Center for Genome Engineering, Institute for Basic Science, Daejeon 34126, Republic of Korea; email:
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA; email:
Annu Rev Biochem. 2019 Jun 20;88:191-220. doi: 10.1146/annurev-biochem-013118-111730. Epub 2019 Mar 18.
Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.
可编程核酸酶和脱氨酶，包括锌指核酸酶、转录激活样效应物核酸酶、CRISPR RNA 指导的核酸酶和 RNA 指导的碱基编辑器，现在被广泛用于细胞和生物体中基因组的靶向修饰。这些基因编辑工具在治疗应用方面具有巨大的潜力。重要的是，这些核酸酶和脱氨酶可能通过识别与其靶位点接近的同源 DNA 序列而表现出脱靶活性，从而导致基因组以局部突变或基因组重排的形式受到附带损伤。对于这些可编程酶的治疗性基因组编辑应用，测量和限制全基因组脱靶活性至关重要。本文讨论了这些系统脱靶活性的关键决定因素。我们描述了用于识别全基因组脱靶位点的各种基于细胞和无细胞的方法，以及为降低可编程基因编辑酶的脱靶活性而开发的各种策略。
