Chapter 25
CRISPR-Cas-Mediated Gene Knockout in Tomato
Gwen Swinnen, Thomas Jacobs, Laurens Pauwels, and Alain Goossens
Abstract
Loss-of-function mutants are crucial for plant functional genomics studies. With the advent of CRISPR-Cas
genome editing, generating null alleles for one or multiple specific gene(s) has become feasible for many
plant species including tomato (Solanum lycopersicum). An easily programmable RNA-guided Cas endonuclease efficiently creates DNA double-strand breaks (DSBs) at targeted genomic sites that can be repaired by
nonhomologous end joining (NHEJ) typically leading to small insertions or deletions that can produce null
mutations. Here, we describe how to utilize CRISPR-Cas genome editing to obtain stable tomato gene
knockout lines.
Key words Genome editing, CRISRP-Cas, Gene knockout, Loss-of-function mutation, Null mutation, Site-directed mutagenesis, Targeted mutagenesis, Tomato, Solanum lycopersicum, Solanaceae
1 Introduction
In recent decades, studying genetic tomato (Solanum lycopersicum)
mutants has allowed functional gene characterization through
reverse genetics. This involved the generation and screening of
mutations randomly introduced in the genome by physical (e.g.,
gamma radiation), chemical (e.g., ethyl methanesulfonate), or
insertional (e.g., T-DNA or transposon insertion) mutagenesis
[1]. The availability of the tomato genome sequence and the advent
of genome editing using sequence-specific nucleases revealed the
possibility of site-directed mutagenesis [2]. With the emergence of
clustered regularly interspaced short palindromic repeats-CRISPR
associated protein (CRISPR-Cas) systems, targeted introduction of
a DNA double-strand break (DSB) to create a gene knockout has
become feasible for Solanaceae species like tomato.
The ease and efficiency by which CRISPR-Cas can be applied
rely on its simple composition and programmable versatility. An
artificial guide RNA (gRNA) directs a generic Cas endonuclease to
the genomic target site through base pairing of the gRNA with the
genomic protospacer sequence, which neighbors a protospacer
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_25,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
321
CRISPR-Cas-Mediated Gene Knockout in Tomato
Gwen Swinnen, Thomas Jacobs, Laurens Pauwels, and Alain Goossens
Abstract
Loss-of-function mutants are crucial for plant functional genomics studies. With the advent of CRISPR-Cas
genome editing, generating null alleles for one or multiple specific gene(s) has become feasible for many
plant species including tomato (Solanum lycopersicum). An easily programmable RNA-guided Cas endonuclease efficiently creates DNA double-strand breaks (DSBs) at targeted genomic sites that can be repaired by
nonhomologous end joining (NHEJ) typically leading to small insertions or deletions that can produce null
mutations. Here, we describe how to utilize CRISPR-Cas genome editing to obtain stable tomato gene
knockout lines.
Key words Genome editing, CRISRP-Cas, Gene knockout, Loss-of-function mutation, Null mutation, Site-directed mutagenesis, Targeted mutagenesis, Tomato, Solanum lycopersicum, Solanaceae
1 Introduction
In recent decades, studying genetic tomato (Solanum lycopersicum)
mutants has allowed functional gene characterization through
reverse genetics. This involved the generation and screening of
mutations randomly introduced in the genome by physical (e.g.,
gamma radiation), chemical (e.g., ethyl methanesulfonate), or
insertional (e.g., T-DNA or transposon insertion) mutagenesis
[1]. The availability of the tomato genome sequence and the advent
of genome editing using sequence-specific nucleases revealed the
possibility of site-directed mutagenesis [2]. With the emergence of
clustered regularly interspaced short palindromic repeats-CRISPR
associated protein (CRISPR-Cas) systems, targeted introduction of
a DNA double-strand break (DSB) to create a gene knockout has
become feasible for Solanaceae species like tomato.
The ease and efficiency by which CRISPR-Cas can be applied
rely on its simple composition and programmable versatility. An
artificial guide RNA (gRNA) directs a generic Cas endonuclease to
the genomic target site through base pairing of the gRNA with the
genomic protospacer sequence, which neighbors a protospacer
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_25,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
321