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Peer-reviewedCC BYNature Biotechnology

Directed evolution of small RNA-stabilizing motifs that improve prime-editing efficiency

  • Holt A. Sakai1,2,3
  • Sarah E. Pierce1,2,3
  • Allen Yujie Jiang1,2,3
  • Ana Cristian1,2,3
  • Meirui An1,2,3
  • Chae Rin Kim4,1
  • Nouraiz Ahmed1,2,3
  • Colin F. Hemez1,2,3
  • Y. Allen Tao1,2,3
  • Emily Zhang1,2,3
  • Sophia J. Wu1,2,3
  • David R. Liu5,6,7
7 affiliations
  1. 1Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA
  2. 2Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA
  3. 3Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of MIT and Harvard, Cambridge, MA, USA
  4. 4Broad Institute of MIT and Harvard, Cambridge, MA, USA
  5. 5Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. [email protected]
  6. 6Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. [email protected]
  7. 7Merkin Institute of Transformative Technologies in Healthcare, The Broad Institute of MIT and Harvard, Cambridge, MA, USA. [email protected]
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Abstract

The performance of prime-editing (PE) systems has been improved by systematic engineering of their protein and small RNA components but the structured RNA motifs appended to the 3′ end of PE guide RNAs (pegRNAs)—a key determinant of pegRNA stability and editing efficiency—have not been extensively studied. We introduce PE-PRISM, a high-throughput pooled screen to identify and optimize these 3′ RNA motifs in human cells. Here, using PE-PRISM, we evaluated 2,858 RNA motifs across four iterative libraries, including natural and engineered pseudoknots, G-quadruplexes and reverse transcriptase recruitment elements. We applied structure-guided mutagenesis and combinatorial variant screening to refine hits, culminating in the engineered and evolved pseudoknot variants tevo2.0, eHAV and eSBRMV1-A. In a screen correcting 847 pathogenic ClinVar variants, the top-performing motifs improved PE efficiency over the widely used tevopreQ 1 motif for >90% of edits. They also increased PE efficiencies for correcting disease-associated mutations in primary human cells and in vivo in mouse brain and liver.

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Cite

BibTeXRIS
BibTeX
@article{sakai2026directed,
  title = {{Directed evolution of small RNA-stabilizing motifs that improve prime-editing efficiency}},
  author = {Holt A. Sakai and Sarah E. Pierce and Allen Yujie Jiang and Ana Cristian and Meirui An and Chae Rin Kim and Nouraiz Ahmed and Colin F. Hemez and Y. Allen Tao and Emily Zhang and Sophia J. Wu and David R. Liu},
  journal = {Nature Biotechnology},
  year = {2026},
  month = may,
  doi = {10.1038/s41587-026-03123-2},
  url = {https://doi.org/10.1038/s41587-026-03123-2},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Directed evolution of small RNA-stabilizing motifs that improve prime-editing efficiency
AU  - Holt A. Sakai
AU  - Sarah E. Pierce
AU  - Allen Yujie Jiang
AU  - Ana Cristian
AU  - Meirui An
AU  - Chae Rin Kim
AU  - Nouraiz Ahmed
AU  - Colin F. Hemez
AU  - Y. Allen Tao
AU  - Emily Zhang
AU  - Sophia J. Wu
AU  - David R. Liu
PY  - 2026
DA  - 2026/05/20
JO  - Nature Biotechnology
DO  - 10.1038/s41587-026-03123-2
UR  - https://doi.org/10.1038/s41587-026-03123-2
AB  - The performance of prime-editing (PE) systems has been improved by systematic engineering of their protein and small RNA components but the structured RNA motifs appended to the 3′ end of PE guide RNAs (pegRNAs)—a key determinant of pegRNA stability and editing efficiency—have not been extensively studied. We introduce PE-PRISM, a high-throughput pooled screen to identify and optimize these 3′ RNA motifs in human cells. Here, using PE-PRISM, we evaluated 2,858 RNA motifs across four iterative libraries, including natural and engineered pseudoknots, G-quadruplexes and reverse transcriptase recruitment elements. We applied structure-guided mutagenesis and combinatorial variant screening to refine hits, culminating in the engineered and evolved pseudoknot variants tevo2.0, eHAV and eSBRMV1-A. In a screen correcting 847 pathogenic ClinVar variants, the top-performing motifs improved PE efficiency over the widely used tevopreQ 1 motif for >90% of edits. They also increased PE efficiencies for correcting disease-associated mutations in primary human cells and in vivo in mouse brain and liver.
SN  - 0733-222X
SN  - 1087-0156
SN  - 1546-1696
SN  - 2331-3684
ER  -