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Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-Gly374Arg mutation in achondroplasia in mice
Kai Miao1,2; Xin Zhang1,3; Sek Man Su1,2; Jianming Zeng1,2; Zebin Huang1,2; Un In Chan1,3; Xiaoling Xu1,2,3; Chu-Xia Deng1,2
2018-11-28
Source PublicationThe Journal of Biological Chemistry
ISSN0021-9258
Abstract

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.

KeywordFgfr3 Crispr/cas9 Protein Targeted Correction Off-target Achondroplasia Dwarfism Genome Editing Gene Therapy
DOIhttp://doi.org/10.1074/jbc.RA118.006496
Language英语
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Document TypeJournal article
CollectionFaculty of Health Sciences
Co-First AuthorKai Miao
Corresponding AuthorXiaoling Xu; Chu-Xia Deng
Affiliation1.Cancer Center, Faculty of Health Sciences, University of Macau, Macau SAR, China
2.Centre for Precision Medicine Research and Training, Faculty of Health Sciences, University of Macau, Macau SAR, China
3.Transgenic and Knockout Core, Faculty of Health Sciences, University of Macau, Macau SAR, China
First Author AffilicationFaculty of Health Sciences
Corresponding Author AffilicationFaculty of Health Sciences
Recommended Citation
GB/T 7714
Kai Miao,Xin Zhang,Sek Man Su,et al. Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-Gly374Arg mutation in achondroplasia in mice[J]. The Journal of Biological Chemistry,2018.
APA Kai Miao.,Xin Zhang.,Sek Man Su.,Jianming Zeng.,Zebin Huang.,...&Chu-Xia Deng.(2018).Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-Gly374Arg mutation in achondroplasia in mice.The Journal of Biological Chemistry.
MLA Kai Miao,et al."Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-Gly374Arg mutation in achondroplasia in mice".The Journal of Biological Chemistry (2018).
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