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Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice
Miao,Kai1,2; Zhang,Xin1,2,3; Su,Sek Man1,2; Zeng,Jianming1,2; Huang,Zebin1,2; Chan,Un In1,3; Xu,Xiaoling1,2,3; Deng,Chu Xia1,2
2019-01-25
Source PublicationJournal of Biological Chemistry
ISSN0021-9258
Volume294Issue:4Pages:1142-1151
AbstractCRISPR/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-G374R), which corresponds to G380R in humans. First, we designed a GFP reporter system that can evaluate the cutting efficiency and specificity of single guide RNAs (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 the Fgfr3-G374R mutation using Cas9 protein rather than Cas9 mRNA. We further demonstrated that targeting oligos of 100 and 200 nucleotides 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.
DOI10.1074/jbc.RA118.006496
URLView the original
Language英语
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Cited Times [WOS]:4   [WOS Record]     [Related Records in WOS]
Document TypeJournal article
CollectionUniversity of Macau
Corresponding AuthorXu,Xiaoling
Affiliation1.Cancer Center,Faculty of Health Sciences,University of Macau,China
2.Centre for Precision Medicine Research and Training,Faculty of Health Sciences,University of Macau,China
3.Transgenic and Knockout Core,Faculty of Health Sciences,University of Macau,China
First Author AffilicationFaculty of Health Sciences
Corresponding Author AffilicationFaculty of Health Sciences
Recommended Citation
GB/T 7714
Miao,Kai,Zhang,Xin,Su,Sek Man,et al. Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice[J]. Journal of Biological Chemistry,2019,294(4):1142-1151.
APA Miao,Kai.,Zhang,Xin.,Su,Sek Man.,Zeng,Jianming.,Huang,Zebin.,...&Deng,Chu Xia.(2019).Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice.Journal of Biological Chemistry,294(4),1142-1151.
MLA Miao,Kai,et al."Optimizing CRISPR/Cas9 technology for precise correction of the Fgfr3-G374R mutation in achondroplasia in mice".Journal of Biological Chemistry 294.4(2019):1142-1151.
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