First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill

Abstract

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.

Keywords: CRISPR/Cas9; European chestnut; phytoene desaturase; protoplast; transgene-free.

Figure 1

Extracted protoplasts. Magnification 40×. Scale bar = 100 µm.

Figure 2

Protoplasts transfected with the pAVA393 plasmid containing the GFP expression cassette. The GFP signal was detected by fluorescence microscopy 72 h after the transfection event, under blue light (a,d,e), white light (b) and fusion of the two images (c). Scale bar = 100 µm.

Figure 3

General overview of protoplast development into embryogenic callus in European chestnut observed on C2 medium. (a) First cellular divisions after 10 days; (b) microcolonies after 30 days; (c) embryogenic callus after 3 months of culture; (d) embryogenic callus after 4 months. Observations were obtained using the stereomicroscope Leica-Wild Heerbrugg M8. Scale bar = 1 mm.

Figure 4

Chestnut protoplast isolation protocol starting from embryogenic callus derived from somatic embryos. Scale bar = 100 µm.

Figure 5

PEG-mediated transfection protocol. (a) Protoplast transfection using GFP marker gene and subsequent visualization using the Nikon Eclipse Ti2 fluorescent microscope. (b) Protoplast transfection using RNP complex by targeting the pds gene, followed by DNA extraction and Sanger sequencing. Scale bar = 100 µm.