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Engineered pegRNAs improve prime editing efficiency
Author(s) -
Jelani 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,
David R. Liu
Publication year - 2021
Publication title -
nature biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 15.358
H-Index - 445
eISSN - 1546-1696
pISSN - 1087-0156
DOI - 10.1038/s41587-021-01039-7
Subject(s) - prime (order theory) , computational biology , rna , computer science , point mutation , dna , genome editing , primer (cosmetics) , biology , rna editing , genetics , chemistry , mutation , crispr , gene , mathematics , combinatorics , organic chemistry
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.

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