Preparation of CRISPR/Cas9 targeting reagents: Single-stranded oligo DNA (ssODN) templates, ALT-R XT CRISPR RNA (crRNA), ALT-R trans-activating crRNA (tracrRNA) and ALT-R Cas9 Nuclease V3 were acquired from Integrated DNA Technologies (IDT).

Prior to use, the crRNA and the tracRNA were resuspended in pH 7.5 TE buffer (IDT # 11-01-02-02) to a final concentration of 100 µM. To prepare crRNA:tracRNA duplexes at 50 µM, equal volumes of each RNA were mixed and heated for 5 minutes at 95 °C, followed by controlled cool-off to 25 °C at ramp rate 0.1 °C/second. The formed duplexes were placed on ice until ready to use.

Ribonucleoproteins were prepared by mixing 5 µl each of crRNA:tracRNA duplex (50 µM) and recombinant Cas9 enzyme (61 µM), followed by incubation at room temperature for 20 minutes. Next, 200 pmol of each HDR template was added to the RNPs prior to delivery into iPSCs.

Nucleofection and luminescence-based screening of CRISPR-targeted iPSCs: Targeting was performed using healthy, subconfluent iPSCs (P51) pretreated with 1X RevitaCell (in E8/F) for 3 hours.The cells were dissociated with StemPro Accutase (Thermo Fisher Scientific # A110501) and 1e6 cells taken forward for nucleofection. Following low-speed centrifugation (100G, 3 minutes), the cell pellet was resuspended in 100 µl nucleofection solution (P3 Nucleofection Kit, Lonza # V4XP-3024). Of this suspension, 85 µl were transferred to the assembled RNPs, resulting in c. 850,000 cells in the final reaction. The nucleofection was carried out with an Amaxa 4D nucleofector, using programme CA137. Immediatley following nucleofection, 500 µl E8/F with 1X RevitaCell were added to the cell suspension and transferred to a Falcon tube containing 4.5 ml E8/F with 1X RevitaCell.