Boosting genome editing in plants with single transcript unit surrogate reporter systems

Abstract

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of edited events. In this study, we introduce multiple Single Transcript Unit Surrogate Reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems utilize the same sgRNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are employed to restore functional reporter genes post-genome editing, including efficient single strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-BE systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are demonstrated to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.