## 2 Materials and Methods
### Evaluation of the Cleavage Activity of RNP  
The RNP complexes were constructed by mixing 1 \(\upmu\) of Cas9 (0.1, 1, 10 pmol), 1 \(\upmu\)L of sgRNA (sgRNA_GFPg1-7U, 10 pmol), 1 \(\upmu\)L of 10 \(\times\) Cas9 buffer (New England Biolabs Inc., Ipswich, MA, USA), and 7 \(\upmu\)L of UltraPure(tm) DNase/RNase-Free Distilled Water (Thermo Fisher Scientific), and incubating at room temperature (RT) for 10 min. We adsorbed Cas9-sgRNA RNPs on the surface of NNA containing tens of thousands of nanoneedles as follows. First, the NNA, in which needles were arrayed squarely at 10 \(\upmu\)m intervals in a 3 mm square area, was cleaned with an O\({}_{2}\) plasma asher, JPA300 (J-SCIENCE Lab Co. Ltd., Kyoto, Japan), at 300 W for 10 min. Then, the NNA was immersed in 1% hydrogen fluoride (HF) solution for 1 min. The NNA was rinsed with ultrapure water, and the RNP solution was poured on it, followed by incubation at 4 \({}^{\circ}\)C for 30 min. Finally, the unadsorbed fraction of the RNP solution left on the NNA was collected, and the NNA was rinsed with PBS and fitted to the array manipulator (detailed information is provided in [19]). Next, the NNA was soaked in 10 \(\upmu\)L of cytosolic simulation buffer (25 mM HEPES, 115 mM CH\({}_{3}\)COOK, 5 mM MgCl\({}_{2}\), 20 v/v% polyethylene glycol 8000) and oscillated to release the RNPs from the NNA surface using fluidic shear force (condition described in the Section 2.6). Following oscillation, we collected the solution as a released fraction. Substrate plasmid DNA containing _EGFP_ (pEGFP-C1, GenBank accession number  
\begin{table}
\begin{tabular}{c c c} \hline \hline  & **Name** & **Sequence 5\({}^{\prime}\)-3\({}^{\prime}\)** \\ \hline \multirow{4}{*}{Oligonucleotide} & sgRNA-(7U) & AAAAAAAGCACGCGCGTGCACCTTTTTCAAGTTGA \\  & TAACGACTAGCCTATTTAACTIGCTATTTCAGTCTAAAAC \\ \cline{2-3}  & T7 sgRNA-GFP-g1 (R332) & TAATACGACTACTATGCCTGAACTTCACC \\  & T7 sgRNA-mNes (F461) & TCGCGGTTTTAGACTAGAATAGCCAAG \\ \cline{2-3}  & T7 sgRNA-mNes (F461) & TAATACGACTACTATAGGCCGCCGACCCT \\  & & GAACCCCCCTTTTTAGAGTAGAATAGCAAG \\ \cline{2-3}  & sgRNA\_GFPg1-7U & GGCCUCGAACUUACCUCCGUUUUAGACCUAGA \\  & sgRNA\_GFPg1-7U & AAUAGCAAGUUAAAAAAGGCUAGUCCGUUUUUUCAA \\  & & CUUGAAAAAAGUGGCACCAGGUCCGUCUCUUUUUUUU \\ \cline{2-3}  & sgRNA\_mNes (F461) & GGCCGCCGACCUGAAACCCGGUUUUUUAGA \\  & & GCUAGAAAUGCCAAGUUAAAAAAGCUCUGACUCCGUUUUUUUU \\  & AACUUGAAAAAAAGUGCACCCAGQUCCGUCUUUUUUUUUU \\ \hline \hline \end{tabular}
\end{table}
Table 1: Sequence of oligo DNA and sgRNA.  
U55763) was used to evaluate the cleavage activity of RNP. Then 50 ng of plasmid DNA was added to the unadsorbed fraction and the released fraction, followed by incubation at 37 \({}^{\circ}\)C for 60 min. The cleaved plasmid DNAs were electrophoresed using a 1% agarose gel in Tris-acetate EDTA buffer and stained with GelGreen Nucleic Acid Gel Stain (Biotium, Fremont, CA, USA). The brightness of the obtained band was measured using Image-Pro Plus (Media Cybernetics). The efficiency was calculated using the following formula:  
\[\begin{array}{l}\text{Cutting efficiency (\%)}=100\times\{1-(\text{brightness of uncut band}/\\ \text{total brightness of cut},\text{ nick and uncut band})\}\end{array} \tag{1}\]