Targeted genome editing in potato protoplast via optical delivery of CRISPR/Cas9 ribonucleoproteins

摘要

Since the development of genome editing tools like CRISPR/Cas9, it is possible to modify the sequences of genes in a very specific manner. The molecular delivery into plant protoplasts to improve the quality of agricultural crops represents a major bottleneck in the routine application of CRISPR/Cas9 in modern plant breeding. To approach this need, we suppose using gold nanoparticle mediated (GNOME) laser transfection for delivery of CR1SPR/Cas9 ribonucleoproteins (RNP) into potato protoplasts with high-throughput. As a proof-of-concept, we aim to reduce the toxic steroidal glykoalkaloid α-solanine in potatoes. GNOME laser transfection utilizes a picosecond Nd:YAG laser operating at 532 nm to excite surface plasmon resonance of membrane-attached gold nanoparticles. The strong absorption of laser light results in a temperature increase, leading to vaporization of the surrounding medium and to the formation of cavitation bubbles, which causes a transient permeabilization of the cell membrane. The challenges modifying protoplasts, in contrast to mammalian cells, include their sensitivity to osmolality stress, the lack of adherence to culture surfaces, the absence of commercial antibodies for nanoparticle targeting, and the low adherence of the applied nanoparticles to the protoplast's membrane. Viability in respect to different conditions was evaluated using a resazurin assay and the delivery of molecules by FITC-dextrane. To facilitate the binding of the nanoparticles, a combination of a cell membrane binding lectin and a linker molecule was investigated. Furthermore, we demonstrate the prototype of a bench-top laser transfection device, which allows conducting the complete workflow within a biological laboratory environment.