Xi Dawn Chen, Zeyu Chen, George Wythes, Yifan Zhang, Benno C. Orr, Gary Sun, Yu-Kai Chao, Andrea Navarro Torres, Ka Thao, Mounica Vallurupalli, Jing Sun, Mehdi Borji, Emre Tkacik, Haiqi Chen, Bradley E. Bernstein, Fei Chen
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引用次数: 0
摘要
破译序列与功能之间的特定环境关系是基因组学的一大挑战。在原生基因组环境中诱导基因座特异性超突变和进化的现有工具非常有限。在这里,我们提出了一种用于长程、位点特异性超突变的可编程平台,称为螺旋酶辅助连续编辑(HACE)。HACE利用CRISPR-Cas9以过程性螺旋酶-脱氨酶融合为目标,在大(大于1000碱基对)基因组区间产生突变。我们应用 HACE 鉴定了使激酶抑制剂产生抗性的丝裂原活化蛋白激酶激酶 1(MEK1)中的突变,剖析了剪接因子 3B 亚基 1(SF3B1)依赖性错误剪接中单个变异的影响,并评估了 CD69 的刺激依赖性免疫增强子中的非编码变异。HACE 为研究编码和非编码变异、揭示序列与功能的组合关系以及开发新的生物功能提供了强大的工具。
Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes
Deciphering the context-specific relationship between sequence and function is a major challenge in genomics. Existing tools for inducing locus-specific hypermutation and evolution in the native genome context are limited. Here we present a programmable platform for long-range, locus-specific hypermutation called helicase-assisted continuous editing (HACE). HACE leverages CRISPR-Cas9 to target a processive helicase-deaminase fusion that incurs mutations across large (>1000–base pair) genomic intervals. We applied HACE to identify mutations in mitogen-activated protein kinase kinase 1 (MEK1) that confer kinase inhibitor resistance, to dissect the impact of individual variants in splicing factor 3B subunit 1 (SF3B1)–dependent missplicing, and to evaluate noncoding variants in a stimulation-dependent immune enhancer of CD69. HACE provides a powerful tool for investigating coding and noncoding variants, uncovering combinatorial sequence-to-function relationships, and evolving new biological functions.
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