Helicase-assisted continuous editing for programmable mutagenesis of endogenous genomes

• 成果类型: Article
• 署名作者: Chen, Xi Dawn; Chen, Zeyu; Wythes, George; Zhang, Yifan; Orr, Benno C.; Sun, Gary; Chao, Yu-Kai; Navarro Torres, Andrea; Thao, Ka; Vallurupalli, Mounica; Sun, Jing; Borji, Mehdi; Tkacik, Emre; Chen, Haiqi; Bernstein, Bradley E.; Chen, Fei
• 署名单位: Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; Harvard University; Harvard University; Harvard University; Harvard University Medical Affiliates; Dana-Farber Cancer Institute; Harvard University; Harvard Medical School; Harvard University; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute; University of Texas System; University of Texas Southwestern Medical Center; University of Texas System; University of Texas Southwestern Medical Center; Harvard University; Massachusetts Institute of Technology (MIT); Broad Institute
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-10953
• DOI: 10.1126/science.adn5876
• 发表日期: 2024-10-01
• 关键词: double-stranded dna; directed evolution; crystal-structure; rna helicase; mutations; base; protein; cd69; CONSEQUENCES; spliceosome

摘要

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.