---
_id: '17233'
abstract:
- lang: eng
  text: CRISPR-Cas9 technology has become an essential tool for plant genome editing.
    Recent advancements have significantly improved the ability to target multiple
    genes simultaneously within the same genetic background through various strategies.
    Additionally, there has been significant progress in developing methods for inducible
    or tissue-specific editing. These advancements offer numerous possibilities for
    tailored genome modifications. Building upon existing research, we have developed
    an optimized and modular strategy allowing the targeting of several genes simultaneously
    in combination with the synchronized expression of the Cas9 endonuclease in the
    egg cell. This system allows significant editing efficiency while avoiding mosaicism.
    In addition, the versatile system we propose allows adaptation to inducible and/or
    tissue-specific edition according to the promoter chosen to drive the expression
    of the Cas9 gene. Here, we describe a step-by-step protocol for generating the
    binary vector necessary for establishing Arabidopsis edited lines using a versatile
    cloning strategy that combines Gateway® and Golden Gate technologies. We describe
    a versatile system that allows the cloning of as many guides as needed to target
    DNA, which can be multiplexed into a polycistronic gene and combined in the same
    construct with sequences for the expression of the Cas9 endonuclease. The expression
    of Cas9 is controlled by selecting from among a collection of promoters, including
    constitutive, inducible, ubiquitous, or tissue-specific promoters. Only one vector
    containing the polycistronic gene (tRNA-sgRNA) needs to be constructed. For that,
    sgRNA (composed of protospacers chosen to target the gene of interest and sgRNA
    scaffold) is cloned in tandem with the pre-tRNA sequence. Then, a single recombination
    reaction is required to assemble the promoter, the zCas9 coding sequence, and
    the tRNA-gRNA polycistronic gene. Each element is cloned in an entry vector and
    finally assembled according to the Multisite Gateway® Technology. Here, we detail
    the process to express zCas9 under the control of egg cell promoter fused to enhancer
    sequence (EC1.2en-EC1.1p) and to simultaneously target two multiple C2 domains
    and transmembrane region protein genes (MCTP3 and MCTP4, respectively at3g57880
    and at1g51570), using one or two sgRNA per gene.
acknowledgement: This work was supported by the European Research Council (ERC) under
  the European Union’s Horizon 2020 research and innovation program (project 772103-BRIDGING
  to E.M.B.).
article_number: e5029
article_processing_charge: Yes
article_type: original
author:
- first_name: Ziqiang
  full_name: Li, Ziqiang
  id: 922e68bb-1727-11ee-857c-966e8cc1b6c3
  last_name: Li
- first_name: Jennifer
  full_name: Huard, Jennifer
  last_name: Huard
- first_name: Emmanuelle M.
  full_name: Bayer, Emmanuelle M.
  last_name: Bayer
- first_name: Valérie
  full_name: Wattelet-Boyer, Valérie
  last_name: Wattelet-Boyer
citation:
  ama: LI Z, Huard J, Bayer EM, Wattelet-Boyer V. Versatile cloning strategy for efficient
    multigene editing in Arabidopsis. <i>Bio-protocol</i>. 2024;14(13). doi:<a href="https://doi.org/10.21769/BioProtoc.5029">10.21769/BioProtoc.5029</a>
  apa: LI, Z., Huard, J., Bayer, E. M., &#38; Wattelet-Boyer, V. (2024). Versatile
    cloning strategy for efficient multigene editing in Arabidopsis. <i>Bio-Protocol</i>.
    Bio-Protocol. <a href="https://doi.org/10.21769/BioProtoc.5029">https://doi.org/10.21769/BioProtoc.5029</a>
  chicago: LI, ZIQIANG, Jennifer Huard, Emmanuelle M. Bayer, and Valérie Wattelet-Boyer.
    “Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis.” <i>Bio-Protocol</i>.
    Bio-Protocol, 2024. <a href="https://doi.org/10.21769/BioProtoc.5029">https://doi.org/10.21769/BioProtoc.5029</a>.
  ieee: Z. LI, J. Huard, E. M. Bayer, and V. Wattelet-Boyer, “Versatile cloning strategy
    for efficient multigene editing in Arabidopsis,” <i>Bio-protocol</i>, vol. 14,
    no. 13. Bio-Protocol, 2024.
  ista: LI Z, Huard J, Bayer EM, Wattelet-Boyer V. 2024. Versatile cloning strategy
    for efficient multigene editing in Arabidopsis. Bio-protocol. 14(13), e5029.
  mla: LI, ZIQIANG, et al. “Versatile Cloning Strategy for Efficient Multigene Editing
    in Arabidopsis.” <i>Bio-Protocol</i>, vol. 14, no. 13, e5029, Bio-Protocol, 2024,
    doi:<a href="https://doi.org/10.21769/BioProtoc.5029">10.21769/BioProtoc.5029</a>.
  short: Z. LI, J. Huard, E.M. Bayer, V. Wattelet-Boyer, Bio-Protocol 14 (2024).
date_created: 2024-07-14T22:01:11Z
date_published: 2024-07-05T00:00:00Z
date_updated: 2025-03-06T10:28:18Z
day: '05'
ddc:
- '570'
department:
- _id: MiSi
doi: 10.21769/BioProtoc.5029
external_id:
  pmid:
  - '39007160'
file:
- access_level: open_access
  checksum: c8671c0ad483da6407cb16cc3fef1990
  content_type: application/pdf
  creator: dernst
  date_created: 2024-07-16T06:16:11Z
  date_updated: 2024-07-16T06:16:11Z
  file_id: '17242'
  file_name: 2024_BioProtocol_Li.pdf
  file_size: 2896048
  relation: main_file
  success: 1
file_date_updated: 2024-07-16T06:16:11Z
has_accepted_license: '1'
intvolume: '        14'
issue: '13'
language:
- iso: eng
license: https://creativecommons.org/licenses/by/4.0/
month: '07'
oa: 1
oa_version: Published Version
pmid: 1
publication: Bio-protocol
publication_identifier:
  eissn:
  - 2331-8325
publication_status: published
publisher: Bio-Protocol
quality_controlled: '1'
scopus_import: '1'
status: public
title: Versatile cloning strategy for efficient multigene editing in Arabidopsis
tmp:
  image: /images/cc_by.png
  legal_code_url: https://creativecommons.org/licenses/by/4.0/legalcode
  name: Creative Commons Attribution 4.0 International Public License (CC-BY 4.0)
  short: CC BY (4.0)
type: journal_article
user_id: 2DF688A6-F248-11E8-B48F-1D18A9856A87
volume: 14
year: '2024'
...