Silenciamiento Genético mediante expresión de proteínas CRISPR–CAS9 en Bentamiana (Nicotiana benthamiana) como modelo de transformación genética.

CRISPR-CAS9 technology has rapidly become a popular tool for genome editing because of its
simplicity, efficiency, low cost, and multiplex editing capability. CAS protein and guide RNAs form
ribonucleoproteins (RNPs) that recognize the target site by complementary of the gRNA on the target
DNA sequence, causing specific DNA double strand breaks. These brakes activate different DNA
repair pathways. If there are DNA fragments homologous to the target site available, homologous
recombination can occur using the cellular homology directed repair mechanism (HR). In the absence
of homologous donor DNA, a non-homologous end joining (NHEJ) repair process occurs, resulting in
small insertion or deletion mutations known as indels.
Genome editing has unparalleled potential for crop breeding, although traditional genome editing
methods rely on the production of a genetically modified T0 generation, which convey problems with
social acceptance. The greatest challenge for gene editing in plants is that plant cells are protected by
robust cell walls that complicate transformation. Therefore, in the present proposal, the induction of
genome editing in Nicotiana benthamiana cells by supplying CAS9/rRNA ribonucleoproteins by
microinjection or biolistic using Crystallized Liposome Acceleration System (CLAS) coupled with
recovery in the absence of selectable markers is presented as an approach for genome editing in
tropical crops.
The partial digestion of cell walls will facilitate protein insertion without excessive damage to cell´s
integrity, leading to faster recovery and tissue regeneration. The incorporation of pre-assembled
ribonucleoprotein into cells by microinjection can result in efficient genome editing events. In addition,
as an alternative strategy, we propose to use crystallised liposomes in biolistic systems to deliver
CRISPR ribonucleoproteins. Crystallised liposomes are solid enough to be used as carriers in biolistic
approaches. The use of CRISPR ribonucleoproteins has proven to induce gene editing on in vivo
assays over target genes such as Phytoene Desaturase (PDs). Therefore, the use of pre-assembled
ribonucleoproteins to induce genome editing of reference gene NbPDs (the phytoene desaturase for
bentamiana) could be achieved by microinjection or biolistic using a Crystallized Liposome
Acceleration System.

Establecer un sistema de silenciamiento genético mediante
expresión de proteínas CRISPR–CAS9 en Bentamiana (Nicotiana benthamiana) como
modelo de transformación genética.

1. Sistemas de producción alternativos sostenibles