The DOI matches its Crossref record; 40 of the 40 references it cites were found in a registry or an index.
DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins
Open accessAbstract
The combined availability of whole genome sequences and genome editing tools is set to revolutionise 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 commercialisation 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 (S-gene) in order to increase resistance to powdery mildew (PM) 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 optimised for each grape and apple cultivar. The targeted mutagenesis insertion and deletion (indel) rate was analysed 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.
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Citations
Cites
DNA targeting specificity of RNA-guided Cas9 nucleases
Hsu, P. D., Scott, D. A., Weinstein, J. A., Ran, F. A., Konermann, S., Agarwala, V., Li, Y., Fine, E. J., Wu, X., Shalem, O., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2647 · Cited by 4,741
Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity
Ran, F. A., Hsu, P. D., Lin, C.-Y., Gootenberg, J. S., Konermann, S., Trevino, A. E., Scott, D. A., Inoue, A., Matoba, S., Zhang, Y., et al. (2013) · Cell · doi:10.1016/j.cell.2013.08.021 · Cited by 3,299
High-Frequency off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells
Fu, Y., Foden, J. A., Khayter, C., Maeder, M. L., Reyon, D., Joung, J. K., & Sander, J. D. (2013) · Nature Biotechnology · doi:10.1038/nbt.2623 · Cited by 3,292
Bae, S., Park, J., & Kim, J.-S. (2014) · Bioinformatics · doi:10.1093/bioinformatics/btu048 · Cited by 2,339
Self-citationWang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014) · Nature Biotechnology · doi:10.1038/nbt.2969 · Cited by 2,072
Quail, M., Smith, M. E., Coupland, P., Otto, T. D., Harris, S. R., Connor, T. R., Bertoni, A., Swerdlow, H. P., & Gu, Y. (2012) · BMC Genomics · doi:10.1186/1471-2164-13-341 · Cited by 2,031 · cited 2×
“Protoplast volume 200 µl (2 × 105 cells) and RNPs for example 3:1 is Cas9 90 µg (stock 10 µg/µl) sgRNA 30 µg (stock 10 µg/µl) is used for transformation.”
MethodImproving CRISPR-Cas nuclease specificity using truncated guide RNAs
Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., & Joung, J. K. (2014) · Nature Biotechnology · doi:10.1038/nbt.2808 · Cited by 1,993 · cited 1×
“Synthesis of unique sgRNA (Cho et al., 2014; Fu et al., 2014; Sung et al., 2014), Cas9 nickases and web based computer programs to identify unique target sites (Mali et al., 2013; Ran et al., 2013; Bae et al., 2014) and CRISPR/Cas9 RNPs (Woo et al., 2015).”
BackgroundTargeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
Cho, S. W., Kim, S., Kim, J. M., & Kim, J.-S. (2013) · Nature Biotechnology · doi:10.1038/nbt.2507 · Cited by 1,863
Self-citationKim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J.-S. (2014) · Genome Research · doi:10.1101/gr.171322.113 · Cited by 1,856
Self-citationTargeted genome modification of crop plants using a CRISPR-Cas system
Shan, Q., Wang, Y., Li, J., Zhang, Y., Chen, K., Liang, Z., Zhang, K., Liu, J., Xi, J. J., Qiu, J.-L., et al. (2013) · Nature Biotechnology · doi:10.1038/nbt.2650 · Cited by 1,810
Cited by
CRISPR for Crop Improvement: An Update Review
Jaganathan, D., Ramasamy, K., Sellamuthu, G., Jayabalan, S., & Venkataraman, G. (2018) · Frontiers in Plant Science · doi:10.3389/fpls.2018.00985 · Cited by 619
CRISPR/Cas system: recent advances and future prospects for genome editing
Manghwar, H., Lindsey, K., Zhang, X., & Jin, S. (2019) · Trends in Plant Science · doi:10.1016/j.tplants.2019.09.006 · Cited by 529
“) [73], apple (Malus pumila) [74], tropical staple cassava (Manihot esculenta) [75], watermelon (Citrullus lanatus) [76], and others (Table 2).”
Applications and potential of genome editing in crop improvement
Zhang, Y., Massel, K., Godwin, I. D., & Gao, C. (2018) · Genome Biology · doi:10.1186/s13059-018-1586-y · Cited by 437
Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing
Zhang, S., Shen, J., Li, D., & Cheng, Y. (2021) · Theranostics · doi:10.7150/thno.47007 · Cited by 430
The emerging and uncultivated potential of CRISPR technology in plant science
Zhang, Y., Malzahn, A. A., Sretenovic, S., & Qi, Y. (2019) · Nature Plants · doi:10.1038/s41477-019-0461-5 · Cited by 394
Metadata
- Authors
- Malnoy, M., Viola, R., Jung, M.-H., Koo, O.-J., Kim, S., Kim, J.-S., Velasco, R., & Nagamangala Kanchiswamy, C.
- Venue
- Frontiers in Plant Science
- Publisher
- Frontiers Media SA
- Published
- 2016-12-20
- Volume
- 7
- Pages
- 1904-1904
- ISSN
- 1664-462X (ISSN-L)
- OpenAlex
- W2567243116
- Cited by
- 756
- References
- 40
- FWCI
- 19.41
- Type
- journal article
- Language
- en
- Open access
- gold
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- Malnoy, M., Viola, R., Jung, M.-H., Koo, O.-J., Kim, S., Kim, J.-S., Velasco, R., & Nagamangala Kanchiswamy, C. (2016). DNA-Free Genetically Edited Grapevine and Apple Protoplast Using CRISPR/Cas9 Ribonucleoproteins. Frontiers in Plant Science. https://doi.org/10.3389/fpls.2016.01904
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Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).
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