Hendel Ayal, Kildebeck Eric J, Fine Eli J, Clark Joseph, Punjya Niraj, Sebastiano Vittorio, Bao Gang, Porteus Matthew H
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
Cell Rep. 2014 Apr 10;7(1):293-305. doi: 10.1016/j.celrep.2014.02.040. Epub 2014 Mar 27.
Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.
利用工程核酸酶进行的靶向基因组编辑已经改变了在基因组内几乎任何位点引入精确序列修饰的能力。探究基因组编辑效率和后果的一个主要障碍是，目前没有任何方法能够在任何内源性基因组位点同时测量细胞群体中不同编辑事件的频率。我们开发了一种利用单分子实时（SMRT）DNA测序来量化任何感兴趣位点的个体基因组编辑结果的方法。我们表明，这种方法可以使用多种工程核酸酶平台，包括转录激活样效应核酸酶（TALENs）、RNA引导的核酸内切酶（CRISPR/Cas9）和锌指核酸酶（ZFNs），应用于各种位点，并在不同细胞系中鉴定出发生所需工程结果的条件和策略。这种方法提供了一种研究双链断裂修复的技术，有助于评估基因编辑技术，并允许在几乎所有使用的实验系统中对编辑结果进行灵敏的量化。
