Engineered pegRNAs improve prime editing efficiency

By James W. Nelson, Peyton B. Randolph, Simon P. Shen, Kelcee A. Everette, Peter J. Chen, Andrew V. Anzalone, Meirui An, Gregory A. Newby, Jonathan C. Chen, Alvin Hsu and 1 more. Nature Biotechnology, 2021

599 citations recorded by the source corpus.

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 show 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 3–4-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 showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations. Stabilizing pegRNAs with 3′ RNA structures increases prime editing efficiency.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/c78a773c404b369bbfa032690619b763a7c15d3a

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In Molecular Biology

Citations in the corpus

CRISPR technology: A decade of genome editing is only the beginning (2023, background): “Refining prime editing tools will require engineering and optimizing the different constituent components, including the pegRNAs (145).”
Enhanced prime editing systems by manipulating cellular determinants of editing outcomes (2021, methodology): “In vitro transcription of prime editor and MLH1dn mRNA used in iPSC and T cell experiments As described previously (Nelson et al., 2021), plasmids were cloned to encode an inactivated T7 promoter followed by a 50 untranslated region (UTR), Kozak sequence, coding sequences of PE2 or MLH1dn, and a 30 ”