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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. doi: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. doi: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
Publisher's Version of Record

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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 referenced in this record:

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journal, October 2018

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Transcript, protein and metabolite temporal dynamics in the CAM plant Agave
journal, November 2016


Phototropin-related NPL1 controls chloroplast relocation induced by blue light
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Overexpression of IbP5CR enhances salt tolerance in transgenic sweetpotato
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Stomatal responses to light in the facultative Crassulacean acid metabolism species, Portulacaria afra
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CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications
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Editing plant genomes with CRISPR/Cas9
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Targeted genome modification of crop plants using a CRISPR-Cas system
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Growth-mediated plant movements: hidden in plain sight
journal, February 2018


Engineering crassulacean acid metabolism to improve water-use efficiency
journal, May 2014


Enzymatic assembly of DNA molecules up to several hundred kilobases
journal, April 2009

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A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world
journal, July 2015

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Phototropins maintain robust circadian oscillation of PSII operating efficiency under blue light
journal, August 2015

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Light quality modulates metabolic synchronization over the diel phases of crassulacean acid metabolism
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High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize
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Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research
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Ecophysiology of constitutive and facultative CAM photosynthesis
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Applying CRISPR/Cas for genome engineering in plants: the best is yet to come
journal, April 2017


An Agrobacterium -delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize
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Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening
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Functional ecology of a blue light photoreceptor: effects of phototropin-1 on root growth enhance drought tolerance in Arabidopsis thaliana
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Efficient genome editing in plants using a CRISPR/Cas system
journal, August 2013

  • Feng, Zhengyan; Zhang, Botao; Ding, Wona
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  • DOI: 10.1038/cr.2013.114

Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth
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The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism
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journal, October 2011


Precision genome engineering through adenine base editing in plants
journal, June 2018


Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion
journal, March 2017

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  • Nature Biotechnology, Vol. 35, Issue 5
  • DOI: 10.1038/nbt.3833

A CRISPR/Cas9 toolkit for multiplex genome editing in plants
journal, November 2014


    Works referencing / citing this record:

    Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system
    journal, March 2015

    • Xie, Kabin; Minkenberg, Bastian; Yang, Yinong
    • Proceedings of the National Academy of Sciences, Vol. 112, Issue 11
    • DOI: 10.1073/pnas.1420294112

    Transgenic Perturbation of the Decarboxylation Phase of Crassulacean Acid Metabolism Alters Physiology and Metabolism But Has Only a Small Effect on Growth
    journal, November 2014

    • Dever, Louisa V.; Boxall, Susanna F.; Kneřová, Jana
    • Plant Physiology, Vol. 167, Issue 1
    • DOI: 10.1104/pp.114.251827

    High-efficiency genome editing using a dmc1 promoter-controlled CRISPR/Cas9 system in maize
    journal, April 2018

    • Feng, Chao; Su, Handong; Bai, Han
    • Plant Biotechnology Journal, Vol. 16, Issue 11
    • DOI: 10.1111/pbi.12920

    Targeted genome modification of crop plants using a CRISPR-Cas system
    journal, August 2013

    • Shan, Qiwei; Wang, Yanpeng; Li, Jun
    • Nature Biotechnology, Vol. 31, Issue 8
    • DOI: 10.1038/nbt.2650

    Functional ecology of a blue light photoreceptor: effects of phototropin-1 on root growth enhance drought tolerance in Arabidopsis thaliana
    journal, October 2006


    Tissue-Autonomous Promotion of Palisade Cell Development by Phototropin 2 in Arabidopsis
    journal, October 2011


    Light quality modulates metabolic synchronization over the diel phases of crassulacean acid metabolism
    journal, May 2014

    • Ceusters, J.; Borland, A. M.; Taybi, T.
    • Journal of Experimental Botany, Vol. 65, Issue 13
    • DOI: 10.1093/jxb/eru185

    A CRISPR/Cas9 toolkit for multiplex genome editing in plants
    journal, November 2014


    Blue Light Induces a Distinct Starch Degradation Pathway in Guard Cells for Stomatal Opening
    journal, February 2016


    Enzymatic assembly of DNA molecules up to several hundred kilobases
    journal, April 2009

    • Gibson, Daniel G.; Young, Lei; Chuang, Ray-Yuan
    • Nature Methods, Vol. 6, Issue 5, p. 343-345
    • DOI: 10.1038/nmeth.1318

    sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites
    journal, June 2014


    Isolation and Characterization of Mutants of Common Ice Plant Deficient in Crassulacean Acid Metabolism
    journal, March 2008

