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Methods Mol Biol. 2019;1917:257-268. doi: 10.1007/978-1-4939-8991-1_19.

Editing a Stomatal Developmental Gene in Rice with CRISPR/Cpf1.

Author information

1
International Rice Research Institute, Manila, Philippines.
2
International Rice Research Institute, Manila, Philippines. anindya.bandyopadhyay@syngenta.com.
3
Syngenta Beijing Innovation Center, Beijing, China. anindya.bandyopadhyay@syngenta.com.

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.

KEYWORDS:

CRISPR/Cpf1; Genome editing; OsEPFL9; Rice

PMID:
30610642
DOI:
10.1007/978-1-4939-8991-1_19
[Indexed for MEDLINE]

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