CRISPR/Cas9-mediated targeted mutagenesis for functional genomics research of crassulacean acid metabolism plants

Degao Liu, Mei Chen, Brian Mendoza, Hua Cheng, Rongbin Hu, Linling Li, Cong T. Trinh, Gerald A. Tuskan, Xiaohan Yang

Research output: Contribution to journalArticlepeer-review

Abstract

Crassulacean acid metabolism (CAM) is an important photosynthetic pathway in diverse lineages of plants featuring high water-use efficiency and drought tolerance. A big challenge facing the CAM research community is to understand the function of the annotated genes in CAM plant genomes. Recently, a new genome editing technology using CRISPR/Cas9 has become a more precise and powerful tool than traditional approaches for functional genomics research in C3 and C4 plants. In this study, we explore the potential of CRISPR/Cas9 to characterize the function of CAM-related genes in the model CAM species Kalanchoë fedtschenkoi. We demonstrate that CRISPR/Cas9 is effective in creating biallelic indel mutagenesis to reveal previously unknown roles of blue light receptor phototropin 2 (KfePHOT2) in the CAM pathway. Knocking out KfePHOT2 reduced stomatal conductance and CO2 fixation in late afternoon and increased stomatal conductance and CO2 fixation during the night, indicating that blue light signaling plays an important role in the CAM pathway. Lastly, we provide a genome-wide guide RNA database targeting 45 183 protein-coding transcripts annotated in the K. fedtschenkoi genome.

Original languageEnglish (US)
Pages (from-to)6621-6629
Number of pages9
JournalJournal of experimental botany
Volume70
Issue number22
DOIs
StatePublished - Nov 29 2019
Externally publishedYes

Bibliographical note

Funding Information:
This research is supported by the Department of Energy (DOE), Office of Science, Genomic Science Program under Award Number DE-SC0008834, and the Center for Bioenergy Innovation (CBI), which is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science.We thank Timothy J. Tschaplinski for critical review and comments on the manuscript. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the US DOE under contract no. DE-AC05-00OR22725. CTT would also like to acknowledge financial support through the DARPA YFA award #D17AP00023.The views, opinions,and/or findings contained in this article are those of the authors and should not be interpreted as representing the official views or policies, either expressed or implied, of the funding agencies. The authors declare no conflicts of interest.

Publisher Copyright:
© 2019 The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Keywords

  • Crassulacean acid metabolism
  • CRISPR/Cas9
  • functional genomics
  • genome editing
  • phototropin 2

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