Seeing is believing: a visualization toolbox to enhance selection efficiency in maize genome editing.

The emerging and promising biotechnology methodology of CRISPR/Cas9 gene-editing is revolutionizing crop improvement. However, the low-efficiency, time-consuming, and labor-intensive nature of positive or/and negative selections during or after transformation and subsequent identification of mutations are major challenges for its agricultural application, from upstream (high-throughput) mutant screening to downstream commercial production (He and Zhao, 2020). Although visual markers, and in particular fluorescent markers including green fluorescent protein (GFP) and red fluorescent protein (RFP), have been employed for the rapid visualization of transgenic material (Qi et al., 2020), the integration of cost-effective and non-invasive CRISPR-separation gene-editing tools is still in its infancy but remains critical for crop genetics and breeding (Callaway, 2018). Furthermore, special light sources are required to visualize fluorescent signals, which increases the cost and inconvenience of the application of fluorescence markers, especially in field conditions. We have developed a visualization toolbox, ViMeBox (VIsual Maize Editing toolBox), for the selection of positive transformants in maize (Zea mays). In the ViMeBox system, the vector expressing Cas9 includes a gene cassette containing a visible marker expressed from a tissue-specific promoter; Cas9-free kernels that are easy to separate by the visible marker are also undergoing gene editing. ViMeBox offers two advantages: 1) It enables enhanced expression of DsRED2, which makes seeds containing Cas9 visible to the naked eye in natural light, and does not affect genome editing efficiency or plant development. 2) It is effective for different seed tissues, for example, using embryo-specific promoters or promoters preferentially expressed in the embryo or aleurone. Furthermore, ViMeBox has potential applications in diverse additional scenarios.