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HUI XU, Sandeep Kumar, Yingbin Fu; Efficient deletion of the IGFBP domain of multifunctional serine protease HTRA1 by CRISPR/Cas9. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2363.
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© ARVO (1962-2015); The Authors (2016-present)
Two synonymous single nucleotide polymorphisms (SNPs) rs1049331 and rs2293870 in IGFBP domain of HTRA1 were reported to be associated with increased risk to neovascular (Nv) type age-related macular degeneration (AMD). The proposed mechanism is that the two SNPs convert common codons for Ala34 and Gly36 to less frequently used codons and affect the folding of the IGFBP domain, which in turn reduced HTRA1’s ability to antagonize insulin-like growth factor 1 (IGF-1)-stimulated signaling events and cellular responses. To investigate the role of the IGFBP domain of HTRA1 in AMD pathogenesis, we generated mice in which the IGFBP domain of HTRA1 was successfully deleted using the CRISPR/Cas9 technology.
We first designed four single-guide RNAs (sgRNAs) targeting the IGFBP domain of HTRA1. Mouse embryonic fibroblasts (MEFs) from C57Bl6 were electroporated with the plasmids encoding sgRNAs and Cas9. The efficiency of each of four sgRNAs were evaluated by SURVEYOR mutation detection kit (Transgenomic). The most efficient three sgRNAs targeting the beginning, middle, and the end of the IGFBP domain were selected for pronuclear injection in C57Bl6 background with either 5 ng or 10 ng of sgRNA plasmids. PCR and DNA sequencing of PCR products were used to determine the modifications of the three sgRNA targets of sixty-five founder mice and their offspring.
Thirty-nine mice from 5 ng and twenty-six from 10 ng injection (total sixty-five) were generated. DNA Sequencing showed highly efficient genomic modification by CRISPR/Cas9, sixty out of sixty-five (92%) founders have modifications in sgRNA targets in IGFBP domain. Moreover, Thirteen out of thirty-nine (33%, 5 ng plasmids) and seven out of twenty-six (26%, 10 ng plasmids) founders showed obvious deletions by PCR using primers covering the IGFBP domain. DNA sequencing showed that four mice had in-frame deletions (6.1%), which deleted the IGFBP domain from HTRA1. Inheritance of IGFBP-domain specific deletion was confirmed by DNA sequencing of the F1 offspring.
We showed that IGFBP domain of HTRA1 can be deleted efficiently using CRISPR/Cas9 technology, which provides a powerful gene modification tool for research in Vision and Ophthalmology.
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