Nucleofection strip

Each nucleofection reaction was prepared by adding 16 µl of cold SG Buffer to 4 µl of the SpCas9 RNP that was assembled as described above. For reactions that used two different guide RNAs, each gRNA was assembled with SpCas9 separately and 4 µl of each RNP solution were combined at this step. 2 µl of the repair oligonucleotide template was added to the SpCas9 RNP diluted in SG buffer. Finally, 2 µl of primed cells were added to the solution with Cas9 RNP and the repair template. The nucleofection reaction was placed in one well of a 16-well nucleofection strip (Lonza; Cat. No. V4XC-2032). The nucleofection strip was placed in the X-unit (Lonza; Cat. No. AAF-1002F) connected to a Nucleofector 4D core unit (Lonza; Cat. No. AAF-1002B), and the EO100 pulse was applied to each well.

Each transfection reaction was prepared by adding 2 µl of “primed” cells resuspended in SG buffer (Lonza) to a mixture of: 16 µl of SG buffer, 2 µl of 20 µg/µl pUC19, 1 µl of 250 mM ATP (pH 7.5), 1 µl of 100 mg/ml sodium heparin, and ≤7 µl of reporter DNA (volume is dependent on the number of constructs transfected). Each transfection reaction was transferred to one well in 16-well nucleofection strip (Lonza; Cat. No. V4XC-2032). The nucleofection strip was placed in the X-unit (Lonza; Cat. No. AAF-1002F) connected to a Nucleofector 4D core unit (Lonza; Cat. No. AAF-1002B), and the EO100 pulse was applied to each well.