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Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1

  • Xiaojia Yin
  • Abhishek Anand
  • Paul Quick
  • Anindya Bandyopadhyay
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1917)

Abstract

CRISPR has arguably been the fastest growing genome editing tool so far. CRISPR/Cas9 (Cas9) has been proved to be efficient and precise in genome editing. However Cas9 has certain limitations. CRISPR/Cpf1 (Cpf1) has been discovered as an alternate approach that can overcome some of those limitations. Cpf1 allows targeting in AT-rich region, creating a staggered cleavage, and cutting at the distal end to the PAM (Protospacer Adjacent Motif) regions. We have successfully tested the efficiency of Cpf1 system in rice using OsEPFL9 which is a developmental gene known to regulate the stomatal density in leaf. Regulation of stomatal density and patterning is an important factor in regulating plant physiology, especially in improving the plant water use efficiency. We targeted the Exon1 of OsEPFL9 and the knockout lines were studied for several generations for establishment of stabilized editing, as well as transmission and segregation of edits through generations. The usage of Cpf1 as a genome editing tool to manipulate stomatal patterning may further help us gain more insight of the physiology of rice in stress conditions.

Key words

CRISPR/Cpf1 OsEPFL9 Genome editing Rice 

Notes

Acknowledgements

Authors would like to acknowledge collaborator Dr. Julie E Gray from University of Sheffield, UK, and funding from Newton fund and IRRI. Authors also gratefully acknowledge the team at the International Rice Research Institute, especially Ms. Florencia Montecillo for performing rice transformation, Ms. Melannie Manguiat-Cabangbang and Ms. Gracetine Magpantay for molecular characterization.

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Copyright information

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

Authors and Affiliations

  • Xiaojia Yin
    • 1
  • Abhishek Anand
    • 1
  • Paul Quick
    • 1
  • Anindya Bandyopadhyay
    • 1
    • 2
  1. 1.International Rice Research InstituteManilaPhilippines
  2. 2.Syngenta Beijing Innovation CenterBeijingChina

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