develop a detailed biophysical model of neurons in the pacemaker network. We then validate the model against experiments using a nonlinear dynamics approach. Our results show that pacemaker precision is due, at least in part, to how individual pacemaker cells generate their activity. This supports the idea that temporal precision in this network is not solely an emergent property of the network but also relies on the dynamics of individual neurons.

neuroscience↗

bioRxiv2016

Nus factors have a widespread regulatory function in bacteria

Nus factors are broadly conserved across bacterial species, and are often essential for viability. A complex of five Nus factors (NusB, NusE, NusA, NusG and SuhB) is considered to be a dedicated regulator of ribosomal RNA folding, and has been shown to prevent Rho-dependent transcription termination. We have established the first cellular function for the Nus factor complex beyond regulation of ribosomal assembly: repression of the Nus factor-encoding gene, suhB. This repression occurs by translation inhibition followed by Rho-dependent transcription termination. Thus, the Nus factor complex can prevent or promote Rho activity depending on the gene context. Extensive conservation of NusB/E binding sites upstream of nus factor genes suggests that Nus factor autoregulation occurs in many species. Putative NusB/E binding sites are also found upstream of many other genes in diverse species, and we demonstrate Nus factor regulation of one such gene in Citrobacter koseri. We conclude that Nus factors have an evolutionarily widespread regulatory function beyond ribosomal RNA, and that they are often autoregulatory.

microbiology↗