The strategy of knock-in with homology-directed genome editing in the model ornamental plant Petunia using CRISPR/Cas9 ribonucleoprotein complex

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system 9 (Cas9) ribonucleoprotein (RNP) complex offers numerous advantages, including increased genome editing efficiency, reduced off-target effects, minimized risk of genomic integration, expanded applicability, and the ability to address specific ethical concerns. These benefits establish the CRISPR/Cas9 RNP complex as a valuable technique for precise genome editing in plant research and applications. Nevertheless, achieving precise knock-in using CRISPR/Cas9-mediated precision knock-in of genes by homology-directed repair (HDR) remains challenging due to the infrequent occurrence of HDR and the limited availability of DNA repair templates near double-strand breaks (DSBs). In this study, we presented the efficient utilization strategy of HDR-mediated gene knock-in through co-infection of the CRISPR/Cas9 RNP complex and donor plasmid in Petunia protoplast. We accomplish this by designing a donor plasmid targeting the chalcone synthase (CHS) in Petunia, consisting of a CRISPR RNA (crRNA)-PAM sequences flanked by a donor DNA cassette with homology arms. These findings offer insights into the application of these techniques in ornamental crops to enhance CRISPR/Cas9 RNA complex-mediated gene knock-in strategies.