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 systemwide losses of gene function.

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Mitotic recombination (MR) between homologous chromosomes is a mutational event that results in loss of heterozygosity in half of the segregants at mitosis. Loss of heterozygosity may have important biological consequences. The purpose of this study was to describe human variation in the spontaneous frequency of MR. Using an immunoselection technique for isolating the somatic mutations that result in loss of expression of one of the codominant alleles at the HLA-A locus, we have measured the frequency and molecular basis of somatic mutations in lymphocytes from a population of young adults. Mutations were classified as being the result of intragenic changes, major deletions, or MR. Here we show that the MR mutation frequency in females was significantly greater than that in males but that intragenic mutation frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed. Furthermore, the observed number of individuals from whom no mutants resulting from MR were obtained was significantly greater than was expected. The endogenous level of MR may be under genetic control. Given the association of loss of heterozygosity with cancer initiation and progression, low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply.