Efficient simultaneous mutagenesis of multiple genes in specific plant tissues by multiplex CRISPR.

CRISPR technology is an established tool for the generation of knockout plants (Zhang et al., 2019), yet limitations remain. First, the manipulation of individual genes may fail to produce phenotypes for groups of genes with redundant or synergistic functions. While this has been partially addressed by multiplexing guide RNAs (gRNAs), there is concern that as the number of targets increases, the chances of obtaining higher-order knockouts diminish (Zhang et al., 2016). Second, knocking out fundamentally important genes can cause severe pleiotropic phenotypes or lethality. Tissue-specific knockout of genes in somatic tissues can overcome this limitation (Decaestecker et al., 2019 ; Wang et al., 2020 ; Liang et al., 2019). However, the efficiency of simultaneously targeting more than three genes in a tissue-specific context is unexplored. Here, by multiplexing gRNAs in Arabidopsis thaliana plants expressing Cas9 either ubiquitously (pPcUBI) or root cap-specifically (pSMB), we show that six genes can be simultaneously mutated with high efficiency, generating higher-order mutant phenotypes already in the first transgenic generation (T1). The mutation frequencies for all target genes were positively correlated and unaffected by the order of the gRNAs in the vector, showing that efficient higher-order mutagenesis in specific plant tissues can be readily achieved.