Abstract
Genome editing with prime editors based on CRISPR-Cas9 is limited by the large size of the system and the requirement for a G/C-rich protospacer-adjacent motif (PAM) sequence. Here, we use the smaller Cas12a protein to develop four circular RNA-mediated prime editor (CPE) systems: nickase-dependent CPE (niCPE), nuclease-dependent CPE (nuCPE), split nickase-dependent CPE (sniCPE) and split nuclease-dependent CPE (snuCPE). CPE systems preferentially recognize T-rich genomic regions and possess a potential multiplexing capacity in comparison to corresponding Cas9-based systems. The efficiencies of the nuclease-based systems are up to 10.42%, whereas niCPE and sniCPE reach editing frequencies of up to 24.89% and 40.75% without positive selection in human cells, respectively. A derivative system, called one-sniCPE, combines all three RNA editing components under a single promoter. By arraying CRISPR RNAs for different targets in one circular RNA, we also demonstrate low-efficiency editing of up to four genes simultaneously with the nickase prime editors niCPE and sniCPE.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
All data supporting the findings of the present study are available in the article, extended data and supplementary figures and tables, or are available from the corresponding author on request. The deep sequencing data have been deposited in an NCBI BioProject database (accession no. PRJNA1048095)42.
Change history
08 February 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41587-024-02160-z
References
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2023).
Eid, A. & Qi, Y. Prime editor integrase systems boost targeted DNA insertion and beyond. Trends Biotechnol. 40, 907–909 (2022).
Gao, C. Genome engineering for crop improvement and future agriculture. Cell 184, 1621–1635 (2021).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR-Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).
Liu, B. et al. A split prime editor with untethered reverse transcriptase and circular RNA template. Nat. Biotechnol. 40, 1388–1393 (2022).
Feng, Y. et al. Enhancing prime editing efficiency and flexibility with tethered and split pegRNAs. Protein Cell 14, 304–308 (2023).
Litke, J. L. & Jaffrey, S. R. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat. Biotechnol. 37, 667–675 (2019).
Liu, C. X. & Chen, L. L. Circular RNAs: characterization, cellular roles, and applications. Cell 185, 2016–2034 (2022).
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021).
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Lin, Q. et al. High-efficiency prime editing with optimized, paired pegRNAs in plants. Nat. Biotechnol. 39, 923–927 (2021).
Zong, Y. et al. An engineered prime editor with enhanced editing efficiency in plants. Nat. Biotechnol. 40, 1394–1402 (2022).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Hirano, S. et al. Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA. Nature 610, 575–581 (2022).
Schuler, G., Hu, C. & Ke, A. Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9. Science 376, 1476–1481 (2022).
Karvelis, T. et al. Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease. Nature 599, 692–696 (2021).
Yan, W. X. et al. Functionally diverse type V CRISPR-Cas systems. Science 363, 88–91 (2019).
Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).
Zetsche, B. et al. Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat. Biotechnol. 35, 31–34 (2017).
Yamano, T. et al. Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165, 949–962 (2016).
Merker, L., Schindele, P., Huang, T. K., Wolter, F. & Puchta, H. Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a. Plant Biotechnol. J. 18, 2382–2384 (2020).
Guo, L. Y. et al. Multiplexed genome regulation in vivo with hyper-efficient Cas12a. Nat. Cell Biol. 24, 590–600 (2022).
Fonfara, I., Richter, H., Bratovič, M., Le Rhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature 532, 517–521 (2016).
Grünewald, J. et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat. Biotechnol. 41, 337–343 (2023).
Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).
Gao, Z. et al. A truncated reverse transcriptase enhances prime editing by split AAV vectors. Mol. Ther. 30, 2942–2951 (2022).
Zheng, C. et al. A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver. Mol. Ther. 30, 1343–1351 (2022).
Davis, J. R. et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01758-z (2023).
Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
Li, X. et al. Development of a versatile nuclease prime editor with upgraded precision. Nat. Commun. 14, 305 (2023).
Peterka, M. et al. Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing. Nat. Commun. 13, 1240 (2022).
Kim, Y. B. et al. A novel mechanistic framework for precise sequence replacement using reverse transcriptase and diverse CRISPR-Cas systems. Preprint at bioRxiv https://doi.org/10.1101/2022.12.13.520319 (2022).
Campa, C. C., Weisbach, N. R., Santinha, A. J., Incarnato, D. & Platt, R. J. Multiplexed genome engineering by Cas12a and CRISPR arrays encoded on single transcripts. Nat. Methods 16, 887–893 (2019).
Breinig, M. et al. Multiplexed orthogonal genome editing and transcriptional activation by Cas12a. Nat. Methods 16, 51–54 (2019).
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
O’Sullivan, J. W. et al. Polygenic risk scores for cardiovascular disease: a scientific statement from the American Heart Association. Circulation 146, e93–e118 (2022).
Bischoff, N., Wimberger, S., Maresca, M. & Brakebusch, C. Improving precise CRISPR genome editing by small molecules: is there a magic potion? Cells 9, 1318 (2020).
Lee, J. et al. Prime editing with genuine Cas9 nickases minimizes unwanted indels. Nat. Commun. 14, 1786 (2023).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Liang, R. et al. Prime editing using CRISPR/Cas12a and circular RNAs in human cells. National Center for Biotechnology Information (NCBI). Available at: https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1048095 (2023).
