Live imaging-assisted domain-specific CRISPR genome editing at single cell resolution in plants

Abstract

AbstractCRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has been widely used for genome engineering in a wide range of organisms1, but much of the development of CRISPR-based genome editing has been aimed toward improving its efficiency and accuracy, so as to obtain genetic materials carrying known and stably heritable genome modifications. Precise spatiotemporal control over genome editing technology at cell type resolution is a key challenge for gene function studies. Some tissue-specific CRISPR genome editing methods relying on phenotypic characterization and fluorescent immune-staining techniques have been developed for biomedical research and gene therapy, they function by spatially controlling expression of Cas9 2. Recent work establishes the presence and location of mutational events at a single cell level in Arabidopsis roots and stomata3,4. Here we present an efficient domain-specific CRISPR-Cas9 system combined with a high resolution live-imaging based screening strategy, applied in the shoot apical meristem of Arabidopsis thaliana. Using the system we investigate PIN-FORMED1 (PIN1) protein functions in tissue morphogenesis and PIN1 mechanical stress response in a cell layer-specific fashion. We find that reported failure to generate new primordia in epidermal PIN1 knockout SAMs is due to a reduction in mechanical stress differences in the sub-epidermal layer. The methods described are applicable to spatial-temporal gene manipulation in plants.