industrial-scale production of secondary metabolites but also of pharmaceutical
proteins. In fact, like undifferentiated cells, they grow in simple, defined media
but are genetically stable and easy to handle, with protocols available to establish
transgenic lines highly scalable, producing significant biomass accumulation
(Guillon et al. 2006).
HR cultures are generated by Agrobacterium rhizogenes infection. This infection
leads to the generation of root clones characterized by an extensive secondary
branching that can be cultivated under contained sterile conditions in hormonefree media (Franconi et al. 2010). Importantly, recombinant proteins can be secreted
in the culture medium facilitating the downstream purification processes (Guillon
et al. 2006).
HR have been used to express a range of recombinant proteins, such as enzymes
(Woods et al. 2008), vaccines (Skarjinskaia et al. 2013), monoclonal antibodies
(Wongsamuth and Doran 1997) and anti-HIV microbicides (Drake et al. 2013).
2.1.5 The CRISPR-Cas9 System
Editing the genome of a plant without introducing foreign DNA into cells is now a
reality. Programmable nucleases, like zinc-finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs) and RNA-guided endonucleases, have
been used for genome editing in plant cells, amplifying the horizon of plant
molecular farming (Li et al. 2013; Shan et al. 2013; Nekrasov et al. 2013). Recently,
the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9
(CRISPR-associated) system has been successfully used in a wide range of plant
species (Bortesi and Fischer 2015) but not in plant suspension cells. The system
is based on a short RNA guide (sgRNA) which associates to the Cas9 endonuclease to create a double-stranded break (DSB) in the target genomic DNA. As a
consequence, mutations are generated through either error-prone non-homologous
end-joining (NHEJ) or homology-directed repair (HDR) of the intended cleavage
site. NHEJ has been used to generate mutagenic insertions/deletions often leading to
gene inactivation. The resulting changes at chromosomal target sites can be indistinguishable from naturally occurring genetic variation. However, the system can be
used to introduce larger modifications and insertion of exogenous sequences. Moreover, similar to “traditional” gene insertion, if A. tumefaciens is used as a delivery
vector of CRISPR-Cas9, the resulting genome-edited plants contain foreign DNA
sequences, in addition to those that encode the programmable nucleases, in the host
genome. On the other hand, the use of episomal plasmids to deliver these nucleases
into plant cells has to take into account that transfected plasmids are rapidly
degraded in cells by endogenous nucleases and that the resulting DNA fragments
can be inserted at off-target sites in host cells (Kim et al. 2014).
The delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into plant
cells has been shown to remove the possibility to introduce recombinant DNA
into the host plant genome (Cho et al. 2013). Furthermore, it as has been shown
that these premixed RNPs cleave target DNA sites immediately after transfection
Engineering Plants for the Future: Farming with Value-Added Harvest
73
proteins. In fact, like undifferentiated cells, they grow in simple, defined media
but are genetically stable and easy to handle, with protocols available to establish
transgenic lines highly scalable, producing significant biomass accumulation
(Guillon et al. 2006).
HR cultures are generated by Agrobacterium rhizogenes infection. This infection
leads to the generation of root clones characterized by an extensive secondary
branching that can be cultivated under contained sterile conditions in hormonefree media (Franconi et al. 2010). Importantly, recombinant proteins can be secreted
in the culture medium facilitating the downstream purification processes (Guillon
et al. 2006).
HR have been used to express a range of recombinant proteins, such as enzymes
(Woods et al. 2008), vaccines (Skarjinskaia et al. 2013), monoclonal antibodies
(Wongsamuth and Doran 1997) and anti-HIV microbicides (Drake et al. 2013).
2.1.5 The CRISPR-Cas9 System
Editing the genome of a plant without introducing foreign DNA into cells is now a
reality. Programmable nucleases, like zinc-finger nucleases (ZFNs), transcription
activator-like effector nucleases (TALENs) and RNA-guided endonucleases, have
been used for genome editing in plant cells, amplifying the horizon of plant
molecular farming (Li et al. 2013; Shan et al. 2013; Nekrasov et al. 2013). Recently,
the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9
(CRISPR-associated) system has been successfully used in a wide range of plant
species (Bortesi and Fischer 2015) but not in plant suspension cells. The system
is based on a short RNA guide (sgRNA) which associates to the Cas9 endonuclease to create a double-stranded break (DSB) in the target genomic DNA. As a
consequence, mutations are generated through either error-prone non-homologous
end-joining (NHEJ) or homology-directed repair (HDR) of the intended cleavage
site. NHEJ has been used to generate mutagenic insertions/deletions often leading to
gene inactivation. The resulting changes at chromosomal target sites can be indistinguishable from naturally occurring genetic variation. However, the system can be
used to introduce larger modifications and insertion of exogenous sequences. Moreover, similar to “traditional” gene insertion, if A. tumefaciens is used as a delivery
vector of CRISPR-Cas9, the resulting genome-edited plants contain foreign DNA
sequences, in addition to those that encode the programmable nucleases, in the host
genome. On the other hand, the use of episomal plasmids to deliver these nucleases
into plant cells has to take into account that transfected plasmids are rapidly
degraded in cells by endogenous nucleases and that the resulting DNA fragments
can be inserted at off-target sites in host cells (Kim et al. 2014).
The delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into plant
cells has been shown to remove the possibility to introduce recombinant DNA
into the host plant genome (Cho et al. 2013). Furthermore, it as has been shown
that these premixed RNPs cleave target DNA sites immediately after transfection
Engineering Plants for the Future: Farming with Value-Added Harvest
73