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Creating Large Chromosomal Deletions in Rice Using CRISPR/Cas9

  • Riqing Li
  • Si Nian Char
  • Bing YangEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

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.

Key words

CRISPR/Cas9 Genome editing Rice Agrobacterium-mediated rice transformation Targeted mutagenesis Large chromosomal deletion 

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

Notes

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Genetics, Development and Cell BiologyIowa State UniversityAmesUSA

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