Abstract
Engineered CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is an efficient and the most popularly used tool for genome engineering in eukaryotic organisms including plants, especially in crop plants. This system has been effectively used to introduce mutations in multiple genes simultaneously, create conditional alleles, and generate endogenously tagged proteins. CRISPR/Cas9 hence presents great value in basic and applied research for improving the performance of crop plants in various aspects such as increasing grain yields, improving nutritional content, and better combating biotic and abiotic stresses. Besides above applications, CRISPR/Cas9 system has been shown to be very effective in creating large chromosomal deletions in plants, which is useful for genetic analysis of chromosomal fragments, functional study of gene clusters in biological processes, and so on. Here, we present a protocol of creating large chromosomal deletions in rice using CRISPR/Cas9 system, including detailed information about single-guide RNA design, vector construction, plant transformation, and large deletion screening processes in rice.
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Abbreviations
- CRISPR/Cas9:
-
Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9
- DSB:
-
Double-stranded DNA break
- HDR:
-
Homology-directed recombination
- hptII :
-
Hygromycin phosphotransferase gene for hygromycin B resistance
- MCS:
-
Multiple cloning sites
- NHEJ:
-
Nonhomologous end joining
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Acknowledgments
The authors acknowledge the funding support from the National Institute of Food and Agriculture of the US Department of Agriculture (2014-67013-21720 to BY).
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Li, R., Char, S.N., Yang, B. (2019). Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9. In: Qi, Y. (eds) Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-8991-1_4
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DOI: https://doi.org/10.1007/978-1-4939-8991-1_4
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