Yu Chuan, Zhang Yaguang, Yao Shaohua, Wei Yuquan
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital and College of Life Science, Sichuan University, Chengdu, Sichuan, People's Republic of China.
PLoS One. 2014 Jun 5;9(6):e98282. doi: 10.1371/journal.pone.0098282. eCollection 2014.
Genome editing techniques such as the zinc-finger nucleases (ZFNs), transcription activator-like effecter nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system Cas9 can induce efficient DNA double strand breaks (DSBs) at the target genomic sequence and result in indel mutations by the error-prone non-homologous end joining (NHEJ) DNA repair system. Several methods including sequence specific endonuclease assay, T7E1 assay and high resolution melting curve assay (HRM) etc have been developed to detect the efficiency of the induced mutations. However, these assays have some limitations in that they either require specific sequences in the target sites or are unable to generate sequencing-ready mutant DNA fragments or unable to distinguish induced mutations from natural nucleotide polymorphism. Here, we developed a simple PCR-based protocol for detecting indel mutations induced by TALEN and Cas9 in zebrafish. We designed 2 pairs of primers for each target locus, with one putative amplicon extending beyond the putative indel site and the other overlapping it. With these primers, we performed a qPCR assay to efficiently detect the frequencies of newly induced mutations, which was accompanied with a T-vector-based colony analysis to generate single-copy mutant fragment clones for subsequent DNA sequencing. Thus, our work has provided a very simple, efficient and fast assay for detecting induced mutations, which we anticipate will be widely used in the area of genome editing.
基因组编辑技术，如锌指核酸酶（ZFNs）、转录激活样效应核酸酶（TALENs）和成簇规律间隔短回文重复序列（CRISPR）/CRISPR相关蛋白（Cas）系统Cas9，能够在目标基因组序列处诱导高效的DNA双链断裂（DSBs），并通过易出错的非同源末端连接（NHEJ）DNA修复系统导致插入缺失突变。已经开发了几种方法，包括序列特异性核酸内切酶测定、T7E1测定和高分辨率熔解曲线分析（HRM）等，以检测诱导突变的效率。然而，这些测定存在一些局限性，即它们要么需要靶位点中的特定序列，要么无法产生可用于测序的突变DNA片段，要么无法将诱导突变与天然核苷酸多态性区分开来。在这里，我们开发了一种基于PCR的简单方案，用于检测斑马鱼中由TALEN和Cas9诱导的插入缺失突变。我们为每个靶位点设计了2对引物，其中一个推定扩增子延伸超过推定的插入缺失位点，另一个与之重叠。使用这些引物，我们进行了qPCR测定，以有效检测新诱导突变的频率，并伴随着基于T载体的菌落分析，以生成单拷贝突变片段克隆用于后续的DNA测序。因此，我们的工作提供了一种非常简单、高效和快速的检测诱导突变的方法，我们预计该方法将在基因组编辑领域得到广泛应用。
