Dear Editor,

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based gene editing has been used to simultaneously generate mutations in multiple target genes in many plant species (Armario Najera et al., 2019; Bai et al., 2020; Luo et al., 2021). Utilization of ribozyme, endogenous tRNA, bacterium-derived Cpf1 endonuclease, and Csy4 ribonuclease to produce multiple guide RNAs (gRNAs) simplifies multiplex gene editing (Gao and Zhao, 2014; Xie et al., 2015; Wang et al., 2017; Ding et al., 2018; Xu et al., 2019; Zhang et al., 2021). However, multiplex gene editing is still labor-intensive and time-consuming. Editing efficiency of gRNAs depends on target sequences that differ in different genes (Ma et al., 2015; Miao et al., 2018). Consequently, a large number of transgenic plants are required for screening in a couple and even several generations to obtain homozygous mutations for all target genes. Further, due to the presence of the CRISPR/Cas9 transgenes, new mutations may be generated along with propagations in generations (Zhang et al., 2018). So far, several technologies have been developed to obtain transgene-free gene-edited plants, including examinations of a large population of plants generated by transiently expressed gene-editing components (Zhang et al., 2016; Chen et al., 2018), or segregations from stable gene-edited plants (Gao et al., 2016); however, screening and genotyping a large number of T0 or T1 plants are labor-intensive (He and Zhao, 2020). Transgenes can also be removed from gene-edited plants by crossing with the wild-type, but the removal is practically difficult in multiplex gene editing, considering the segregations of multiple loci including the CRISPR/Cas9 transgene and mutation loci in the F2 generation. Recently, the Transgene Killer CRISPR (TKC) technology was developed that relies on spatial–temporal expressions of suicide cassettes of p35S:: Cytoplasmic Male Sterility 2 (CMS2) and pREG2::BARNASE to kill all transgene-containing sperms and embryos, respectively (He et al., 2018). In this study, we developed a Customized Assembly and Simplified Editing (CASE) toolkit in rice (Oryza sativa) that combines TKC technology with multiplex gene editing. The toolkit provides an easy and efficient way to obtain transgene-free gene-edited plants for multiple genes in the T1 generation.