July 2019
Volume 60, Issue 9
Free
ARVO Annual Meeting Abstract  |   July 2019
A Patient-Specific Point Mutation Mouse Model of X-Linked Retinoschisis by CRISPR/Cas9 System
Author Affiliations & Notes
  • Mao Shengru
    Aier School of Ophthalmology, Central South University, Changsha, Hunan, China
    Aier Eye Institute, Changsha, Hunan, China
  • Yalan Zhou
    Aier School of Ophthalmology, Central South University, Changsha, Hunan, China
    Aier Eye Institute, Changsha, Hunan, China
  • Xin Yan
    Aier School of Ophthalmology, Central South University, Changsha, Hunan, China
    Aier Eye Institute, Changsha, Hunan, China
  • Yuzhuo Li
    Institute of Neuroscience, Chinese Academy of Sciences (CAS) Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Shanghai, Shanghai, China
  • Qiang Sun
    Institute of Neuroscience, Chinese Academy of Sciences (CAS) Key Laboratory of Primate Neurobiology, CAS Center for Excellence in Brain Science and Intelligence Technology, Shanghai Institutes for Biological Sciences, Shanghai, Shanghai, China
  • Jiansu Chen
    Aier Eye Institute, Changsha, Hunan, China
    Institute of Ophthalmology, Medical College, Jinan University, Guangzhou, Guangdong, China
  • Shibo Tang
    Aier School of Ophthalmology, Central South University, Changsha, Hunan, China
    Aier Eye Institute, Changsha, Hunan, China
  • Footnotes
    Commercial Relationships   Mao Shengru, None; Yalan Zhou, None; Xin Yan, None; Yuzhuo Li, None; Qiang Sun, None; Jiansu Chen, None; Shibo Tang, None
  • Footnotes
    Support  National Natural Scientific Fund of China (81570876)
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 4211. doi:
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      Mao Shengru, Yalan Zhou, Xin Yan, Yuzhuo Li, Qiang Sun, Jiansu Chen, Shibo Tang; A Patient-Specific Point Mutation Mouse Model of X-Linked Retinoschisis by CRISPR/Cas9 System. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4211.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : X-linked retinoschisis (XLRS) is a one of most common retinal genetic diseases of juvenile progressive vitreoretinal degeneration in males. Clinical studies have identified that more than 200 mutations of RS1 gene associated with XLRS. However, Rs1 knock-out mouse disease models are not consistent with XLRS patient specific characteristics exactly. Therefore, knock-in(KI) mouse models that carrying exact mutations which identified in patients are actively demanded. This study is to characterize a knock-in mouse model with patient-specific mutation c.304C>T (p.R102W) of XLRS.

Methods : The point mutation knock-in mice were created by co-injection of zygotes with Cas9 mRNA and sgRNAs in CRISPR/Cas9 system targeting Rs1. The specificity of gene editing was confirmed by deep sequencing and whole-genome sequencing. All eyes of KI and wild-type(WT) mice underwent ophthalmologic examinations such as fundus photography, optical coherence tomography (OCT) and electroretinogram (ERG) in postnatal 4-8 weeks. Retinas were isolated for HE staining in postnatal 12 weeks.

Results : We successfully produce the knock-in mice with patient-specific point mutation c.304C>T, (p.R102W) by using CRISPR/Cas9 system (Fig.1). The images of OCT and HE staining show cyst-like cavities and schisis in the retinal inner nuclear layer (INL) with outer plexiform layer(OPL) indistinction of our Rs1-R102W-KI model mice in postnatal 4 weeks (Fig.2A,B). ERG result displays that the b-wave enormously reduces in the tests and a-wave reduces slightly as well in Rs1-R102W-KI model mice when compared to those in WT mice (Fig.2C).

Conclusions : CRISPR/Cas9 system is highly efficient and precise in vivo, making it feasible to precise point XLRS model with XLRS patient characteristics. This model is a valuable for preclinical evaluation of XLRS disease mechanism and studies of new treatments.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Fig1. Genetic tests of one patient and the Rs1-R102W-KI mouse.(A) A missense mutation was found in a XLRS patient family.(B)DNA sequencing shows that the Rs1-R102W-KI mice have patient-specific point mutation.

Fig1. Genetic tests of one patient and the Rs1-R102W-KI mouse.(A) A missense mutation was found in a XLRS patient family.(B)DNA sequencing shows that the Rs1-R102W-KI mice have patient-specific point mutation.

 

Fig2: OCT, HE staining and Electrophysiological Properties. (A) OCT shows the typical retinoschisis in retinal INL of Rs1-R102W-KI mice compared with WT mice(blue arrows). (B) HE staining demonstraits schisis in INL(red arrows). (C) ERG displays the decreased change in the b- waves in Rs1-R102W-KI mice compared with WT mice.

Fig2: OCT, HE staining and Electrophysiological Properties. (A) OCT shows the typical retinoschisis in retinal INL of Rs1-R102W-KI mice compared with WT mice(blue arrows). (B) HE staining demonstraits schisis in INL(red arrows). (C) ERG displays the decreased change in the b- waves in Rs1-R102W-KI mice compared with WT mice.

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