must be harbored simultaneously with Cas9 expression cassette on
a same T-DNA construct. The efficiency and heritability of
CRISPR/Cas9-mediated genome editing depends on the expression of Cas9 and sgRNAs in the cells. Driving the expression of
Cas9 by germline-specific promoters increases the efficiency of
heritable gene mutations in Arabidopsis as previously shown for
egg cell-specific DD45 [13, 14], meiosis-specific MGE1 [15],
male gametocyte-specific SPL [14], pollen-specific Lat52 [14],
cell division-specific YAO [16] and CDC45 [11], and all developmental stage-specific RPS5A [17].
On the other hand, homology-directed repair (HDR) is
exploited for gene targeting (GT) and makes precise modifications
in genome such as DNA knock-in and gene replacement (Fig. 1a).
However, HDR-mediated GT is extremely inefficient in plants,
thus limiting its widespread application. We recently developed a
simple, efficient, and precise method of gene targeting in Arabidopsis by using a combination of germline and early embryo-specific
promoter DD45 to drive Cas9 expression and sequential transformation strategy [18]. By using this method we achieved a heritable
GT efficiency of ~9%, and GT plants were easily identified by
regular PCR.
Another alternative approach that enables precise base editing
without DSB is the use of “base editors” allowing the direct conversion of target bases into different ones [19]. Two members of
base editors are reported in plants, which include cytidine base
editors (CBEs) and adenine base editors (ABEs). CBEs efficiently
induce cytosine to thymine (C-to-T) or guanine to adenine
(G-to-A) conversions, while adenine base editors (ABEs) convert
A-to-G or T-to-C (Fig. 1c). The vector construct is prepared by
fusing base editors with enzymatically dead Cas9 (dCas9, mutations at D10A and H840A domains) or nickase Cas9 (nCas9,
mutation at D10A) and sgRNA expression cassette that enable
binding to DNA in a precise manner [20–23]. These base editors
can be utilized to edit one or a few single nucleotide(s) with high
accuracy and efficiency (Fig. 1b), and have applicability where
off-target editing of adjacent nucleotides is not tolerable and phenotype is controlled by a single nucleotide variation.
In this chapter, we discuss the applicability of CRISPR/Cas9
system to generate single/multiple knockout mutants, and perform
precise base edits (including knock-in and replacement) in the
genome of Arabidopsis thaliana (Fig. 2). The protocol is easy to
follow and practical steps of target gene selection, sgRNA design,
vector construction, transformation, and analysis of transgenic lines
are described in detail. The principles of this protocol can potentially be adapted to perform genome editing in other plant species
such as rice.
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Daisuke Miki et al.
a same T-DNA construct. The efficiency and heritability of
CRISPR/Cas9-mediated genome editing depends on the expression of Cas9 and sgRNAs in the cells. Driving the expression of
Cas9 by germline-specific promoters increases the efficiency of
heritable gene mutations in Arabidopsis as previously shown for
egg cell-specific DD45 [13, 14], meiosis-specific MGE1 [15],
male gametocyte-specific SPL [14], pollen-specific Lat52 [14],
cell division-specific YAO [16] and CDC45 [11], and all developmental stage-specific RPS5A [17].
On the other hand, homology-directed repair (HDR) is
exploited for gene targeting (GT) and makes precise modifications
in genome such as DNA knock-in and gene replacement (Fig. 1a).
However, HDR-mediated GT is extremely inefficient in plants,
thus limiting its widespread application. We recently developed a
simple, efficient, and precise method of gene targeting in Arabidopsis by using a combination of germline and early embryo-specific
promoter DD45 to drive Cas9 expression and sequential transformation strategy [18]. By using this method we achieved a heritable
GT efficiency of ~9%, and GT plants were easily identified by
regular PCR.
Another alternative approach that enables precise base editing
without DSB is the use of “base editors” allowing the direct conversion of target bases into different ones [19]. Two members of
base editors are reported in plants, which include cytidine base
editors (CBEs) and adenine base editors (ABEs). CBEs efficiently
induce cytosine to thymine (C-to-T) or guanine to adenine
(G-to-A) conversions, while adenine base editors (ABEs) convert
A-to-G or T-to-C (Fig. 1c). The vector construct is prepared by
fusing base editors with enzymatically dead Cas9 (dCas9, mutations at D10A and H840A domains) or nickase Cas9 (nCas9,
mutation at D10A) and sgRNA expression cassette that enable
binding to DNA in a precise manner [20–23]. These base editors
can be utilized to edit one or a few single nucleotide(s) with high
accuracy and efficiency (Fig. 1b), and have applicability where
off-target editing of adjacent nucleotides is not tolerable and phenotype is controlled by a single nucleotide variation.
In this chapter, we discuss the applicability of CRISPR/Cas9
system to generate single/multiple knockout mutants, and perform
precise base edits (including knock-in and replacement) in the
genome of Arabidopsis thaliana (Fig. 2). The protocol is easy to
follow and practical steps of target gene selection, sgRNA design,
vector construction, transformation, and analysis of transgenic lines
are described in detail. The principles of this protocol can potentially be adapted to perform genome editing in other plant species
such as rice.
124
Daisuke Miki et al.