The Japanese Biochemical Society

[1BS1-1] Highly efficient genome editing by direct delivery of CRISPR/Cas9 into the nucleus using the cutting-edge intracellular delivery technology SU10

Ryo Kuniba (Kamakura Research Group, Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science)

Genome editing, Intracellular/intranuclear delivery, Single cell operation

When editing genome by using CRISPR/Cas9, the off-target effect and immune response can be reduced by directly delivering Cas9 protein and sgRNA complex (Ribonucleoprotein: RNP) into the cell. On the other hand, the improvement of intracellular transfection efficiency of Cas9 RNP is a current challenge, and there is a need to develop a highly efficient and reliable intracellular delivery method that is independent of the cell type. In this study, we evaluated the genome editing efficiency of direct Cas9 RNP delivery into the nucleus using Single CellomeTM Unit SU10 (Yokogawa Electric Corporation), a novel intracellular delivery technology. The SU10 is a new platform that can deliver the target substance directly into the cytoplasm or nucleus at the single-cell level. Cells are selected under microscopy, and a solution filled in a nanopipette, which isa glass capillary with minimum tip outer diameter of several tens of nanometers, is automatically delivered into the cell by electrochemical mechanisms. When Cas9 RNP for targeting the GFP gene was delivered into the cell lines with stable GFP expression, a genome editing knockout efficiency of more than 70% was observed. Conversion of the GFP gene to the BFP gene by homologous-directed repair (HDR) was also successfully performed. These results suggest that SU10 is an efficient intracellular deliver technology for Cas9 RNP.