## 3 Results and Discussion
### Nestin Knockout by Direct RNP Delivery  
To further demonstrate the usefulness of our method of delivering Cas9-sgRNA via an NNA, we performed a gene knockout targeting the type VI intermediate filament nestin in a chromosome of FP10 SC2 mouse breast cancer cells [24]. An RNP complex solution, consisting of Cas9-mEmGFP and sgRNA, targeting the rod domain of nestin, was poured on the NNA and allowed to dry completely to avoid loss of RNP (Figure 3C). Nestin deficient cells were counted according to their fluorescence intensity after immunostaining (Figure 3D). A previously established nestin knockout cell line 5 NK-H10 (H10) was used as the negative control [26] and an average fluorescence intensity of H10 cells plus five SD was set as a threshold to determine nestin negative cells. Neither H10 cells with a fluorescence higher than the threshold nor wild type cells with a fluorescence lower than the threshold were detected (Figure 3E). NNA-treated cells with a fluorescence intensity under the threshold value were considered  
Figure 3: Gene disruption in mammalian cells by direct delivery of the RNP complex. (**A**) Schematic of the EGFP disruption. (**B**) EGFP disruption in reporter cells (\(n=3\)). Left panel shows bright field and fluorescence images of the portion of cells that were cultured for 48 h after the insertion of bare NNA (mock) or RNP delivery using NNA. Scale bar: 200 μm. Right panel shows the ratio of EGFP-depressed cells. RNP was introduced by lipofection (red) or NNA (yellow), respectively and the mock-treated cells was showed in blue. (**C**) Schematic representation of the mouse nestin gene and the sgRNA targeting sequence. (**D**) Immuno-stained FP10 SC2 cells after delivery of RNP with the NNA (right) and negative control (left). Green indicates nestin, and blue indicates the nucleus stained with DAPI. Scale bar: 20 μm. (**E**) Fluorescence intensity threshold for estimating nestin deficient cells. Parental cells (SC2; green; positive control), nestin knockout cell line 5 NK-H10 (H10; red; negative control), and RNP introduced cells by NNA (blue). Red line indicates the average fluorescence intensity of H10 plus five SD. The demonstrated plot and threshold were based on an experiment replicated 7 times. The target sequence of sgRNA and protospacer adjacent motif (PAM) are depicted in pink and light blue. Black arrowheads indicate cleavage sites. Red arrowheads indicate target-protein-deficient cells. as nestin deficient cells. The results indicated that the nestin disruption efficiency of this method was 15.4 \(\pm\) 8.2% (\(n\) = 7), while transfection of a plasmid vector expressing Cas9 and similar amounts of sgRNA resulted in a disruption efficiency of 20.3 \(\pm\) 4.7% (\(n\) = 3) [26]. Moreover, we also detected nestin gene disruption in genomic DNA via a conventional T/E1 assay (data not shown). We selected 5 loci similar to the nestin target sequence with higher than 75% homologies and investigated off-target gene disruption, and determined that neither insertion nor deletion occurred at all in the candidate sites of even knockout cells generated by the plasmid-based method. This indicated that the sgRNA sequence used in this study recognized target sequence by high specificity. In further studies, off-target effect must be studied. When all RNPs were retained on the NNA surface via drying, the gene disruption efficiency of this method reached levels comparable to that of plasmid-based genome editing. Although the efficiency of RNP introduction needs to be improved, we were able to successfully demonstrate genome editing by the direct delivery of Cas9-sgRNA using NNA. The lower efficiency of this method compared to introduction via lipofection is presumed to be due to the loss of RNPs on the NNA surface and the insertion efficiency of nanoneedles into cells.