Multiplexed CRISPR/Cas9 mutagenesis of rice PSBS1 non-coding sequences for transgene-free overexpression

Understanding CRISPR/Cas9s capacity to generate native overexpression (OX) alleles would accelerate agronomic gains achievable by gene editing. To generate OX alleles with increased RNA and protein abundance, we leveraged multiplexed CRISPR/Cas9 mutagenesis of non-coding DNA sequences located upstream of the rice PSBS1 gene. We isolated 120 transgene-free, gene-edited alleles with varying NPQ capacity in vivo --ranging from complete knockout to overexpression, using a high-throughput phenotyping and transgene screening pipeline. Overexpression of OsPSBS1 increased protein abundance 2-3-fold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced non-photochemical quenching capacity and improved water-use efficiency. Across our resolved genetic variation, we identify the role of 5UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252kb duplication/inversion generated in this study, evidence the potential of CRISPR/Cas9 to facilitate significant genomic changes with negligible off-target transcriptomic perturbations. Our results may inform future gene-editing strategies for hypermorphic alleles and have opened the door to the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.