Multiplexed CRISPR-Cas9 mutagenesis of rice PSBS1 noncoding sequences for transgene-free overexpression.

Understanding CRISPR-Cas9's capacity to produce 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 noncoding sequences upstream of the rice PSBS1 gene. We isolated 120 gene-edited alleles with varying non-photochemical quenching (NPQ) capacity in vivo-from knockout to overexpression-using a high-throughput screening pipeline. Overexpression increased OsPsbS1 protein abundance two- to threefold, matching fold changes obtained by transgenesis. Increased PsbS protein abundance enhanced NPQ capacity and water-use efficiency. Across our resolved genetic variation, we identify the role of 5'UTR indels and inversions in driving knockout/knockdown and overexpression phenotypes, respectively. Complex structural variants, such as the 252-kb duplication/inversion generated here, 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 advanced the pursuit of gene-edited, non-transgenic rice plants with accelerated relaxation of photoprotection.