Transgene-Killer-CRISPR version 2 (TKC2) eliminates occasional transgene escape by coupling with a RUBY reporter

A critical step in generating gene-edited plants is the removal of CRISPR-related transgenes from T₀ plants and their progenies, a process that is generally time-consuming and labour-intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self-elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T₁ plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY-CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T₀ generation and its progenies. We then combined the RUBY-CRISPR unit with several TKC variants to develop four RUBY-TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non-TKC, TKC alone and RUBY-CRISPR, our TKC2s were much more efficient in the generation of transgene-free edited progenies by up to 100% in the T₀ generation. TKC2s not only omit the need for screening of the plants with transgenes in the T₀ generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene-free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.