DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins Highly Cited Paper

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Title
DNA-free genetically edited grapevine and apple protoplast using CRISPR/Cas9 ribonucleoproteins
Author(s)
Mickael Malnoy; Robert Viola; Min-Hee Jung; Ok-Jae Koo; Seokjoong Kim; Jin-Soo Kim; Riccardo Velasco; Chidananda Nagamangala Kanchiswamy
Publication Date
2016-12
Journal
Frontiers in Plant Science, v.7, no., pp.1904 -
Publisher
FRONTIERS MEDIA SA
Abstract
The combined availability of whole genome sequences and genome editing tools is set to revolutionize the field of fruit biotechnology by enabling the introduction of targeted genetic changes with unprecedented control and accuracy, both to explore emergent phenotypes and to introduce new functionalities. Although plasmid-mediated delivery of genome editing components to plant cells is very efficient, it also presents some drawbacks, such as possible random integration of plasmid sequences in the host genome. Additionally, it may well be intercepted by current process-based GMO regulations, complicating the path to commercialization of improved varieties. Here, we explore direct delivery of purified CRISPR/Cas9 ribonucleoproteins (RNPs) to the protoplast of grape cultivar Chardonnay and apple cultivar such as Golden delicious fruit crop plants for efficient targeted mutagenesis. We targeted MLO-7, a susceptible gene in order to increase resistance to powdery mildew in grape cultivar and DIPM-1, DIPM-2, and DIPM-4 in the apple to increase resistance to fire blight disease. Furthermore, efficient protoplast transformation, the molar ratio of Cas9 and sgRNAs were optimized for each grape and apple cultivar. The targeted mutagenesis insertion and deletion rate was analyzed using targeted deep sequencing. Our results demonstrate that direct delivery of CRISPR/Cas9 RNPs to the protoplast system enables targeted gene editing and paves the way to the generation of DNA-free genome edited grapevine and apple plants. © 2016 Malnoy, Viola, Jung, Koo, Kim, Kim, Velasco and Nagamangala Kanchiswamy
URI
https://pr.ibs.re.kr/handle/8788114/3220
ISSN
1664-462X
Appears in Collections:
Center for Genome Engineering(유전체 교정 연구단) > Journal Papers (저널논문)
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