Extended Data Fig. 10: Twin prime editing mediated insertion in CCR5 region 2 in HEK293T cells, twin prime editing in multiple human cell lines, and editing activity of Cas9 nickase and PE2-dead RT variants.

(a) TwinPE-mediated endogenous sequence replacement with Bxb1 attB attachment site in CCR5 region 2 in HEK293T cells. (b) TwinPE-mediated endogenous sequence replacement with attP, attB, or 22-nt DNA sequences in multiple human cell lines. Six different pegRNA pairs targeting five loci were tested in HEK293T, HeLa, U2OS and K562 cells. HEK293T and HeLa cell were transfected with PE2 and pegRNA plasmids via Lipofectamine 2000 (Thermo Fisher) and TransIT-HeLaMonster (Mirus), respectively. U2OS and K562 cells were nucleofected using Lonza 4D-Nucleofector and SE kit. DNA loci and the specified insertion edits are shown in the x-axis. (c) HEK293T cells were transfected with twinPE pegRNA pairs and either Cas9–H840A nickase (nCas9), PE2-dRT (a PE2 variant that contains K103L and R110S inactivating mutations to the RT domain), or PE2. Treatment with either nCas9 or PE2-dRT did not result in desired edits, while PE2 installed the specified edits as indicated. Values and error bars in (a) and (c) reflect the mean and s.d. of three independent biological replicates. Values and error bars in (b) reflect the mean and s.d. of at least two independent biological replicates except editing in IDS2 in HeLa cells, editing in U2OS cells, and editing in MYC in K562 cells, which represent two independent biological replicates.