Engineered pegRNAs improve prime editing efficiency
Open accessAbstract
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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Cloning pegRNA sequences in the evolved PegRNA backbone pU6-tevopreq1-GG-acceptor includes PegRNA extensions that further stabilize the guide and result in higher editing efficiencies.
“We recommend cloning PegRNA sequences in evolved PegRNA backbone pU6-tevopreq1-GG-acceptor (Addgene #174038) as this will include PegRNA extensions that further stabilize the guide which results in higher editing efficiencies.48”
From Protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools (2024) · cites this paper as [48]
Nelson et al. reported that structured RNA motifs on pegRNAs protect from exonuclease degradation, increase stability, and enhance efficiency.
“While our paper was in review, Nelson et al.30 reported that the inclusion of structured RNA motifs on prime editing gRNAs (pegRNAs) led to protection from exonuclease degradation, increased stability, and enhanced efficiency.”
From Efficient combinatorial targeting of RNA transcripts in single cells with Cas13 RNA Perturb-seq (2023) · cites this paper as [30]
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Volodina, O. V., Fabrichnikova, A. R., Anuchina, A. A., Mishina, O. S., Lavrov, A. V., & Smirnikhina, S. A. (2025) · Current Gene Therapy · doi:10.2174/0115665232295117240405070809 · Cited by 1 · see citing works
- Authors
- Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A., Chen, P. J., Anzalone, A. V., An, M., Newby, G. A., Chen, J. C., Hsu, A., & Liu, D. R.
- Affiliations
- Broad Institute (US)
- Corresponding author
- Liu, David R.
- ORCID
- Venue
- Nature Biotechnology
- Published
- 2021-10-04
- Volume
- 40
- Issue
- 3
- Pages
- 402-410
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC8930418
- OpenAlex
- W3202931255
- Cited by
- 727
- References
- 65
- Citations by year
- 2026: 133 · 2025: 167 · 2024: 160 · 2023: 155 · 2022: 94 · 2021: 18
- FWCI
- 33.79
- Type
- journal article
- Language
- en
- Update notices
- correction (2021-12-08)
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (acceptedVersion)
- Funders
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases (U01Al142756)
- U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (RM1HG009490)
- U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (R01EB031172)
- U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (R35GM118062)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Keywords
- prime editing, RNA stability, primer binding site, off-target editing, point mutation correction, genome editing efficiency
- MeSH terms
- Gene Editing, RNA, Guide, CRISPR-Cas Systems, DNA, Humans, RNA-Directed DNA Polymerase, CRISPR-Cas Systems
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