BACKGROUND:TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity.METHODS:We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy–associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation.RESULTS:CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts.CONCLUSIONS:TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.

中文翻译：

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CRISPR 激活可逆转 TTN 截短变异引起的单倍体不足和功能缺陷

背景：TTN截短变异 (TTNtvs) 是扩张型心肌病患者中最常见的遗传病变，扩张型心肌病是一种发病率和死亡率很高的疾病。 TTNtvs 降低正常 TTN（肌联蛋白）蛋白水平，产生截短的蛋白，并损害肌节内容和功能。由于 TTN 的巨大尺寸、特定 TTNtv 的稀有性以及对 TTNtv 致病性的不完全了解，针对 TTNtv 的治疗一直难以捉摸。方法：我们使用 dCas9-VPR 调整 CRISPR 激活来功能性地询问 TTNtv 致病性，并开发一种针对人类心肌细胞和 TTNtv 的治疗方法。由诱导多能干细胞模型改​​造而成的 3 维心脏微组织，含有与扩张型心肌病相关的 TTNtv。我们使用定制 TTN 报告基因检测、琼脂糖凝胶电泳来量化 TTN 蛋白水平和亚型，以及 RNA 测序来识别 TTN 激活的分子后果，进行引导 RNA 筛选。心肌细胞表观遗传测定也被用来指定 DNA 调控元件，以实现心肌细胞特异性 TTN 激活。结果：使用单引导 RNA CRISPR 激活 TTN，通过 3 维染色质相互作用靶向TTN启动子或空间邻近TTN启动子的调控元件挽救了受 TTNtvs 干扰的 TTN 蛋白缺陷。尽管增加了截短的 TTN 蛋白，但增加 TTN 蛋白水平可使肌小节含量和收缩功能正常化。除了TTN转录本外，CRISPR 激活还增加了肌原纤维组装相关和肌节相关转录本的水平。 结论：尽管增加了截短的 TTN 水平，但 TTN CRISPR 激活挽救了 TTNtv 相关的功能缺陷，这提供了支持单倍体不足作为相关遗传机制的证据潜在杂合 TTNtvs。 CRISPR 激活可以开发为治疗大部分 TTNtv 的疗法。