Neon transfection system r resuspension buffer

2.5 µM of NSUN2 exon 2 crRNA was combined with equimolar amounts of NSUN2 exon 19 crRNA and annealed with 5 µM Alt-R CRISPR-Cas9 trans-activating CRISPR (tracr)RNA, ATTO 550 (IDT) in 10 µl of 1 × IDT Duplex Buffer. The ribonuclear protein (RNP) assembly reaction was then performed by combining 0.575 µM of the annealed crRNA:tracrRNA with 30.5 pmol of IDT Alt-R S.p. Cas9 Nuclease V3 in 2.2 µl Neon Transfection System R resuspension buffer (Invitrogen) for 5 min at 37 °C; the resultant mixture was kept at room temperature until transfection.

Electroporation was conducted using Neon Transfection System (Invitrogen) and following the manufacturer’s protocol, with the following modifications. HeLa cells were resuspended in Neon Transfection System R resuspension buffer (Invitrogen) to a concentration of 2.8 × 10⁷ per ml. For each electroporation reaction, 2 × 10⁵ cells prepared as above were incubated with 1 × v/v RNP at 37 °C for 5 min, before being electroporated at 1,005 volts, 35 milliseconds, with 2 pulses. Two reactions were seeded per well of a 24-well plate. Cells were recovered in complete medium under standard incubation conditions of 37 °C and 5% v/v CO₂ for 24 to 36 h.