以CRISPR-Cas9系統建立模擬人類CRYAA突變之先天性白內障小鼠疾病模式

Generation of Congenital Cataract Mouse that Recapitulates Human CRYAA Mutations via the CRISPR-Cas9 System

蘇家賢(Chia-Hsien Su)

指導教授 ： 吳信志

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201704246

摘要

先天性白內障是一種可能造成視力損傷或失明的先天性遺傳眼科疾病。先前研究對先天性白內障患者進行定序和連鎖分析，以確認可能存在於白內障相關基因的DNA序列變異，結果發現一些患者帶有CRYAA基因突變，然而該基因突變之致病機制尚未完全明瞭。基因工程技術為研究基因功能時重要的研究方法之一。CRISPR-Cas9系統是近年來新興的基因編輯工具，由短嚮導RNA (single guide RNA, sgRNA) 和Cas9核酸酶組成，兩者協同作用誘發DNA雙股斷裂，引發細胞修復機制，進而造成缺失突變或特定序列的準確編輯。
本試驗擬利用 CRISPR-Cas9 系統將已知存在於人類先天性白內障患者的Cryaa 突變序列，導入小鼠基因組中。體外試驗部分，將能夠同時表現 sgRNA 和 Cas9 核酸酶的質體轉染至小鼠胚胎纖維母細胞 (mouse embryonic fibroblast, MEF) 中，利用質體含有的報導基因 mCherry 的特性，以流式細胞分選儀篩選出轉染成功的 MEF，並從分析結果比較 sgRNA 之效率。接著，將體外試驗中三組作用效率最佳 sgRNA、Cas9 mRNA 及單股 DNA 模板，顯微注射至一細胞期小鼠胚之細胞質中，於二細胞期或囊胚期進行胚移置，共產下 8 隻小鼠，其中 2 隻以裂隙燈觀察拍照發現具有先天性白內障病徵，也利用仔鼠耳刻萃取基因組 DNA 進行定序，定序結果發現帶有白內障的小鼠皆為具有 Cryaa 基因突變的個體，而不具白內障病徵的小鼠與野生型 C57BL/6j 品系小鼠的 Cryaa 基因序列相同。另外，預計於 F0 繁殖子代後再進行眼球組織切片染色。
目前尚未完全瞭解小鼠 Cryaa 突變與先天性白內障之間的分子機制與疾病機轉，本研究藉由將sgRNA、Cas9 mRNA 及單股 DNA 模板共同注射到小鼠一細胞期受精卵中，產製出具有 Cryaa 基因突變的先天性白内障疾病模式小鼠，以模擬人類先天性白內障患者的狀況。此疾病模式可作為未來研究者欲深入探討時的動物材料，望能為此基因的功能和分子機制提供研究基礎。

關鍵字

白內障 ； 先天性白內障 ； CRISPR-Cas9系統 ； Cryaa ； 先天性白内障疾病模式

並列摘要

Congenital cataract is a disease caused by congenital hereditary opacity of the lens which may lead to visual impairment or blindness. Previous studies performed sequencing and linkage analysis of congenital cataract patients to confirm the candidate causative variants in cataract-related genes, and found that these patients had CRYAA mutations. However, the pathogenesis of these gene mutations are not fully understood. Genetic engineering technology is one of the important research methods for studying gene function, and the CRISPR-Cas9 system is an emerging genetic editing tool in recent years. The system consists of two components, single guided RNA (sgRNA) and Cas9 nuclease which initiate DNA double strand breaks. Then, the cellular DNA repair system gives rise to insertions or deletion mutations or precise editing.
The aim of this study is to introduce mutations identified in human CRYAA gene into mouse genome via CRISPR-Cas9 system. In the in vitro study, the plasmid expressing sgRNA and Cas9 nuclease were transfected into mouse embryonic fibroblast (MEF). The transfected cells were sorted by cell sorter using the characteristics of the reporter gene, and we could compare the efficiency of sgRNA from the analysis results. Next, three efficient sgRNA, Cas9 mRNA and single-strand DNA template were injected into the cytoplasm of one-cell mouse embryos by microinjection. When these embryos were cultured to two-cell stage or blastocyst stage, we performed embryo transfer into pseudo-pregnant mice. They currently gave birth 8 pups, 2 were found to have congenital cataract symptoms by slit lamp biomicroscopy. The genomic DNA was extracted from the ear of the pups for sequencing, and the sequencing results showed that mice with cataracts had Cryaa mutations. The F0 mice that showing no cataract phenotype had the same Cryaa sequence as the wild-type C57BL/6j mice. In addition, lens tissue sections and hematoxylin and eosin staining will be performed after the F0 mice breeding progeny.
The molecular mechanism and pathogenesis of mouse Cryaa mutations and congenital cataracts are not fully understood. This study generated congenital cataract mice with Cryaa gene mutation by co-injection of sgRNA, Cas9 mRNA and single-strand DNA templates into mouse one-cell embryos. This disease model can be used by future researchers to investigate the gene function of Cryaa, and it is expected to provide a research basis for the function and molecular mechanism of this gene.

並列關鍵字

cataract ； congenital cataract ； CRISPR-Cas9 system ； Cryaa ； congenital cataract disease model

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