Tracking Transgenes with Color: RUBY as a Visual Marker in CRISPR-Edited Mutant Plants in Two Triticum Species

Abstract

CRISPR-Cas9 is a powerful tool for precise genome editing in plants, but the presence of foreign DNA, such as T-DNA, raises regulatory concerns and complicates mutant screening and field studies of edited material. Detecting plants with good transgene expression and later removing the T-DNA from edited plants is both time-consuming and costly. To address this, we developed a system that uses the non-destructive RUBY reporter, linked to the CRISPR-Cas9 cassette, and expressed under the ZmUbi1 promoter. To assess the applicability of the system, it was tested on two Triticum species, targeting three genes in either tetraploid or hexaploid wheat. Strong correlations were observed in both T0 and T1 plants between betalain content and Cas9 expression, allowing for the quick identification of plants likely to be edited. Furthermore, the RUBY reporter could be used to select against the transgenic CRISPR-Cas9 cassette in subsequent generations at both the seed and seedling stages, thereby reducing the number of plants that need to be screened to identify edited lines without a T-DNA. This approach, using a nondestructive reporter, enabled rapid distinction between transgene expression in primary transgenics and served as a negative selection in the T1 generation, streamlining selection towards edited and T-DNA-free progeny.