CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

Detailed functional analyses of many fundamentally important genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. In particular, mutations in genes that are required for basic cellular functions or plant reproduction often interfere with the generation of homozygous mutant plants precluding further functional studies. To overcome this limitation, we devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling us to generate mutations in particular plant cell types, tissues, and organs. In Arabidopsis, resulting somatic mutations even in essential genes caused well-defined, localized phenotypes with high efficiency in the root cap, stomatal lineage, or in entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to analyze gene functions in virtually any given spatial and temporal context of plant life while avoiding pleiotropic effects of system-wide loss of gene function.