Abstract

The field of gene editing is rapidly moving towards the implementation of CRISPR/Cas9–based approaches to tackle human disease. The development of novel Cas9 variants is a crucial step to achieve precise and effective gene therapy.To validate the safety of modified Cas9 versions, standardized experimental strategies for the evaluation of on-target endonuclease efficacy and potential toxicity, in vivo, are necessary since in vitro approaches are not predictive of the impact that such complex molecules can have on a living organism. Here, we propose a zebrafish-based pipeline for quality control (QC) of two Cas9 protein variants, by evaluating, in a single assay, Cas9-induced toxicity, teratogenicity and on-target mutagenesis. To perform this comparative analysis, we established a multistep approach composed by: 1) dose-range finding of Cas9/sgRNA complexes based on mortality evaluation at different timepoints after injection; 2) assessment of seven teratogenic phenotypes (heart edema, scoliosis, body deformity, craniofacial edema, defects in body axis curvature, eye diameter, pigmentation) to uncover putative developmental defects induced by the injection of Cas9/sgRNA complexes 3) high-throughput analysis of loss-of-function phenotypes by employing a fully automated microfluidic system 4) evaluation of Cas9 mutagenesis efficacy based on INDEL analysis of each analyzed larva for accurate correlation between phenotype and genotype. As proof of principle, we targeted the tyrosinase locus, since biallelic disruption of this gene results in loss of pigmentation, thus providing a direct readout of Cas9/sgRNA efficacy. Overall, our results provide evidence supporting the use of zebrafish as a robust system to perform high throughput QC of new Cas9 protein variants. This study paves the ground for the simultaneous validation of several gene therapy approaches employing innovative gene editing tools targeted to different therapeutic areas.More about ZeClinics