First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue

Abstract

Genome editing using CRISPR/Cas is a key technology for speeding up breeding for climate-resilient, high-yielding crops (Scheben et al., 2017). However, efficient targeted mutagenesis requires implementing stable transformation methods and establishing a CRISPR/Cas setup suitable for the species of interest (Shan et al., 2020). The availability of such methods is a significant bottleneck to advancing many promising, albeit under-researched, crops. Testing and establishing vectors for efficient application of CRISPR/Cas in non-model crops could boost research and breeding of new valuable crops (Ye and Fan, 2021). We edited key pathway genes in the betalain biosynthesis pathway of grain amaranth, i.e., A. hypochondriacus L., to prove how targeted mutagenesis can be implemented in an orphan crop using the CasCADE modular cloning system (Hoffie, 2022). Grain amaranth is a resilient C4 dicot orphan crop with excellent nutritional composition. These properties make amaranth a well-suited candidate to be bred as a climate-resilient crop (Joshi et al., 2018). However, no efficient and reproducible protocol for successful application of CRISPR/Cas9 or stable transformation and regeneration, has been demonstrated in A. hypochondriacus (Castellanos-Arevalo et al., 2020).