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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 Laboratory (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 = {Sat Sep 28 00:00:00 EDT 2019},
month = {Sat Sep 28 00:00:00 EDT 2019}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 15 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:

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

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


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

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


Stomatal Biology of CAM Plants
journal, February 2017


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

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


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

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

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


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


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


Editing plant genomes with CRISPR/Cas9
journal, April 2015


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

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

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

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

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

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

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

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

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


Ecophysiology of constitutive and facultative CAM photosynthesis
journal, February 2019


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
journal, September 2016

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

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


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


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

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

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


Tissue-Autonomous Promotion of Palisade Cell Development by Phototropin 2 in Arabidopsis
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

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

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


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


Overexpression of Rubisco subunits with RAF1 increases Rubisco content in maize.
text, January 2018

  • Salesse-Smith, Coralie E.; Sharwood, Robert E.; Busch, Florian A.
  • Apollo - University of Cambridge Repository
  • DOI: 10.17863/cam.38299

Stomatal biology of CAM plants
text, January 2017

  • Males, Jamie; Griffiths, Howard
  • Apollo - University of Cambridge Repository
  • DOI: 10.17863/cam.9618

Editing plant genomes with CRISPR/Cas9
journal, April 2015


Plant Physiology: Redefining the Enigma of Metabolism in Stomatal Movement
journal, February 2016


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


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


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


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


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

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

Systems biology: A new CAM era
journal, November 2016


Overexpressing the most abundant enzyme
journal, October 2018


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

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

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

Stomatal Biology of CAM Plants
journal, February 2017


Viewpoints – a new addition to the New Phytologist Forum
journal, July 2015

  • Hetherington, Alistair M.; Lennon, Sarah
  • New Phytologist, Vol. 207, Issue 3
  • DOI: 10.1111/nph.13541

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

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

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


Works referencing / citing this record:

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

New perspectives on crassulacean acid metabolism biology
journal, November 2019

  • Hultine, Kevin R.; Cushman, John C.; Williams, David G.
  • Journal of Experimental Botany, Vol. 70, Issue 22
  • DOI: 10.1093/jxb/erz465

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