and are degraded rapidly by endogenous proteases, thus reducing the frequency of
off-target effects in regenerated plants (Kim et al. 2014; Cho et al. 2013). Moreover,
when using protein-and-RNA-only systems, there is no need to optimize codon
usage or to find promoters that will express Cas9 and gRNAs. In 2015, the delivery
of RNPs into protoplasts of Arabidopsis thaliana, Nicotiana attenuata and Oryza
sativa and the induction of targeted genome modifications in regenerated plants were
reported. Purified Cas9 protein was mixed in molar excess with gRNAs targeting
genes from the three plant species in vitro to obtain preassembled RNPs. The RNPs
were incubated with protoplasts in the presence of polyethylene glycol. Indels were
detected at the expected positions, with various frequencies (Woo et al. 2015).
2.2 Transient Expression (Delivery of Genes to Somatic
Tissues)
The alternative plant-based technology to stable transformation of genomes is
transient expression. As mentioned, stable transformation is not suitable for the
rapid production of pharmaceuticals. This feature would be an obstacle to the use
of plant-based technologies particularly in specific fields like those related to the
production of biologics to combat emerging diseases.
The transient technological platforms imply the introduction of episomal vectors
into plant tissues. There are two main transient expression strategies. The first is
agro-infiltration, where leaves are infiltrated with A. tumefaciens (by injection or
by applying vacuum to transfect whole plants at a glance). The second implies
the infection of plant tissues directly with recombinant plant viruses that infect
cells, replicate and spread from cell to cell and systemically guide the expression
of recombinant protein in every cell. The majority of plant viral vectors used are
based on single-stranded RNA viruses, such as tobacco mosaic virus, potato virus X
and cowpea mosaic virus (CPMV) (Loh et al. 2017).
The use of deconstructed virus genomes delivered by A. tumefaciens is a methodology that is in between agro-infiltration and viral infection. The target gene
is cloned into a modified plant viral vector which can be integrated into the
Agrobacterium vector and delivered into the plant tissues by infiltrating with the
corresponding transformed Agrobacterium. This facilitates the transfer of T-DNA
to a very high number of cells. With the use of a deconstructed virus, the issue
of instability of viral genome due to the introduction of large target genes can be
resolved. Moreover, a certain degree of containment can be obtained because it
implies the transfection of many cells with the virus genome followed by its cell-tocell movement but no systemic spreading (Peyret and Lomonossoff 2015).
Icon Genetics, a German plant biotechnology company, has adapted this technology as MagnICON™ for the manufacturing of various plant-based vaccines,
including those based on the hepatitis B virus surface antigen (300 mg/kg
N. benthamiana fresh leaves) in the form of VLP (Huang et al. 2006), norovirus
74
S. Massa et al.
off-target effects in regenerated plants (Kim et al. 2014; Cho et al. 2013). Moreover,
when using protein-and-RNA-only systems, there is no need to optimize codon
usage or to find promoters that will express Cas9 and gRNAs. In 2015, the delivery
of RNPs into protoplasts of Arabidopsis thaliana, Nicotiana attenuata and Oryza
sativa and the induction of targeted genome modifications in regenerated plants were
reported. Purified Cas9 protein was mixed in molar excess with gRNAs targeting
genes from the three plant species in vitro to obtain preassembled RNPs. The RNPs
were incubated with protoplasts in the presence of polyethylene glycol. Indels were
detected at the expected positions, with various frequencies (Woo et al. 2015).
2.2 Transient Expression (Delivery of Genes to Somatic
Tissues)
The alternative plant-based technology to stable transformation of genomes is
transient expression. As mentioned, stable transformation is not suitable for the
rapid production of pharmaceuticals. This feature would be an obstacle to the use
of plant-based technologies particularly in specific fields like those related to the
production of biologics to combat emerging diseases.
The transient technological platforms imply the introduction of episomal vectors
into plant tissues. There are two main transient expression strategies. The first is
agro-infiltration, where leaves are infiltrated with A. tumefaciens (by injection or
by applying vacuum to transfect whole plants at a glance). The second implies
the infection of plant tissues directly with recombinant plant viruses that infect
cells, replicate and spread from cell to cell and systemically guide the expression
of recombinant protein in every cell. The majority of plant viral vectors used are
based on single-stranded RNA viruses, such as tobacco mosaic virus, potato virus X
and cowpea mosaic virus (CPMV) (Loh et al. 2017).
The use of deconstructed virus genomes delivered by A. tumefaciens is a methodology that is in between agro-infiltration and viral infection. The target gene
is cloned into a modified plant viral vector which can be integrated into the
Agrobacterium vector and delivered into the plant tissues by infiltrating with the
corresponding transformed Agrobacterium. This facilitates the transfer of T-DNA
to a very high number of cells. With the use of a deconstructed virus, the issue
of instability of viral genome due to the introduction of large target genes can be
resolved. Moreover, a certain degree of containment can be obtained because it
implies the transfection of many cells with the virus genome followed by its cell-tocell movement but no systemic spreading (Peyret and Lomonossoff 2015).
Icon Genetics, a German plant biotechnology company, has adapted this technology as MagnICON™ for the manufacturing of various plant-based vaccines,
including those based on the hepatitis B virus surface antigen (300 mg/kg
N. benthamiana fresh leaves) in the form of VLP (Huang et al. 2006), norovirus
74
S. Massa et al.