The obtained UCNPs@SiO2 (100 mg) was treated with 20 μl of TEDATS for 4.5 hours to acquire the carboxylic group first (UCNPs@SiO2-COOH). Then, the nanoparticles were transferred into 10 ml of dry tetrahydrofuran, including 100 μg of ONA for esterification catalyzed by DCC and 4-dimethylaminopyridine. After 12 hours, the nanoparticles were washed three times with tetrahydrofuran and then resolved in tetrahydrofuran with 200 μg of DCC to further incubate for activation of the carboxyl group. After 8 hours, the particles were collected and transferred into PBS. At the same time, Cas9 protein and sgRNA (1:2) were mixed in PBS for 5 min to form the CRISPR-Cas9 system. Afterward, the CRISPR-Cas9 system was mixed with the particles overnight at 4°C. After centrifugation, PEI was coated onto the UCNPs-Cas9 at a PEI:sgRNA weight ratio of 5:1 and needed further equilibration at room temperature for 5 min. The nanovehicles were further diluted to the concentration of Cas9 (0.36 μg/ml) (before centrifugation) in the in vitro experiment. To visualize the internalization process of UCNPs-Cas9@PEI by CLSM (LSM 710, Zeiss), the Cas9 was conjugated with Cy3-NHS.

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