Figure 3.

Cold shock enhances Prime editing rates in human cells. (A, C) RNP-mediated PE2 editing efficiency at the specified positions with varying pegRNA PBS length for (A) FANCF (+5 G to T) and (C) MECP2 (+4+5 TG to CC) loci in HEK293T cells. (B, D) RNP-mediated PE2 editing efficiency at the specified positions for (B) FANCF (+5 G to T) and (D) HEK4 (+5 G to T) loci in different cell lines (HEK293T, U2OS, RPE-1). 50 pmol PEmax protein and 100 pmol pegRNA were used for electroporation. Immediately after nucleofection, cells were incubated 3 days at 37°C or for cold shock, 12–16 h at 30°C followed by 2 days at 37°C. Editing efficiency reflects the frequency of sequencing reads that contain the intended precise edit among all amplicon deep sequencing reads. ‘Untreat’ indicates untreated cells. Values and error bars reflect mean ± s.d. of n = 3 independent biological replicates. Two-way ANOVA statistical analysis was used to determine the significance of prime editing at different temperature in different cell lines, ns indicates P > 0.05, ** indicates P ≤ 0.01, and **** indicates P ≤ 0.0001 (also see Supplementary table).