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RSS FeedsIJMS, Vol. 23, Pages 5762: First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill (International Journal of Molecular Sciences)

 
 

20 may 2022 18:50:20

 
IJMS, Vol. 23, Pages 5762: First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill (International Journal of Molecular Sciences)
 


Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.


 
61 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
IJMS, Vol. 23, Pages 5760: Extracellular Vesicles Released from Stem Cells as a New Therapeutic Strategy for Primary and Secondary Glomerulonephritis (International Journal of Molecular Sciences)
IJMS, Vol. 23, Pages 5761: Biomolecules under Pressure: Phase Diagrams, Volume Changes, and High Pressure Spectroscopic Techniques (International Journal of Molecular Sciences)
 
 
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