Biology subjects

Despres, P. C.

Publications and source records attributed to Despres, P. C..

2 recordsLinked to original sources

bioRxiv2018

beditor: A computational workflow for designing libraries of guide RNAs for CRISPR base editing

CRISPR-mediated base editors have opened unique avenues for scar-free genome-wide mutagenesis. Here, we describe a comprehensive computational workflow called beditor that can be broadly adapted for designing guide RNA libraries with a range of CRISPR-mediated base editors, PAM recognition sequences and genomes of many species. Additionally, in order to assist users in selecting the best sets of guide RNAs for their experiments, a priori estimates, called beditor scores are calculated. These beditor scores are intended to select guide RNAs that conform to requirements for optimal base editing: editable base falls within maximum activity window of the CRISPR-mediated base editor and produces non-confounding mutational effects with minimal off-target effects. We demonstrate the utility of the software by designing guide RNAs for base-editing mediated edition of thousands of clinically important human disease mutations.

synthetic biology↗

bioRxiv2018

Highly efficient CRISPR gene editing in yeast enabled by double selection

CRISPR-Cas9 loss of function (LOF) and base editing screens are powerful tools in genetics and genomics. Yeast is one of the main models in genetics and genomics, yet large-scale approaches remain to be developed in this species because of low mutagenesis rates without donor DNA. We developed a double selection strategy based on co-selection that increases LOF mutation rates, both for CRISPR-Cas9 and the Target-AID base editor. We constructed the pDYSCKO vector, which is amenable to high throughput double selection for both approaches. Using modeling, we show that this improvement provides the required increased in detection power to measure the fitness effects of thousands of mutations in typical yeast pooled screens. We also show that multiplex genome editing with Cas9 causes programmable chromosomal translocations at high frequency, suggesting that multiplex editing should be performed with caution and that base-editors could be preferable tools for LOF screens.

synthetic biology↗