genes in the DNA sequences to be used for
transformation. Since this is the first application
of the CRISPR-Cas9 technology in duckweed,
we would like to describe how genome editing
works and how to apply genome editing in
duckweed.
Nearly all available genome editing technologies rely on endonucleases, which create a
double-strand break (DSB) in the target DNA
(Osakabe and Osakabe 2015). DSBs, like any
other DNA damage, are repaired in vivo through
different repair mechanisms. The two major
mechanisms are the non-homologous end joining
(NHEJ) and the homology directed repair
(HDR).
NHEJ can be described as an erroneous repair
mechanism. There are two pathways in which
NHEJ can repair a DSB. First, the proteins
Ku70/Ku80 can lead a ligase and its cofactor to
the DSB. This option usually leads to small base
insertions or deletions (Deriano and Roth 2013).
The second, often preferred pathway is based on
microhomologies (Vu et al. 2014). Exonucleases
digest the ends of the broken DNA until short
homologies on both ends are available, which are
then re-ligated (Crespan et al. 2012; Deriano and
Roth 2013). Therefore, this pathway causes
deletions instead of insertions. NHEJ is the main
repair mechanism for DSBs in eukaryotic cells
(Sonoda et al. 2006).
The other type of repair mechanism is available only in a small number of cell types and is
named homology directed repair (HDR). HDR is
based on homologous recombination (HR) and
works in the same fashion. The process requires
homologous DNA sequences as a repair template. This template DNA is used to repair the
DSB. The HDR process allows the integration or
substitution of bases if there are flanking,
homologous sequences around the DSB. If the
DNA to be integrated is flanked by homologous
DNA sequences (each about 1000 bp), an integration at the site of the broken dsDNA strand
will occur (Osakabe and Osakabe 2015). It was
shown that whole genes can be inserted using
this method in plants (Fauser et al. 2014; Schiml
et al. 2014).
Today, two methods of genome editing are
used: TALEN and CRISPR/Cas9 (Osakabe and
Osakabe 2015; Singh et al. 2015; Samanta et al.
2016; Schiml and Puchta 2016). We have chosen
the second procedure, the CRISPR/Cas9 system.
Originally, CRISPR (clustered regularly
interspaced short palindromic repeat) was
described as a defense mechanism in bacteria and
archaea to fight phages (Wiedenheft et al. 2012).
Genomes of these organisms contain a cluster of
foreign (phage) DNA sequences, the so-called
CRISPR array (Barrangou et al. 2007). Each
repeat code for two RNAs: the crRNA represents
the foreign DNA sequences, and the tracrRNA
represents an integral element of the bacterial
genome. The crRNA binds to foreign DNA,
delivered by the phage in the case of an infection,
whereas the tracrRNA binds to the crRNA. This
binding causes a hairpin structure, which can be
detected by the Cas9 protein, an endonuclease
that creates a DSB in the target DNA (Garneau
et al. 2010; Sternberg et al. 2014).
There is another prerequisite needed by the
Cas9 nuclease; it is the so-called protospacer
adjacent motif (PAM). The PAM consists of
three bases (NGG) and must be located next to
the sequence to be cut (Garneau et al. 2010;
Sternberg et al. 2014). For practical reasons, the
two RNAs involved in the CRISPR/Cas9 mechanism are combined into one so-called single
guide RNA (sgRNA or gRNA) (Jinek et al.
2013), when used for genome editing.
CRISPR/Cas9 has successfully been applied
to edit the genome of various organisms (e.g.,
Cong et al. 2013; Jiang et al. 2013a, 2013b; Mali
et al. 2013; Dicarlo et al. 2013; Kim et al. 2014;
Tang et al. 2017), but has yet to be demonstrated
in Lemnaceae.
Cloning of sgRNAs can be performed relatively simply using the Golden Gate cloning
technology (Engler et al. 2008). The tracrRNA
section of the sgRNAs is already present in the
plasmids used for genome editing, and the same
is true for the Cas9 gene. Therefore, only the
cloning of the homologous section of the sgRNA
has to be cloned. These parts can be constructed
from two 20 bp long oligonucleotides, which are
17 Editing the Genome of Wolffia australiana
171
transformation. Since this is the first application
of the CRISPR-Cas9 technology in duckweed,
we would like to describe how genome editing
works and how to apply genome editing in
duckweed.
