Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucl
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ORIGINAL ARTICLE
Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR‑Cas9 ribonucleoproteins Jihyeon Yu1,2 · Luhua Tu3 · Saminathan Subburaj3 · Sangsu Bae1,2 · Geung‑Joo Lee3 Received: 27 March 2020 / Accepted: 26 August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Key message We obtained a complete mutant line of Petunia having mutations in both F3H genes 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 in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar ‘Madness Midnight’ has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T 0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knockout plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. 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. Keywords CRISPR-Cas9 ribonucleoproteins · DNA-free gene editing · One step generation · Protoplast regeneration · Petunia × hybrida
Introduction Communicated by Neal Stewart. Jihyeon Yu, Luhua Tu, and Saminathan Subburaj have contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00299-020-02593-1) contains supplementary material, which is available to authorized users. * Sangsu Bae [email protected] * Geung‑Joo Lee [email protected] 1
Department of Chemistry, Hanyang University, Seoul 04763, South Korea
2
Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, South Korea
3
Department of Horticulture & Department of Smart Agriculture systems, Chungnam National University, Daejeon 34134, South Korea
The CRISPR-Cas system is a versatile and cutting-edge technology that allows researchers to generate various desired mutations in plants at will, enabling the development of plant mutants. In contrast with classical genetic methods that require long-term breeding processes, CRISPR modifies a target t
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