Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-Gly374Arg mutation in achondroplasia in mice.

CRISPR/Cas9 is a powerful technology widely used for genome editing, with the potential to be used for correcting a wide variety of deleterious disease-causing mutations. However, the technique tends to generate more indels (insertions and deletions) than precise modifications at the target sites, which might not resolve the mutation and could instead exacerbate the initial genetic disruption. We sought to develop an improved protocol for CRISPR/Cas9 that would correct mutations without unintended consequences. As a case study, we focused on achondroplasia, a common genetic form of dwarfism defined by missense mutation in the Fgfr3 gene that results in glycine-to-arginine substitution at position 374 in mice in fibroblast growth factor receptor 3 (Fgfr3-Gly374Arg), which corresponds to Gly380Arg in humans. First, we designed a GFP reporter system that can evaluate the cutting efficiency and specificity of sgRNAs. Using the sgRNA selected based on our GFP reporter system, we conducted targeted therapy of achondroplasia in mice. We found that we achieved higher frequency of precise correction of Fgfr3-Gly374Arg mutation using Cas9 protein rather than Cas9 mRNA. We further demonstrated that targeting oligos of 100nt and 200nt precisely corrected the mutation at equal efficiency. We showed that our strategy completely suppressed phenotypes of achondroplasia and whole genome sequencing detected no off-target effects. These data indicate that improved protocols can enable the precise CRISPR/Cas9 mediated correction of individual mutations with high fidelity.