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Title: CRISPR/Cas9-mediated Targeted Mutagenesis for Functional Genomics Research of Crassulacean Acid Metabolism Plants

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 seek to realize 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 to create biallelic indel mutagenesis to reveal previously unknown roles of blue light receptor phototropin 2 (KfePHOT2) in the CAM pathway. Knocking-out of the KfePHOT2 reduced stomatal conductance and CO2 fixation in the late afternoon and increased stomatal conductance and CO2 fixation in the early evening, indicating that blue light signaling plays an important role in the CAM pathway. Lastly, we provide a genome-wide gRNA database targeting 45,183 protein-coding genes annotated in the K. fedtschenkoi genome.

Authors:
 [1];  [2];  [3];  [4];  [4];  [4];  [5];  [1];  [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-Center for Bioenergy Innovation (CBI)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Southwest Univ. of Science and Technology, Mianyang, Sichuan (China). School of Life Science and Engineering
  3. Univ. of Tennessee, Knoxville, TN (United States). Dept.t of Chemical and Biomolecular Engineering
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
  5. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). DOE-Center for Bioenergy Innovation (CBI); Univ. of Tennessee, Knoxville, TN (United States). Dept.t of Chemical and Biomolecular Engineering
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1569393
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Experimental Botany
Additional Journal Information:
Journal Volume: 70; Journal Issue: 22; Journal ID: ISSN 0022-0957
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CRISPR/Cas9; crassulacean acid metabolism; phototropin 2; functional genomics; genome-editing

Citation Formats

Liu, Degao, Chen, Mei, Mendoza, Brian, Cheng, Hua, Hu, Rongbin, Li, Linling, Trinh, Cong T., Tuskan, Gerald A., and Yang, Xiaohan. CRISPR/Cas9-mediated Targeted Mutagenesis for Functional Genomics Research of Crassulacean Acid Metabolism Plants. United States: N. p., 2019. Web. doi:10.1093/jxb/erz415.
Liu, Degao, Chen, Mei, Mendoza, Brian, Cheng, Hua, Hu, Rongbin, Li, Linling, Trinh, Cong T., Tuskan, Gerald A., & Yang, Xiaohan. CRISPR/Cas9-mediated Targeted Mutagenesis for Functional Genomics Research of Crassulacean Acid Metabolism Plants. United States. https://doi.org/10.1093/jxb/erz415
Liu, Degao, Chen, Mei, Mendoza, Brian, Cheng, Hua, Hu, Rongbin, Li, Linling, Trinh, Cong T., Tuskan, Gerald A., and Yang, Xiaohan. Sat . "CRISPR/Cas9-mediated Targeted Mutagenesis for Functional Genomics Research of Crassulacean Acid Metabolism Plants". United States. https://doi.org/10.1093/jxb/erz415. https://www.osti.gov/servlets/purl/1569393.
@article{osti_1569393,
title = {CRISPR/Cas9-mediated Targeted Mutagenesis for Functional Genomics Research of Crassulacean Acid Metabolism Plants},
author = {Liu, Degao and Chen, Mei and Mendoza, Brian and Cheng, Hua and Hu, Rongbin and Li, Linling and Trinh, Cong T. and Tuskan, Gerald A. and Yang, Xiaohan},
abstractNote = {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 seek to realize 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 to create biallelic indel mutagenesis to reveal previously unknown roles of blue light receptor phototropin 2 (KfePHOT2) in the CAM pathway. Knocking-out of the KfePHOT2 reduced stomatal conductance and CO2 fixation in the late afternoon and increased stomatal conductance and CO2 fixation in the early evening, indicating that blue light signaling plays an important role in the CAM pathway. Lastly, we provide a genome-wide gRNA database targeting 45,183 protein-coding genes annotated in the K. fedtschenkoi genome.},
doi = {10.1093/jxb/erz415},
journal = {Journal of Experimental Botany},
number = 22,
volume = 70,
place = {United States},
year = {2019},
month = {9}
}

Journal Article:
Free Publicly Available Full Text
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Cited by: 4 works
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Figures / Tables:

Table 1 Table 1: Off-target analysis of sgRNAs in the mutant lines.

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Works referencing / citing this record:

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Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.