Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.
Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.
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DOI：
10.1093/nar/gky548
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发表时间：
2018-09-19
影响因子：
14.9
通讯作者：
VandenDriessche T
中科院分区：
文献类型：
--
作者：
Dastidar S;Ardui S;Singh K;Majumdar D;Nair N;Fu Y;Reyon D;Samara E;Gerli MFM;Klein AF;De Schrijver W;Tipanee J;Seneca S;Tulalamba W;Wang H;Chai YC;In't Veld P;Furling D;Tedesco FS;Vermeesch JR;Joung JK;Chuah MK;VandenDriessche T
CRISPR/Cas9是一个有吸引力的平台，可以通过基因编辑以前所未有的精度纠正显性遗传疾病。在目前的原理验证研究中，我们探索了CRISPR/Cas9在强直性肌营养不良1型（DM 1）（一种常染色体显性肌肉疾病）中的基因编辑的用途，方法是切除DM 1患者特异性诱导多能干细胞中人强直性肌营养不良蛋白激酶（DMPK）基因3′-非编码区（UTR）中的CTG重复扩增在一些实施方案中，所述细胞包括DM 1-iPSC、DM 1-iPSC衍生的肌源性细胞和DM 1患者特异性成肌细胞。为了消除致病性功能获得性突变体DMPK转录物，我们设计了一种基于双指导RNA的策略，其以高效率切除CTG重复扩增，如通过Southern印迹和单分子实时（SMRT）测序所证实的。在CRISPR/Cas9组分的核糖核蛋白（RNP）转染后，在DM 1-iPSC中可以达到高达90%的校正效率，如在克隆水平上证实的，而不需要选择性富集。在校正的DM 1-iPSC、DM 1-iPSC衍生的肌原性细胞和DM 1成肌细胞中，扩大的CTG重复序列切除导致核糖核病灶（DM 1的典型细胞表型）消失。因此，肌盲样剪接调节因子1（MBNL 1）的正常细胞内定位得以恢复，导致SERCA 1剪接模式的正常化。该研究验证了CRISPR/Cas9用于重复扩增的基因编辑的用途。
CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3′-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.
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