Prime editing enables the installation of virtually any combination of point mutations, small insertions, or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we demonstrate that degradation of the 3′ region of the pegRNA that contains the reverse transcriptase template and the primer-binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3′ terminus of pegRNAs that enhance their stability and prevent degradation of the 3′ extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3 to 4-fold in HeLa, U2OS, and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3′ structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3′ motifs. Finally, we demonstrated that epegRNAs enhance the efficiency of the installation or correction disease-relevant mutations.

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