Multiplexed CRISPR-Cas9 mutagenesis of rice <i>PSBS1</i> noncoding sequences for transgene-free overexpression.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 38848363.
- Also identified by DOI 10.1126/sciadv.adm7452 and PMC identifier 11160471.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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 <i>PSBS1</i> 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.
Medical subject headings
- Oryza
- CRISPR-Cas Systems
- Gene Editing
- Mutagenesis