eGFP disruption assay. Cas9 (10 pmol) and gRNA (10 pmol) were mixed and incubated at RT for 5 min. U2OS.eGFP.PEST cells were transfected with the ribonucleoprotein (RNP) complex using the SE Cell Line 4D-Nucleofector kit (Lonza) following the pulse program of DN-100. After transfection, the cells were suspended in the culture media and transferred to a 96-well plate (20,000 cell per well). 

HiBiT sequence knockin by nucleofection. U2OS cells stably expressing eGFP. PEST, MDA-MB-231 cells, hiPSC, and HDFn were transfected with Cas9 RNP and
ssODN using the cell-specific 4D-Nucleofector kit (Lonza). For U2OS cells, the SE Cell Line kit and the program DN-100 were used. For MDA-MB-231 cells, the SE Cell Line kit and the program CH-125 were used. For hiPSC, the P3 Primary Cell kit and the program CA-137 were used. For HDFn, the P3 Primary Cell kit and the program EN-150 were used. For U2OS and MDA-MB-231 cells, 10 pmol of Cas9, gRNA, and ssODN were used. For hiPSC and HDFn, 20 pmol of Cas9, gRNA, and ssODN were used. For the Cas9:ssODN conjugates, the Cas9-adaptor was premixed with ssODN and incubated at RT for 15–30 min prior to RNP formation to ensure Cas9:ssODN conjugate formation. Then gRNA was added, and the final mixture was incubated for 5–10 min at RT. When Cas9 would not specifically bind ssODNs, the RNP was formed first because it is known that nonspecific Cas9-DNA interactions hamper the RNP formation. After incubating Cas9 and gRNA at RT for 5–10 min, ssODN was added to the mixture. Approximately 200,000 cells (U2OS and MDA-MB-231) or 500,000 cells (hiPSC and HDFn) were transfected with the above mixtures in a well of the nucleofection kit, and 20,000 transfected cells were seeded in each well of a 96-well plate in the case of U2OS and MDA-MB- 231 cells. For hiPSC and HDFn, all the cells were plated in a well of a 24-well plate. Cells were incubated for 24 h (U2OS and MDA-MB-231) or 48 h (hiPSC and HDFn) at 37 °C, and cell viability was measured using the PrestoBlue Cell Viability Reagent (Thermo) with SpectraMax M5 and SoftMax Pro 7.0 (Molecular Devices).

Conversion of eGFP to BFP. U2OS.eGFP.PEST cells were transfected with RNP complex and ssODNs using the SE Cell Line 4D-Nucleofector kit (Lonza) following the DN-100 program. First, 30 pmol of Cas9 (or Cas9-adaptor) and 33 pmol of gRNA were mixed and incubated at RT for 5–10 min, then 60 pmol of ssODN was added, and the final mixture was incubated at RT for 15–30 min. After transfection, cells were suspended in the culture medium and transferred to a 96-well plate (20,000 cell per well). 

HiBiT sequence knockin by nucleofection in INS-1E cells. INS-1E cells were transfected with Cas9 RNP and ssODN using the SF Cell Line 4D-Nucleofector kit (Lonza) following the pulse program of DE-130. For Cas9:ssODN conjugates, 20 pmol of Cas9-adaptor were premixed with 20 pmol of ssODN and incubated at RT for 15–30 min prior to RNP formation to ensure Cas9:ssODN conjugate formation. Then 20 pmol of gRNA was added, and the final mixture was incubated for 5–10 min at RT. In cases where Cas9 did not specifically bind ssODNs, the RNP was formed first because nonspecific Cas9-DNA interactions can hamper the RNP formation. After incubating Cas9 and gRNA at RT for 5–10 min, 20 pmol of ssODN were added to the mixture. Approximately 200,000 cells were transfected with the above mixtures in a well of the nucleofection kit, and cells were seeded in a well of a 24-well plate.