Nearly all available genome editing technologies rely on endonucleases, which create a
double-strand break (DSB) in the target DNA
(Osakabe and Osakabe 2015). DSBs, like any
other DNA damage, are repaired in vivo through
different repair mechanisms. The two major
mechanisms are the non-homologous end joining
(NHEJ) and the homology directed repair
(HDR).
NHEJ can be described as an erroneous repair
mechanism. There are two pathways in which
NHEJ can repair a DSB. First, the proteins
Ku70/Ku80 can lead a ligase and its cofactor to
the DSB. This option usually leads to small base
insertions or deletions (Deriano and Roth 2013).
The second, often preferred pathway is based on
microhomologies (Vu et al. 2014). Exonucleases
digest the ends of the broken DNA until short
homologies on both ends are available, which are
then re-ligated (Crespan et al. 2012; Deriano and
Roth 2013). Therefore, this pathway causes
deletions instead of insertions. NHEJ is the main
repair mechanism for DSBs in eukaryotic cells
(Sonoda et al. 2006).
The other type of repair mechanism is available only in a small number of cell types and is
named homology directed repair (HDR). HDR is
based on homologous recombination (HR) and
works in the same fashion. The process requires
homologous DNA sequences as a repair template. This template DNA is used to repair the
DSB. The HDR process allows the integration or
substitution of bases if there are flanking,
homologous sequences around the DSB. If the
DNA to be integrated is flanked by homologous
DNA sequences (each about 1000 bp), an integration at the site of the broken dsDNA strand
will occur (Osakabe and Osakabe 2015). It was
shown that whole genes can be inserted using
this method in plants (Fauser et al. 2014; Schiml
et al. 2014).
Today, two methods of genome editing are
used: TALEN and CRISPR/Cas9 (Osakabe and
Osakabe 2015; Singh et al. 2015; Samanta et al.
2016; Schiml and Puchta 2016). We have chosen
the second procedure, the CRISPR/Cas9 system.
Originally, CRISPR (clustered regularly
interspaced short palindromic repeat) was
described as a defense mechanism in bacteria and
archaea to fight phages (Wiedenheft et al. 2012).
Genomes of these organisms contain a cluster of
foreign (phage) DNA sequences, the so-called
CRISPR array (Barrangou et al. 2007). Each
repeat code for two RNAs: the crRNA represents
the foreign DNA sequences, and the tracrRNA
represents an integral element of the bacterial
genome. The crRNA binds to foreign DNA,
delivered by the phage in the case of an infection,
whereas the tracrRNA binds to the crRNA. This
binding causes a hairpin structure, which can be
detected by the Cas9 protein, an endonuclease
that creates a DSB in the target DNA (Garneau
et al. 2010; Sternberg et al. 2014).
There is another prerequisite needed by the
Cas9 nuclease; it is the so-called protospacer
adjacent motif (PAM). The PAM consists of
three bases (NGG) and must be located next to
the sequence to be cut (Garneau et al. 2010;
Sternberg et al. 2014). For practical reasons, the
two RNAs involved in the CRISPR/Cas9 mechanism are combined into one so-called single
guide RNA (sgRNA or gRNA) (Jinek et al.
2013), when used for genome editing.
CRISPR/Cas9 has successfully been applied
to edit the genome of various organisms (e.g.,
Cong et al. 2013; Jiang et al. 2013a, 2013b; Mali
et al. 2013; Dicarlo et al. 2013; Kim et al. 2014;
Tang et al. 2017), but has yet to be demonstrated
in Lemnaceae.
Cloning of sgRNAs can be performed relatively simply using the Golden Gate cloning
technology (Engler et al. 2008). The tracrRNA
section of the sgRNAs is already present in the
plasmids used for genome editing, and the same
is true for the Cas9 gene. Therefore, only the
cloning of the homologous section of the sgRNA
has to be cloned. These parts can be constructed
from two 20 bp long oligonucleotides, which are
17 Editing the Genome of Wolffia australiana
171