---
_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. Bio-protocol. 2024;14(13). doi:10.21769/BioProtoc.5029
apa: LI, Z., Huard, J., Bayer, E. M., & Wattelet-Boyer, V. (2024). Versatile
cloning strategy for efficient multigene editing in Arabidopsis. Bio-Protocol.
Bio-Protocol. https://doi.org/10.21769/BioProtoc.5029
chicago: LI, ZIQIANG, Jennifer Huard, Emmanuelle M. Bayer, and Valérie Wattelet-Boyer.
“Versatile Cloning Strategy for Efficient Multigene Editing in Arabidopsis.” Bio-Protocol.
Bio-Protocol, 2024. https://doi.org/10.21769/BioProtoc.5029.
ieee: Z. LI, J. Huard, E. M. Bayer, and V. Wattelet-Boyer, “Versatile cloning strategy
for efficient multigene editing in Arabidopsis,” Bio-protocol, 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.” Bio-Protocol, vol. 14, no. 13, e5029, Bio-Protocol, 2024,
doi:10.21769/BioProtoc.5029.
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'
...
---
_id: '442'
abstract:
- lang: eng
text: The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the
nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the
apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the
method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin
response in hypocotyl segments as well as the determination of relative values
of the cell wall pH.
acknowledgement: 'This protocol was adapted from Fendrych et al., 2016. This project
has received funding from the European Union’s Horizon 2020 research and innovation
programme under the Marie Skłodowska-Curie Grant Agreement No. 665385, and Austrian
Science Fund (FWF) [M 2128-B21]. '
article_processing_charge: No
article_type: original
author:
- first_name: Lanxin
full_name: Li, Lanxin
id: 367EF8FA-F248-11E8-B48F-1D18A9856A87
last_name: Li
orcid: 0000-0002-5607-272X
- first_name: Gabriel
full_name: Krens, Gabriel
id: 2B819732-F248-11E8-B48F-1D18A9856A87
last_name: Krens
orcid: 0000-0003-4761-5996
- first_name: Matyas
full_name: Fendrych, Matyas
id: 43905548-F248-11E8-B48F-1D18A9856A87
last_name: Fendrych
orcid: 0000-0002-9767-8699
- first_name: Jirí
full_name: Friml, Jirí
id: 4159519E-F248-11E8-B48F-1D18A9856A87
last_name: Friml
orcid: 0000-0002-8302-7596
citation:
ama: Li L, Krens G, Fendrych M, Friml J. Real-time analysis of auxin response, cell
wall pH and elongation in Arabidopsis thaliana Hypocotyls. Bio-protocol.
2018;8(1). doi:10.21769/BioProtoc.2685
apa: Li, L., Krens, G., Fendrych, M., & Friml, J. (2018). Real-time analysis
of auxin response, cell wall pH and elongation in Arabidopsis thaliana Hypocotyls.
Bio-Protocol. Bio-protocol. https://doi.org/10.21769/BioProtoc.2685
chicago: Li, Lanxin, Gabriel Krens, Matyas Fendrych, and Jiří Friml. “Real-Time
Analysis of Auxin Response, Cell Wall PH and Elongation in Arabidopsis Thaliana
Hypocotyls.” Bio-Protocol. Bio-protocol, 2018. https://doi.org/10.21769/BioProtoc.2685.
ieee: L. Li, G. Krens, M. Fendrych, and J. Friml, “Real-time analysis of auxin response,
cell wall pH and elongation in Arabidopsis thaliana Hypocotyls,” Bio-protocol,
vol. 8, no. 1. Bio-protocol, 2018.
ista: Li L, Krens G, Fendrych M, Friml J. 2018. Real-time analysis of auxin response,
cell wall pH and elongation in Arabidopsis thaliana Hypocotyls. Bio-protocol.
8(1).
mla: Li, Lanxin, et al. “Real-Time Analysis of Auxin Response, Cell Wall PH and
Elongation in Arabidopsis Thaliana Hypocotyls.” Bio-Protocol, vol. 8, no.
1, Bio-protocol, 2018, doi:10.21769/BioProtoc.2685.
short: L. Li, G. Krens, M. Fendrych, J. Friml, Bio-Protocol 8 (2018).
corr_author: '1'
date_created: 2018-12-11T11:46:30Z
date_published: 2018-01-05T00:00:00Z
date_updated: 2026-05-26T22:31:19Z
day: '05'
ddc:
- '576'
- '581'
department:
- _id: JiFr
- _id: Bio
doi: 10.21769/BioProtoc.2685
ec_funded: 1
file:
- access_level: open_access
checksum: 6644ba698206eda32b0abf09128e63e3
content_type: application/pdf
creator: system
date_created: 2018-12-12T10:17:43Z
date_updated: 2020-07-14T12:46:29Z
file_id: '5299'
file_name: IST-2018-970-v1+1_2018_Lanxin_Real-time_analysis.pdf
file_size: 11352389
relation: main_file
file_date_updated: 2020-07-14T12:46:29Z
has_accepted_license: '1'
intvolume: ' 8'
issue: '1'
language:
- iso: eng
month: '01'
oa: 1
oa_version: Published Version
project:
- _id: 2564DBCA-B435-11E9-9278-68D0E5697425
call_identifier: H2020
grant_number: '665385'
name: International IST Doctoral Program
publication: Bio-protocol
publication_identifier:
eissn:
- 2331-8325
publication_status: published
publisher: Bio-protocol
publist_id: '7381'
pubrep_id: '970'
quality_controlled: '1'
related_material:
record:
- id: '10083'
relation: dissertation_contains
status: public
status: public
title: Real-time analysis of auxin response, cell wall pH and elongation in Arabidopsis
thaliana Hypocotyls
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: 8
year: '2018'
...
