Versatile strategy for isolating TALEN-mediated knockout mutants in Caenorhabditis elegans.
Versatile strategy for isolating TALEN-mediated knockout mutants in Caenorhabditis elegans.
复制标题
在秀丽隐杆线虫中分离 TALEN 介导的敲除突变体的多功能策略。
DOI：
10.1111/dgd.12108
复制
发表时间：
2014
影响因子：
2.5
通讯作者：
Ohtani T and Yamamoto T
中科院分区：
文献类型：
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作者：
Sugi T;Sakuma T;Ohtani T and Yamamoto T
使用转录激活因子样效应物核酸酶（TALEN）和成簇的规则间隔短回文重复序列（CRISPR）/Cas9系统的靶向基因组编辑最近已经成为在秀丽隐杆线虫中创建基因座特异性突变的潜在强大方法。由于突变频率低，使用这些技术的关键步骤之一是筛选携带靶向突变的动物。在以前的研究中，鉴定秀丽隐杆线虫中的靶向突变通常依赖于对荧光标记物如绿色荧光蛋白或可见表型如矮胖和不协调表型的观察。然而，这种策略在实践中是有限的，因为靶向突变引起的表型，如感觉行为的缺陷往往是明显不可见的。在这里，我们描述了一种通过TALEN介导的基因组编辑和异源双链体迁移率测定分离秀丽隐杆线虫敲除突变体的通用策略。我们应用TALENs来设计神经基因glr-1的位点，glr-1是一种已知在各种感觉行为中起关键作用的秀丽隐杆线虫AMPA-型受体直系同源物。通过异源双链体迁移率测定，有效地鉴定了glr-1基因座中的敲除突变，其导致缺陷的机械感觉行为。因此，我们证明了基于TALEN的敲除策略用于创建具有导致不可见表型的突变的秀丽隐杆线虫的实用性。
Targeted genome editing using transcription activator‐like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus‐specific mutations inCaenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations inC. elegansusually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolatingC. elegansknockout mutants by TALEN‐mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr‐1, which is aC. elegansAMPA‐type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in theglr‐1locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN‐based knockout strategy for creatingC. eleganswith mutations that cause invisible phenotypes.