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.
Highlights
Grape and apple fruit crop plants are a major source of fiber, nutrients, and antioxidants, all essential for a healthy diet
Embryogenic calli provided a higher yield of up to 3.6 × 106 with 90% viability when using 1.5% cellulase R-10 and 0.4% macerozyme R-10, with 20 min of vacuum infiltration followed by 3 h incubation with gentle shaking
CRISPR/Cas9 or other genome editing tools mediated protoplast transfection system has been successfully adopted in Arabidopsis, rice, wheat, maize, tobacco, lettuce, and petunia (Jiang et al, 2013; Li et al, 2013; Shan et al, 2013; Wang et al, 2014; Gao et al, 2015; Woo et al, 2015; Subburaj et al, 2016), a similar system has not been developed for the grapevine and apple
Summary
Grape and apple fruit crop plants are a major source of fiber, nutrients, and antioxidants, all essential for a healthy diet These crops play a key role in the economy of many developed and developing countries and considerable efforts are being made to improve commercial traits using both conventional breeding and genetic engineering. US Department of Agriculture (USDA) does not impose any GMO regulations to the plants with targeted mutagenesis generated by self-repair mechanisms, if they are free from Agrobacterium or any transgenic or foreign genetic materials; we assume there is high probability that CRISPR/Cas RNPs could be exempted from current GMO regulations (Waltz, 2012; Ledford, 2013; Jones, 2015). The EU is uncertain to approve them and has yet to provide information on whether targeted mutation made by CRISPR/Cas or CRISPR/Cas RNPs fall outside regulatory criteria (Waltz, 2012; Jones, 2015)
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