Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function.

中文翻译：

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CRISPR-TSKO：一种在拟南芥中特定细胞类型、组织或器官中进行有效诱变的技术。

通过传统的功能丧失方法对许多根本上重要的植物基因进行详细的功能分析受到这些损失导致的严重多效性表型的阻碍。特别是，基本细胞功能和/或繁殖所需的基因突变通常会干扰纯合突变植物的产生，从而阻碍进一步的功能研究。为了克服这一限制，我们设计了一种基于成簇规则间隔短回文重复序列 (CRISPR) 的组织特异性敲除系统 CRISPR-TSKO，能够在特定植物细胞类型、组织和器官中产生体细胞突变。在拟南芥（Arabidopsis thaliana）中，必需基因中的 CRISPR-TSKO 突变导致根冠、气孔谱系或整个侧根出现明确的局部表型。本研究开发的模块化克隆系统可以直接在第一代转基因中对突变株系进行有效的选择、鉴定和功能分析。CRISPR-TSKO 的功效为发现和分析植物生命时空背景下的基因功能开辟了途径，同时避免了全系统基因功能丧失的多效性影响。