Biology subjects

Baccouche, A.

Publications and source records attributed to Baccouche, A..

4 recordsLinked to original sources

bioRxiv2022

License to cut: Smart RNA guides for conditional control of CRISPR-Cas9

The Cas9 enzyme is a programmable endonuclease, whose target sequence is directed by a companion RNA guide. Cas9 and RNA guides have revolutionized biology, enabling facile editing of the genome in almost all organisms. Controlling where and when Cas9 and the guide operate is indispensable for many fields ranging from developmental biology to therapeutics, but it remains a challenge. Most methods focus on controlling Cas9 with physico-chemical means (which lack finesse, precision or multiplexing), or transcriptional tools (which are slow and difficult to design). Rather than directly engineering Cas9, engineering the RNA guide itself has emerged as a more general and potent way to manage the activity of Cas9. Here we report smart RNA guides that are conditionally activated by the presence of a specific RNA opener. Contrary to most previous approaches, the design affords ample freedom as spacer and the opener are independent. We demonstrate this flexibility by operating SmartGuides activated by a panel of miRNA relevant for human health, and by composing SmartGuides in Boolean logic circuits. Lastly, we test the SmartGuides in mammalian cells - validating the basics tenets of the design, but also highlighting the challenges that remain to be lifted for in-vivo operation.

bioengineering↗

bioRxiv2022

Silicon as a microfluidic material for imaging and incubation of droplets

Droplet microfluidics has become a powerful tool in life sciences, underlying digital assays, single-cell sequencing or directed evolution, and it is making foray in physical sciences as well. Imaging and incubation of droplets are crucial, yet they are encumbered by the poor optical, thermal and mechanical properties of PDMS - the de facto material for microfluidics. Here we show that silicon is an ideal material for droplet chambers. Si chambers pack droplets in a crystalline and immobile monolayer, are immune to evaporation or sagging, boost the number of collected photons, and tightly control the temperature field sensed by droplets. We use the mechanical and optical benefits of Si chambers to image [~]1 million of droplets from a multiplexed digital assay - with an acquisition rate similar to the best in-line methods. Lastly, we demonstrate their applicability with a demanding assay that maps the thermal dependence of Michaelis-Menten constants with an array of [~]150,000. The design of the Si chambers is streamlined to avoid complicated fabrication and improve reproducibility, which makes Silicon a complementary material to PDMS in the toolbox of droplet microfluidics. Significance StatementAs the technological engine behind single-cell sequencing and digital assays, droplets microfluidics has revolutionized life science and molecular diagnosis, and is making foray into physical sciences as well. Observing droplets in a controlled manner is becoming crucial, but PDMS - the de facto material of microfluidics - hampers imaging and incubation. Here we revisit silicon as a microfluidic material and show that its superior mechanical, optical and thermal performances improve the throughput and operation of droplets assay.

biochemistry↗

bioRxiv2022

Quantitative assaying of SpCas9-NG with fluorescent reporters

The Cas9 enzyme has revolutionized biology in less than a decade. Engineering Cas9 to expand its functionality has become a major research goal, yet assaying variants of Cas9 remains a laborious task that is commonly performed with gel electrophoresis. Fluorescence assays have been reported for Cas9 but their utility for assaying variants of Cas9 has not been investigated in detail. Here we use a simple fluorescent assay to resolve differences of activity between the wild type Streptococcus pyogenes Cas9 (SpCas9) and SpCas9-NG, a variant with an expanded PAM repertoire. We compare the kinetics of the two enzymes on dozens of mutated RNA guides - highlighting the benefits of fluorescence such as quantitativity, sensitivity, multiplexing, non-invasiveness and real-timeness. This validates fluorescence as a tool for engineering Cas9 and lays the groundwork for directly evolving Cas9 in microfluidic compartments.

synthetic biology↗

bioRxiv2022

Neural coding of temperature with a DNA-based spiking chemical neuron

Complex organisms perceive their surroundings with sensory neurons which encode physical stimuli into spikes of electrical activities. The past decade has seen reports of DNA-based chemical neurons that mimic artificial neural networks with chemical reactions. Yet, they lack the physical sensing and temporal coding of sensory biological neurons. Here we report a thermosensory chemical neuron based on DNA and enzymes that spikes with chemical activity when exposed to cold. Surprisingly, this chemical neuron shares deep mathematical similarities with a toy model of a cold nociceptive neuron: they follow a similar bifurcation route between rest and oscillations and avoid artefacts associated with canonical bifurcations (such as irreversibility, damping or untimely spiking). We experimentally demonstrate this robustness by encoding - digitally and analogically - thermal messages into chemical waveforms. This chemical neuron could pave the way for implementing in DNA the third generation of neural network models (spiking networks), and opens the door for associative learning. One-Sentence SummaryA DNA-based chemical network mathematically mimics the sensing of cold by a biological neuron.

biochemistry↗