For standard conditions, CD34+ HSPCs were thawed and cultured for 24 h in the presence of cytokines before nucleofection. CD34+ HSPCs were electroporated using the P3 Primary Cell 4D-Nucleofector X Kit S(Lonza, catalog no. V4XP-3032) according to the manufacturer’s recommendations. Unless otherwise indicated, 2.5 × 105 cells were electroporated with 2,000 ng prime editor mRNA, 200 pmol (e)pegRNA and 100 pmol nicking sgRNA using pulse code DS-130. For TwinPE conditions, 2.5 × 105 cells were electroporated with 2,000 ng PEmax mRNA and 150 pmol of each (e)pegRNA using pulse code DS-130. For Vpx mRNA co-delivery, Vpx:PEmax mRNAs were electroporated at an equimolar ratio.

K562 cells (2 × 105 cells) were electroporated with 750 ng PEmax vector, 250 ng epegRNA vector and 100 ng nicking sgRNA vector using the SF Cell Line 4D-Nucleofector X Kit S (Lonza, catalog no. V4XC-2032) and pulse code FF-120. Jurkat cells (5 × 105 cells) were electroporated with 500 ng PEmax vector, 250 ng epegRNA vector and 100 ng nicking sgRNA vector using the SE Cell Line 4D-Nucleofector X Kit S (Lonza, catalog no. V4XC-1032) and pulse code CL-120. K562 and Jurkat cells
were cultured for 72 h before genotyping.