Gene Regulation Observatory, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
Science. 2024 Oct 11;386(6718):eadn5876. doi: 10.1126/science.adn5876.
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.
解析序列与功能之间的特定关系是基因组学面临的主要挑战。目前在天然基因组背景下诱导基因座特异性超突变和进化的工具是有限的。在这里，我们提出了一种称为解旋酶辅助连续编辑（HACE）的长程、基因座特异性超突变可编程平台。HACE 利用 CRISPR-Cas9 靶向一个连续的解旋酶-脱氨酶融合，在大 (>1000 碱基对)基因组间隔区引发突变。我们应用 HACE 来鉴定丝裂原活化蛋白激酶激酶 1 (MEK1)中的突变，这些突变赋予激酶抑制剂抗性，剖析剪接因子 3B 亚基 1 (SF3B1)依赖性错剪接中单个变体的影响，并评估 CD69 刺激依赖性免疫增强子中的非编码变体。HACE 为研究编码和非编码变体、揭示组合序列-功能关系以及进化新的生物学功能提供了强大的工具。
