Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins

Key message We obtained a complete mutant line ofPetuniahaving mutations in bothF3Hgenes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis inPetuniato engineer flower color modifications. We determined that the commercialPetuniacultivar 'Madness Midnight' has twoF3Hcoding genes and designed one guide RNA that targets bothF3Hgenes at once. Among 67 T(0)plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in eitherF3HAorF3HBgene and one complete mutant line having mutations in bothF3Hgenes without any selectable markers. It is noteworthy that only thef3ha f3hbexhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-typePetunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.

Automated summary

Utilizing the CRISPR-Cas system, we engineered flower color in Petunia successfully. The DNA-free genome editing method is efficient and avoids GMO regulations. Our study sets a precedent for ornamental crop engineering, potentially accelerating the transition of the technology from the laboratory to the field.