Chapter 5
CRISPR/Cas9-Based Genome Editing Toolbox
for Arabidopsis thaliana
Daisuke Miki, Gaurav Zinta, Wenxin Zhang, Fangnan Peng,
Zhengyan Feng, and Jian-Kang Zhu
Abstract
CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and
improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to
generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by
error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways
can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused
to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without
generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene
mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system.
Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and
analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing
in other plant species such as rice.
Keywords CRISPR/Cas9, Sequence-specific nucleases, Targeted gene editing, Homologous recombination, Base editors, Genetic manipulation
1 Introduction
Genome sequencing technologies have revolutionized the
biological sciences. With the availability of genome sequences of
various living organisms including plants, the focus is now shifted
to uncover gene function [1]. In this regard, both forward and
reverse genetic approaches have contributed immensely to plant
functional genomics [2]. Geneticists initially used natural mutants
to elucidate the function of genes, but later on artificial mutants
were created by using physical, chemical, and biological agents.
However, these methods have some limitations. For instance,
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Daisuke Miki and Gaurav Zinta contributed equally with all other contributors.
121
CRISPR/Cas9-Based Genome Editing Toolbox
for Arabidopsis thaliana
Daisuke Miki, Gaurav Zinta, Wenxin Zhang, Fangnan Peng,
Zhengyan Feng, and Jian-Kang Zhu
Abstract
CRISPR/Cas9 system has emerged as a powerful genome engineering tool to study gene function and
improve plant traits. Genome editing is achieved at a specific genome sequence by Cas9 endonuclease to
generate double standard breaks (DSBs) directed by short guide RNAs (sgRNAs). The DSB is repaired by
error-prone nonhomologous end joining (NHEJ) or error-free homology-directed repair (HDR) pathways, resulting in gene mutation or sequence replacement, respectively. These cellular DSB repair pathways
can be exploited to knock out or replace genes. Also, cytidine or adenine base editors (CBEs or ABEs) fused
to catalytically dead Cas9 (dCas9) or nickase Cas9 (nCas9) are used to perform precise base editing without
generating DSBs. In this chapter, we describe a detailed procedure to carry out single/multiple gene
mutations and precise base editing in the Arabidopsis genome by using CRISPR/Cas9-based system.
Specifically, the steps of target gene selection, sgRNA design, vector construction, transformation, and
analysis of transgenic lines are described. The protocol is potentially adaptable to perform genome editing
in other plant species such as rice.
Keywords CRISPR/Cas9, Sequence-specific nucleases, Targeted gene editing, Homologous recombination, Base editors, Genetic manipulation
1 Introduction
Genome sequencing technologies have revolutionized the
biological sciences. With the availability of genome sequences of
various living organisms including plants, the focus is now shifted
to uncover gene function [1]. In this regard, both forward and
reverse genetic approaches have contributed immensely to plant
functional genomics [2]. Geneticists initially used natural mutants
to elucidate the function of genes, but later on artificial mutants
were created by using physical, chemical, and biological agents.
However, these methods have some limitations. For instance,
Jose J. Sanchez-Serrano and Julio Salinas (eds.), Arabidopsis Protocols, Methods in Molecular Biology, vol. 2200,
https://doi.org/10.1007/978-1-0716-0880-7_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
Daisuke Miki and Gaurav Zinta contributed equally with all other contributors.
121