---
DOAJ_listed: '1'
OA_place: publisher
OA_type: gold
_id: '832'
abstract:
- lang: eng
text: Plants maintain capacity to form new organs such as leaves, flowers, lateral
shoots and roots throughout their postembryonic lifetime. Lateral roots (LRs)
originate from a few pericycle cells that acquire attributes of founder cells
(FCs), undergo series of anticlinal divisions, and give rise to a few short initial
cells. After initiation, coordinated cell division and differentiation occur,
giving rise to lateral root primordia (LRP). Primordia continue to grow, emerge
through the cortex and epidermal layers of the primary root, and finally a new
apical meristem is established taking over the responsibility for growth of mature
lateral roots [for detailed description of the individual stages of lateral root
organogenesis see Malamy and Benfey (1997)]. To examine this highly dynamic developmental
process and to investigate a role of various hormonal, genetic and environmental
factors in the regulation of lateral root organogenesis, the real time imaging
based analyses represent extremely powerful tools (Laskowski et al., 2008; De
Smet et al., 2012; Marhavy et al., 2013 and 2014). Herein, we describe a protocol
for real time lateral root primordia (LRP) analysis, which enables the monitoring
of an onset of the specific gene expression and subcellular protein localization
during primordia organogenesis, as well as the evaluation of the impact of genetic
and environmental perturbations on LRP organogenesis.
acknowledgement: "European Research Council with a Starting Independent Research grant:
ERC-2007-Stg-207362-HCPO, Czech Science Foundation: GA13-39982S\r\nWe thank Matyas
Fendrych for critical reading and comments. The protocol was developed based on
previously published work of De Rybel et al. (2010) and Laskowski et al. (2008). "
article_processing_charge: No
article_type: original
author:
- first_name: Peter
full_name: Marhavy, Peter
id: 3F45B078-F248-11E8-B48F-1D18A9856A87
last_name: Marhavy
orcid: 0000-0001-5227-5741
- first_name: Eva
full_name: Benková, Eva
id: 38F4F166-F248-11E8-B48F-1D18A9856A87
last_name: Benková
orcid: 0000-0002-8510-9739
citation:
ama: Marhavý P, Benková E. Real time analysis of lateral root organogenesis in arabidopsis.
Bio-protocol. 2015;5(8). doi:10.21769/BioProtoc.1446
apa: Marhavý, P., & Benková, E. (2015). Real time analysis of lateral root organogenesis
in arabidopsis. Bio-Protocol. Bio-protocol LLC. https://doi.org/10.21769/BioProtoc.1446
chicago: Marhavý, Peter, and Eva Benková. “Real Time Analysis of Lateral Root Organogenesis
in Arabidopsis.” Bio-Protocol. Bio-protocol LLC, 2015. https://doi.org/10.21769/BioProtoc.1446.
ieee: P. Marhavý and E. Benková, “Real time analysis of lateral root organogenesis
in arabidopsis,” Bio-protocol, vol. 5, no. 8. Bio-protocol LLC, 2015.
ista: Marhavý P, Benková E. 2015. Real time analysis of lateral root organogenesis
in arabidopsis. Bio-protocol. 5(8).
mla: Marhavý, Peter, and Eva Benková. “Real Time Analysis of Lateral Root Organogenesis
in Arabidopsis.” Bio-Protocol, vol. 5, no. 8, Bio-protocol LLC, 2015, doi:10.21769/BioProtoc.1446.
short: P. Marhavý, E. Benková, Bio-Protocol 5 (2015).
date_created: 2018-12-11T11:48:44Z
date_published: 2015-04-20T00:00:00Z
date_updated: 2026-05-19T09:39:29Z
day: '20'
doi: 10.21769/BioProtoc.1446
extern: '1'
external_id:
pmid:
- '27331080'
intvolume: ' 5'
issue: '8'
language:
- iso: eng
main_file_link:
- open_access: '1'
url: https://doi.org/10.21769/BioProtoc.1446
month: '04'
oa: 1
oa_version: Published Version
pmid: 1
publication: Bio-protocol
publication_identifier:
eissn:
- 2331-8325
publication_status: published
publisher: Bio-protocol LLC
publist_id: '6816'
quality_controlled: '1'
status: public
title: Real time analysis of lateral root organogenesis 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: ba8df636-2132-11f1-aed0-ed93e2281fdd
volume: 5
year: '2015'
...