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Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions

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Plant Gametogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2484))

Abstract

Investigating the process of gamete formation in plants often requires the use of mutants of selected genes in various genetic backgrounds. For example, analysis of meiotic recombination based on sequencing or genotyping requires the generation of hybrids between two lines. Although T-DNA mutant collections of Arabidopsis thaliana are vast and easily accessible, they are largely confined to Col-0 background. This chapter describes how to efficiently generate knock-out mutants in different Arabidopsis accessions using CRISPR/Cas9 technology. The presented system is based on designing two single-guide RNAs (sgRNAs), which direct the Cas9 endonuclease to generate double-strand breaks at two sites, leading to genomic deletion in targeted gene. The presence of seed-expressed dsRed fluorescence cassette in the CRISPR construct facilitates preselection of genome-edited and transgene-free plants by monitoring the seed fluorescence under the epifluorescent microscope. The protocol provides the detailed information about all steps required to perform genome editing and to obtain loss-of-function mutants in different Arabidopsis accessions within merely two generations.

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Acknowledgments

The authors thank Dr. Jian-Kang Zhu for the 3×Flag-NLS-hSpCas9-NLS::NosT cassette which was used to construct pFGC-I2Cas9 plasmid. This work was supported by Polish National Science Centre grants 2016/22/E/NZ2/00455 to P.A.Z., and a Foundation for Polish Science grant POIR.04.04.00-00-5C0F/17-00 to P.A.Z. M.Sz.-L. is the Adam Mickiewicz University Foundation scholarship holder in the academic year 2021/2022.

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Authors and Affiliations

  1. Laboratory of Genome Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland

    Tomasz Bieluszewski, Maja Szymanska-Lejman, Wojciech Dziegielewski, Longfei Zhu & Piotr A. Ziolkowski

Authors
  1. Tomasz Bieluszewski
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  2. Maja Szymanska-Lejman
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  3. Wojciech Dziegielewski
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  4. Longfei Zhu
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  5. Piotr A. Ziolkowski
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Corresponding authors

Correspondence to Tomasz Bieluszewski or Piotr A. Ziolkowski .

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Editors and Affiliations

  1. Department of Plant Sciences, University of Cambridge, Cambridge, UK

    Christophe Lambing

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© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Bieluszewski, T., Szymanska-Lejman, M., Dziegielewski, W., Zhu, L., Ziolkowski, P.A. (2022). Efficient Generation of CRISPR/Cas9-Based Mutants Supported by Fluorescent Seed Selection in Different Arabidopsis Accessions. In: Lambing, C. (eds) Plant Gametogenesis. Methods in Molecular Biology, vol 2484. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2253-7_13

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  • DOI: https://doi.org/10.1007/978-1-0716-2253-7_13

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2252-0

  • Online ISBN: 978-1-0716-2253-7

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