TY - GEN

T1 - Targeted genome editing in potato protoplast via optical delivery of CRISPR/Cas9 ribonucleoproteins

AU - Londenberg, Anke

AU - Bartels, Frederik Matti

AU - Kqakpo Quaye, Joseph

AU - Boch, Jens

AU - Ripken, Tammo

AU - Heinemann, Dag

N1 - Funding information: This work is funded by the Federal Ministry of Education and Research, Germany, Grant no. FKZ 031B0542. We thank Dr. Annette Barchanski for kind guidance with the development of a conjugate, Prof. Andrea Hoffmann for technical support with the centrifugation experiments, and Björn Wünschmann for his major contribution in realizing the GNOME prototype.

PY - 2020/4/1

Y1 - 2020/4/1

N2 - 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 CRISPR/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.

AB - 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 CRISPR/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.

KW - A-solanine

KW - CRISPR/Cas9 ribonucleoproteins (RNP)

KW - Gold nanoparticle mediated (GNOME) laser transfection

KW - Nd:YAG laser

KW - Optoporation

KW - Plant protoplasts

KW - Plasmon resonance

UR - http://www.scopus.com/inward/record.url?scp=85094149324&partnerID=8YFLogxK

U2 - 10.1117/12.2555288

DO - 10.1117/12.2555288

M3 - Conference contribution

AN - SCOPUS:85094149324

T3 - Proceedings of SPIE - The International Society for Optical Engineering

BT - Nanophotonics VIII

A2 - Andrews, David L.

A2 - Bain, Angus J.

A2 - Kauranen, Martti

A2 - Nunzi, Jean-Michel

PB - SPIE

T2 - Nanophotonics VIII 2020

Y2 - 6 April 2020 through 10 April 2020

ER -