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. 2022 Jul 15;14(4):304–308. doi: 10.1093/procel/pwac014

Figure 2.

Figure 2.

Split pegRNA prime editing maintains PE activity by tethering prime RNA to Cas9. (A) Schematics of split pegRNA prime editors. The split pegRNA prime editor (SnPE) consists of a Cas9 nickase-H840A fused with C-terminal MMLV and N-terminal RNA binding proteins (RBPs), a sgRNA, and a separated prime RNA (pRNA). pRNA consists of a prime binding site (PBS), a reverse transcription template (RTT) and a RNA stem-loop aptamer such as MS2 or PP7. SnPE-5ʹ or 3ʹ-MS2 or PP7 consists of RBP-Cas9 nickase-MMLV, a pRNA bearing MS2 or PP7 and a sgRNA. RBP at the N-terminal Cas9 binds the RNA aptamer MS2 or PP7. SnPE-c-MS2 or PP7 includes the circular pRNA (cpRNA) bearing MS2 or PP7 generated by the tornado circular RNA expression system. Efficiency of SnPE-5ʹ-MS2 or PP7, SnPE-3ʹ-MS2 or PP7, SnPE-c-MS2, or PP7 were tested at RUNX1_+5 G·C to T·A using PE3 in U2OS cells (B), HEK293FT cells (C), and HeLa cells (D). (E) Efficiency of PE, SnPE, SnPE-5ʹ-Com, SnPE-5ʹ-BoxB, and SnPE-5ʹ-Csy4 at RUNX1_+5 G·C to T·A using PE3 in HEK293FT cells. (F) Efficiency of SnPE-5ʹ-MS2 or PP7, SnPE-5ʹ-MS2 or PP7, SnPE-5ʹ-Csy4 and SnPE-5ʹ-BoxB were tested at RUNX1_+1 ATG insertion using PE3 HEK293FT cells. (G) Efficiency of SnPE-5ʹ-MS2 or PP7, SnPE-5ʹ-MS2 or PP7, SnPE-5ʹ-Csy4, and SnPE-5ʹ-BoxB were tested at RUNX1_+1 CGA deletion using PE3 HEK293FT cells. Data and error bars in (B–G) indicate the mean and standard deviation of three independent biological replicates.