Figure 1.

The RT RNase H domain in PE3 is dispensable for prime editing

(A) Top: crystal structure of M-MLV RT (PDB: 5dmq.1). Bottom: schematic representations of RT variants with C-terminal truncations. (B) Top: schematic of an mCherry reporter containing a premature stop codon in HEK293T cells. PEs can generate a TAG-to-CAA transition to correct the premature stop codon and trigger mCherry expression. Bottom: editing efficiencies of RT variants (RT362, RT418, RT474, and RT497) and full-length PE3 were measured by flow cytometry. Results were obtained from six independent experiments, shown as mean ± SD. (C) TLR-MCV1 HEK293T cells containing a GFP with a 39-bp insertion, P2A, and out-of-frame mCherry. A precise insertion of 18 bp to replace the 39-bp insertion leads to GFP expression, while indels cause a frameshift that leads to mCherry expression. The frequencies of precise insertion (GFP+) or indel (mCherry+) were measured by flow cytometry. Results were obtained from five independent experiments, shown as mean ± SD. (D) TLR HEK293T cells containing GFP with a 47-bp insertion. Deletion of the 47-bp insertion leads to GFP expression, while indels cause a frameshift that leads to mCherry expression. Frequencies of precise deletion (GFP+) or indel (mCherry+) were measured by flow cytometry. Results were obtained from four independent experiments, shown as mean ± SD. NS, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.