creating mutants by physical (radiation) and chemical (EMS, ethyl
methanesulfonate) agents causes random mutations in the genome
[3]. Thus, to establish a causal relationship between genotype and
phenotype, large-scale genetic screening is required, which is labor
intensive, costly, and time-consuming. The reverse genetics methods, including T-DNA insertion lines (e.g., SALK T-DNA insertion
library), RNA interference (RNAi), and virus-induced gene silencing (VIGS), offer direct ways to elucidate gene function, which
involve reduction of transcript levels of endogenous genes to generate knockdown mutants [4]. However, sometimes appropriate
T-DNA insertion lines are not available in the libraries, the RNA
silencing methods are not highly specific, and reductions achieved
in the gene expression are variable and not stably inherited to next
generation.
Sequence-specific nucleases (SSNs) generate target site-specific
double strand breaks (DSBs) in the genome of numerous organisms [5–8]. Due to this property, SSNs including zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases
(TALENs), and clustered regularly interspaced short palindromic
repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) have
emerged as versatile tools for genome engineering. Particularly,
CRISPR/Cas9 system is the most frequently used genome editing
tool in plants due to its simplicity, high specificity, efficiency, and
multiplexing capacity [9]. The Cas9 endonuclease is directed to
specific genomic loci by a 20-nt single-guide RNA (sgRNA)
(Fig. 1a). Type II Streptococcus pyogenes Cas9 (SpCas9) is the most
widely used Cas9 that recognize NGG as a protospacer adjacent
motif (PAM) sequence. The activity of Cas9 generates a double
strand break (DSB) at 3–4 base pairs distal to the PAM sequence
(Fig. 1b). DSBs are subsequently repaired by either error-prone
nonhomologous end joining (NHEJ) or error-free homologydirected repair (HDR), if certain homologous DNA repair donor
template is provided, resulting in gene mutations or knock-in/
replacement, respectively (Fig. 1a).
The imprecise repair of DSBs by NHEJ causes random insertion or deletion of nucleotides at the target site. This results into
generation of null mutants by frameshift mutation, which can be
used for functional gene analysis. However, genetic redundancy is a
formidable problem to understand gene function when members of
a gene family perform the same function. The CRISPR/Cas9
system provides opportunities to overcome this issue by directing
Cas9 to multiple genetic loci by co-expressing multiple sgRNAs,
and facilitating multiplex gene targeting [10–12]. When targeting a
single genetic locus in the genome, the expression sgRNA is driven
by AtU6–26 Pol III promoter, whereas for targeting multiple
genetic loci to obtain high-order mutants, a combination of other
Pol III promoters such as AtU3b and At7SL-2 is used
[10, 11]. This implies that multiple sgRNA expression cassettes
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Daisuke Miki et al.
methanesulfonate) agents causes random mutations in the genome
[3]. Thus, to establish a causal relationship between genotype and
phenotype, large-scale genetic screening is required, which is labor
intensive, costly, and time-consuming. The reverse genetics methods, including T-DNA insertion lines (e.g., SALK T-DNA insertion
library), RNA interference (RNAi), and virus-induced gene silencing (VIGS), offer direct ways to elucidate gene function, which
involve reduction of transcript levels of endogenous genes to generate knockdown mutants [4]. However, sometimes appropriate
T-DNA insertion lines are not available in the libraries, the RNA
silencing methods are not highly specific, and reductions achieved
in the gene expression are variable and not stably inherited to next
generation.
Sequence-specific nucleases (SSNs) generate target site-specific
double strand breaks (DSBs) in the genome of numerous organisms [5–8]. Due to this property, SSNs including zinc finger
nucleases (ZFNs), transcription activator-like effector nucleases
(TALENs), and clustered regularly interspaced short palindromic
repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) have
emerged as versatile tools for genome engineering. Particularly,
CRISPR/Cas9 system is the most frequently used genome editing
tool in plants due to its simplicity, high specificity, efficiency, and
multiplexing capacity [9]. The Cas9 endonuclease is directed to
specific genomic loci by a 20-nt single-guide RNA (sgRNA)
(Fig. 1a). Type II Streptococcus pyogenes Cas9 (SpCas9) is the most
widely used Cas9 that recognize NGG as a protospacer adjacent
motif (PAM) sequence. The activity of Cas9 generates a double
strand break (DSB) at 3–4 base pairs distal to the PAM sequence
(Fig. 1b). DSBs are subsequently repaired by either error-prone
nonhomologous end joining (NHEJ) or error-free homologydirected repair (HDR), if certain homologous DNA repair donor
template is provided, resulting in gene mutations or knock-in/
replacement, respectively (Fig. 1a).
The imprecise repair of DSBs by NHEJ causes random insertion or deletion of nucleotides at the target site. This results into
generation of null mutants by frameshift mutation, which can be
used for functional gene analysis. However, genetic redundancy is a
formidable problem to understand gene function when members of
a gene family perform the same function. The CRISPR/Cas9
system provides opportunities to overcome this issue by directing
Cas9 to multiple genetic loci by co-expressing multiple sgRNAs,
and facilitating multiplex gene targeting [10–12]. When targeting a
single genetic locus in the genome, the expression sgRNA is driven
by AtU6–26 Pol III promoter, whereas for targeting multiple
genetic loci to obtain high-order mutants, a combination of other
Pol III promoters such as AtU3b and At7SL-2 is used
[10, 11]. This implies that multiple sgRNA expression cassettes
122
Daisuke Miki et al.