    • Cushman, John C.; Agarie, Sakae; Albion, Rebecca L.
    • Plant Physiology, Vol. 147, Issue 1
    • DOI: 10.1104/pp.108.116889

    Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research
    journal, April 2016


    Growth-mediated plant movements: hidden in plain sight
    journal, February 2018


    Precision genome engineering through adenine base editing in plants
    journal, June 2018


    CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications
    journal, July 2016


    The Kalanchoë genome provides insights into convergent evolution and building blocks of crassulacean acid metabolism
    journal, December 2017


    Phototropin 2 is involved in blue light-induced anthocyanin accumulation in Fragaria x ananassa fruits
    journal, August 2013

    • Kadomura-Ishikawa, Yasuko; Miyawaki, Katsuyuki; Noji, Sumihare
    • Journal of Plant Research, Vol. 126, Issue 6
    • DOI: 10.1007/s10265-013-0582-2

    Stomatal response to blue light in crassulacean acid metabolism plants Kalanchoe pinnata and Kalanchoe daigremontiana
    journal, December 2018

    • Gotoh, Eiji; Oiwamoto, Kohei; Inoue, Shin-ichiro
    • Journal of Experimental Botany, Vol. 70, Issue 4
    • DOI: 10.1093/jxb/ery450

    Overexpression of IbP5CR enhances salt tolerance in transgenic sweetpotato
    journal, December 2013

    • Liu, Degao; He, Shaozhen; Zhai, Hong
    • Plant Cell, Tissue and Organ Culture (PCTOC), Vol. 117, Issue 1
    • DOI: 10.1007/s11240-013-0415-y

    Applying CRISPR/Cas for genome engineering in plants: the best is yet to come
    journal, April 2017


    Efficient genome editing in plants using a CRISPR/Cas system
    journal, August 2013

    • Feng, Zhengyan; Zhang, Botao; Ding, Wona
    • Cell Research, Vol. 23, Issue 10
    • DOI: 10.1038/cr.2013.114

    A roadmap for research on crassulacean acid metabolism (CAM) to enhance sustainable food and bioenergy production in a hotter, drier world
    journal, July 2015

    • Yang, Xiaohan; Cushman, John C.; Borland, Anne M.
    • New Phytologist, Vol. 207, Issue 3
    • DOI: 10.1111/nph.13393

    Engineering crassulacean acid metabolism to improve water-use efficiency
    journal, May 2014


    Stomatal Biology of CAM Plants
    journal, February 2017


    Transcript, protein and metabolite temporal dynamics in the CAM plant Agave
    journal, November 2016


    Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize
    journal, October 2018

    • Salesse-Smith, Coralie E.; Sharwood, Robert E.; Busch, Florian A.
    • Nature Plants, Vol. 4, Issue 10
    • DOI: 10.1038/s41477-018-0252-4

    Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion
    journal, March 2017

    • Shimatani, Zenpei; Kashojiya, Sachiko; Takayama, Mariko
    • Nature Biotechnology, Vol. 35, Issue 5
    • DOI: 10.1038/nbt.3833

    Orchestration of carbohydrate processing for crassulacean acid metabolism
    journal, June 2016


    An Agrobacterium -delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize
    journal, September 2016

    • Char, Si Nian; Neelakandan, Anjanasree K.; Nahampun, Hartinio
    • Plant Biotechnology Journal, Vol. 15, Issue 2
    • DOI: 10.1111/pbi.12611

    Phototropin-related NPL1 controls chloroplast relocation induced by blue light
    journal, April 2001

    • Jarillo, Jose A.; Gabrys, Halina; Capel, Juan
    • Nature, Vol. 410, Issue 6831
    • DOI: 10.1038/35073622

    Ecophysiology of constitutive and facultative CAM photosynthesis
    journal, February 2019


    Phototropins maintain robust circadian oscillation of PSII operating efficiency under blue light
    journal, August 2015

    • Litthauer, Suzanne; Battle, Martin W.; Lawson, Tracy
    • The Plant Journal, Vol. 83, Issue 6
    • DOI: 10.1111/tpj.12947

    Stomatal responses to light in the facultative Crassulacean acid metabolism species, Portulacaria afra
    journal, May 1992


    Editing plant genomes with CRISPR/Cas9
    journal, April 2015


    Perspectives on the basic and applied aspects of crassulacean acid metabolism (CAM) research
    journal, September 2018


    Comparative genomics can provide new insights into the evolutionary mechanisms and gene function in CAM plants
    journal, September 2019

    • Yang, Xiaohan; Liu, Degao; Tschaplinski, Timothy J.
    • Journal of Experimental Botany, Vol. 70, Issue 22
    • DOI: 10.1093/jxb/erz408

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