skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction

Abstract

Single guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established. We developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase ( HCT), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the HCT target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of HCT. For the targeting of the second gene, Golgi-localized nucleotide sugar transporter2 ( GONST2), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation. This screening methodmore » is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.« less

Authors:
 [1];  [1];  [1];  [1];  [1];  [2];  [1];  [1];  [1]; ORCiD logo [3];  [3]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States)
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1526557
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 12; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CRISPR/Cas9; Guide RNA efficiency; sgRNA; Constrained editing; HCT; GONST2; Genome editing; Lignin; Arabidopsis

Citation Formats

Liang, Yan, Eudes, Aymerick, Yogiswara, Sasha, Jing, Beibei, Benites, Veronica T., Yamanaka, Reo, Cheng-Yue, Clarabelle, Baidoo, Edward E., Mortimer, Jenny C., Scheller, Henrik V., and Loqué, Dominique. A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction. United States: N. p., 2019. Web. doi:10.1186/s13068-019-1467-y.
Liang, Yan, Eudes, Aymerick, Yogiswara, Sasha, Jing, Beibei, Benites, Veronica T., Yamanaka, Reo, Cheng-Yue, Clarabelle, Baidoo, Edward E., Mortimer, Jenny C., Scheller, Henrik V., & Loqué, Dominique. A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction. United States. doi:10.1186/s13068-019-1467-y.
Liang, Yan, Eudes, Aymerick, Yogiswara, Sasha, Jing, Beibei, Benites, Veronica T., Yamanaka, Reo, Cheng-Yue, Clarabelle, Baidoo, Edward E., Mortimer, Jenny C., Scheller, Henrik V., and Loqué, Dominique. Thu . "A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction". United States. doi:10.1186/s13068-019-1467-y. https://www.osti.gov/servlets/purl/1526557.
@article{osti_1526557,
title = {A screening method to identify efficient sgRNAs in Arabidopsis, used in conjunction with cell-specific lignin reduction},
author = {Liang, Yan and Eudes, Aymerick and Yogiswara, Sasha and Jing, Beibei and Benites, Veronica T. and Yamanaka, Reo and Cheng-Yue, Clarabelle and Baidoo, Edward E. and Mortimer, Jenny C. and Scheller, Henrik V. and Loqué, Dominique},
abstractNote = {Single guide RNA (sgRNA) selection is important for the efficiency of CRISPR/Cas9-mediated genome editing. However, in plants, the rules governing selection are not well established. We developed a facile transient assay to screen sgRNA efficiency. We then used it to test top-performing bioinformatically predicted sgRNAs for two different Arabidopsis genes. In our assay, these sgRNAs had vastly different editing efficiencies, and these efficiencies were replicated in stably transformed Arabidopsis lines. One of the genes, hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT), is an essential gene, required for lignin biosynthesis. Previously, HCT function has been studied using gene silencing. Here, to avoid the negative growth effects that are due to the loss of HCT activity in xylem vessels, we used a fiber-specific promoter to drive CAS9 expression. Two independent transgenic lines showed the expected lignin decrease. Successful editing was confirmed via the observation of mutations at the HCT target loci, as well as an approximately 90% decrease in HCT activity. Histochemical analysis and a normal growth phenotype support the fiber-specific knockout of HCT. For the targeting of the second gene, Golgi-localized nucleotide sugar transporter2 (GONST2), a highly efficient sgRNA drastically increased the rate of germline editing in T1 generation. This screening method is widely applicable, and the selection and use of efficient sgRNAs will accelerate the process of expanding germplasm for both molecular breeding and research. In addition, this, to the best of our knowledge, is the first application of constrained genome editing to obtain chimeric plants of essential genes, thereby providing a dominant method to avoid lethal growth phenotypes.},
doi = {10.1186/s13068-019-1467-y},
journal = {Biotechnology for Biofuels},
issn = {1754-6834},
number = 1,
volume = 12,
place = {United States},
year = {2019},
month = {5}
}

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

Save / Share:

Works referenced in this record:

CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes
journal, July 2013


Lignin modification improves fermentable sugar yields for biofuel production
journal, June 2007

  • Chen, Fang; Dixon, Richard A.
  • Nature Biotechnology, Vol. 25, Issue 7, p. 759-761
  • DOI: 10.1038/nbt1316

Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease
journal, September 2010

  • Haurwitz, R. E.; Jinek, M.; Wiedenheft, B.
  • Science, Vol. 329, Issue 5997, p. 1355-1358
  • DOI: 10.1126/science.1192272

High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants
journal, August 2002

  • Klahre, U.; Crete, P.; Leuenberger, S. A.
  • Proceedings of the National Academy of Sciences, Vol. 99, Issue 18, p. 11981-11986
  • DOI: 10.1073/pnas.182204199

Engineering of plants with improved properties as biofuels feedstocks by vessel-specific complementation of xylan biosynthesis mutants
journal, January 2012

  • Petersen, Pia Damm; Lau, Jane; Ebert, Berit
  • Biotechnology for Biofuels, Vol. 5, Issue 1, Article No. 84
  • DOI: 10.1186/1754-6834-5-84

RNA-programmed genome editing in human cells
journal, January 2013

  • Jinek, Martin; East, Alexandra; Cheng, Aaron
  • eLife, Vol. 2, Article No. e00471
  • DOI: 10.7554/eLife.00471

Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration
journal, April 2012

  • Thorvaldsdottir, H.; Robinson, J. T.; Mesirov, J. P.
  • Briefings in Bioinformatics, Vol. 14, Issue 2, p. 178-192
  • DOI: 10.1093/bib/bbs017

A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity
journal, June 2012


Collapsed Xylem Phenotype of Arabidopsis Identifies Mutants Deficient in Cellulose Deposition in the Secondary Cell Wall
journal, May 1997

  • Turner, S. R.; Somerville, C. R.
  • The Plant Cell, Vol. 9, Issue 5, p. 689-701
  • DOI: 10.1105/tpc.9.5.689

NAC Transcription Factors, NST1 and NST3, Are Key Regulators of the Formation of Secondary Walls in Woody Tissues of Arabidopsis
journal, January 2007

  • Mitsuda, N.; Iwase, A.; Yamamoto, H.
  • The Plant Cell, Vol. 19, Issue 1, p. 270-280
  • DOI: 10.1105/tpc.106.047043

Flavonoid Accumulation in Arabidopsis Repressed in Lignin Synthesis Affects Auxin Transport and Plant Growth
journal, January 2007

  • Besseau, S.; Hoffmann, L.; Geoffroy, P.
  • THE PLANT CELL ONLINE, Vol. 19, Issue 1, p. 148-162
  • DOI: 10.1105/tpc.106.044495

Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana
journal, December 1998


Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant
journal, March 2014

  • Bonawitz, Nicholas D.; Kim, Jeong Im; Tobimatsu, Yuki
  • Nature, Vol. 509, Issue 7500, p. 376-380
  • DOI: 10.1038/nature13084