## 1 Introduction  
Genome editing technology has advanced rapidly following the introduction of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, which is composed of the Cas9 protein, which cleaves the target DNA and single guide RNA (sgRNA) for recognizing the target DNA sequence [1; 2; 3; 4; 5; 6; 7]. A recent study showed that a minimal protospacer adjacent motif (PAM) sequence (5\({}^{\prime}\)-NNG-3\({}^{\prime}\)), which is needed for binding Cas9-sgRNA ribonucleoproteins (RNPs) to the target sequence, was sufficient for the activation of Cas9 from _Streptococcusanis_, indicating the possibility of expanding the target range for genome editing in this system [8]. Although the CRISPR/Cas9 system is a powerful tool for genome engineering, the overexpression of Cas9, derived from a plasmid vector, may cause off-target effects such as the cleavage of non-target sites. In addition, the plasmid-based introduction of Cas9 requires an optimum promoter for gene expression. For _Arabidopsis thaliana_, a constitutively active promoter from an early embryonic stage can be used to obtain a mutant, because the egg cell-specific expression of Cas9 limits the chance of mutation [9]. This suggests that a promoter that is effective for editing a particular genome should be identified before being used in an organism. Recently, direct delivery of Cas9-sgRNA RNPs was performed using electroporation, or lipofection using lipid nanoparticles [10; 11; 12; 13]. These studies demonstrated that direct delivery of RNPs may reduce off-target effects compared with plasmid-based DNA delivery. Direct delivery of Cas9 via electroporation leads to Cas9 facilitated cleavage of the genome DNA at the target site almost immediately, and results in the mutation frequency reaching its plateau a day after electroporation [10]. While the direct-delivered Cas9 proteins completely disappear from cells via protein degradation, plasmid DNA continues to constitutively expresses Cas9 for several days, even after mutation frequency at the target site reaches its plateau. Such continuous expression of Cas9 and sgRNA in cells causes off-target mutations. Therefore, introducing only the required quantity of Cas9 protein is important. In addition, direct delivery of Cas9-sgRNA is more versatile, because the use of a specific promoter becomes unnecessary. However, electroporation has disadvantages, such as low cell viability and low throughput. In the case of lipid nanoparticle delivery, many RNPs are needed to obtain a high genome editing efficiency because the ratio of the endosomal escape of Cas9-sgRNA affects its activity in the cells. Therefore, it has become necessary to develop efficient direct Cas9-sgRNA delivery tools that ensure safe genome editing technology. The delivery of macromolecules into living cells via the insertion of nano-scale acicular materials, such as carbon nanofiber arrays, silicon nanowires, diamond nanoneedle arrays (NNAs), and porous silicon nanoneedle arrays, has been reported as a new delivery platform [14; 15; 16; 17]. Although these studies have demonstrated the usefulness of nanomaterials as a delivery method for nucleic acids, successful delivery of RNPs using acicular materials has not been reported. We have been developing cell manipulation techniques using 200 nm diameter needle materials, such as the AFM cantilever type nanoneedle (AFM-NN) and the silicon NNA [18; 19; 20; 21]. Our original NNA has several tens of thousands of nanoneedles in a 3 mm square area. Each nanoneedle has a near regular configuration of 200 nm in diameter and 25 \(\upmu\)m in length (Figure 1A). Cell doubling time did not change, even after 50 insertions of AFM-NN, and neural stem cells manipulated with an antibody-functionalized NNA were capable of differentiating into neurons [22; 23]. These results led us to assume that damage due to the insertion of 200 nm nanoneedles into a cell was negligible.