Smart RNA guides for conditional control of CRISPR/Cas9 – SmartGuideRNA

In the past 5 years, a protein from prokaryotes has revolutionized biology in ways not seen since PCR in the 80s. CRISPR (Clustered regularly interspaced short palindromic repeats) is an adaptive immunity system for bacteria and archaea that fights off viruses by keeping a DNA record of past infections. This CRISPR array is transcribed, matured into short RNAs and loaded into a programmable nuclease (Cas9) that continuously searches for and cleaves DNA matching the loaded RNA guide. Type II CRISPR stands out by its sheer simplicity as it comprises only two parts: a programmable nuclease (Cas9) and a RNA guide (either a chimeric RNA guide or a duplex crRNA:tracrRNA). Gene editing with Cas9 has fast become a plug-and-play routine, and Cas9 is so versatile that it has been retooled to cut-and-paste DNA, activate or repress gene expression, scout around the genome for particular loci, modulate epigenetic activities or fluorescently illuminate chromosomal regions. Beyond gene editing, CRISPR is poised to be a tool of choice for genome-wide screening, gene and cellular therapy, disease modelling or xenograft transplantation. Cas9 has even been proposed to serve as a molecular recorder to continuously log cellular events in a DNA register.

Nagging problems remain for safe, efficient and versatile deployment of CRISPR/Cas9 in life sciences, biotechnology and medicine. Specifically controlling Cas9 remains challenging. Once Cas9 and a guide RNA are both present in a cell, the tandem will inevitably cut its target loci, irrespectively of the type, environment or developmental status of the cell. But mistimed or misplaced action of Cas9 could prove detrimental. Distinct cellular types follow distinct developmental programs, and premature intervention by Cas9 could wreak havoc on cellular progeny. Various strategies have been suggested to control Cas9 (namely split enzymes, small molecule induction or photo-activation), but they are rather crude, lack single-cell precision and are not autonomous because they rely on an external operator. Lastly, the delivery of the inducing signal is limiting: optogenetic methods are hindered by the low penetrability of visible light into deep tissues, and the timed delivery of chemical signals to cells with high specificity is challenging.

The goal of this project is to conditionally control the cutting of Cas9 in response to internal, tissular or environmental stimuli. To do this, we will bring about the large repertoire of molecular processing tools developed by the community of DNA nanotechnology: not only nanostructured shapes, but also logic circuits, diagnostics systems, autonomous walkers, pattern classifiers... We will adopt a high-throughput approach thanks to the massive parallelism allowed by droplet microfluidics and next generation sequencing.


(see confidential part of the application for further details)