Acknowledgements
This work was supported by the STI 2030-Major Projects (2023ZD04074), the National Key Research and Development Program (2023YFD1202904, 2023YFF1001601 and 2023YFF1001602), and the New Cornerstone Science Foundation.
Author information
Authors and Affiliations
Contributions
C.G. and K.T.Z. designed the experiments and supervised the project. R.L., Z.H. and M.L. performed experiments. J.H., G.L., Q.G. and R.Z. performed the MiSeq data analysis. C.G., R.L., Z.H., H.Z. and J-L.Q. wrote the manuscript. All authors reviewed the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors have submitted one patent application based on the results reported in this article. K.T.Z. is a founder and employee of Qi Biodesign. Q.G. is an employee of Qi Biodesign.
Peer review
Peer review information
Nature Biotechnology thanks Daesik Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data
Extended Data Fig. 1 Comparison of the editing efficiencies of Cas12a and Cas12a-D156R.
a, b, Editing efficiencies of Cas12a and Cas12a-D156R at the DNMT1-T3 site (a) and RUNX1-T4 site (b) in HEK293T cells. Frequencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in a, b.
Extended Data Fig. 2 The editing efficiencies of various prime editors.
a, Efficiencies of prime editing of various prime editors with Cas9 or nCas9 (H840A) and linear RNA (LinRNA) or circular RNA (CircRNA) at the FANCF site in HEK293T cells. b, Efficiencies of prime editing of these prime editors with Cas12a (D156R) or nCas12a (D156R-R1138A) and LinRNA or CircRNA at the DNMT1-T3 site. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in a, b. RTΔR, M-MLV RTΔRNase H.
Extended Data Fig. 3 Visualization of editing by various prime editors using the GFP reporter system.
Fields of cells labeled GFP (a), untreated sample (b), and only reporter (c) are controls. d–o, Microscope views of reconstitution of GFP by 12 prime editors. Scale bars, 200 µm. One of three independent experiments is shown (n = 3) in a-o. rCas12a, D156R-H759A-LbCas12a; nCas12a, D156R-R1138A-LbCas12a; rnCas12a, D156R-H759A-R1138A-LbCas12a; CircRNA, circular RNA; RTΔR, M-MLV RTΔRNase H.
Extended Data Fig. 4 Editing efficiencies of CPEs containing 5′ MS2-CircRNA, 3′ MS2-CircRNA, and 5′+3′ MS2-CircRNA.
a–c, Schematic diagram of 5′ MS2-CircRTT-PBS (a), 3′ MS2-CircRTT-PBS (b), and 5′+3′ MS2-CircRTT-PBS (c). d–f, Schematic diagram of CPEs using 5′ MS2-CircRTT-PBS (d), 3′ MS2-CircRTT-PBS (e), and 5′+3′ MS2-CircRTT-PBS (f). g, Comparison of the editing efficiencies of CPEs containing 5′ MS2-CircRTT-PBS, 3′ MS2-CircRTT-PBS, and 5′+3′ MS2-CircRTT-PBS at different sites in HEK293T cells. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in g. Del, deletion; Sub, substitution; CircRNA, circular RNA.
Extended Data Fig. 5 Frequencies of InDels induced by CPEs.
a, The frequencies of InDel byproducts produced by CPEs at the four sites of Fig. 2i. b, The frequencies of InDel byproducts produced by CPEs at the eight sites in Fig. 2j. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in a, b. InDel editing includes imprecise prime editing byproducts.
Extended Data Fig. 6 Analysis of InDels induced by CPEs.
a, Analysis of InDels induced by CPEs at the DNMT1-T1 site in HEK293T cells. b, Analysis of InDels induced by one-CPEs at the HBB-T1 site in HeLa cells. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in a, b.
Extended Data Fig. 7 Precise editing using twin CPE2.
The editing efficiencies of CPE2-Forward, CPE2-Reverse, CPE2-two CircRNA and CPE2-one CircRNA at the SITE7 site. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3).
Extended Data Fig. 8 Comparison of the editing efficiencies of CPE2-5.
a, The editing efficiencies of niCPE2-5 and sniCPE2-5 at DNMT1-T1, DNMT3B, and HDAC1 sites. b, The editing efficiencies of one-niCPE2-5 and one-sniCPE2-5 at DNMT1-T1, HBB-T1, and HDAC1 sites. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in a, b.
Extended Data Fig. 9 Comparison of the editing efficiencies of Cas12a-CPE2 and Cas9-PE2.
The editing efficiencies of Cas9-PE2, niCPE2, sniCPE2, one-niCPE2, and one-sniCPE2. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3).
Extended Data Fig. 10 Design and delivery sniCPE2 with dual AAVs.
a, Design and packaging dual AAV vectors of sniCPE2. b, Schematic diagram of AAV delivery. c, The editing efficiencies of HEK2 and HBB-T1 double prime editings with dual AAV delivery of sniCPE2. Efficiencies (mean ± s.d.) were calculated from three independent experiments (n = 3) in c.
Supplementary information
Supplementary Information
Supplementary Figures 1–5.
Supplementary Tables
Supplementary Table1. Target sites, RT templates, PBS sequnences, desired edits and nicking crRNAs in this study. Supplementary Table 2. Sequences used for vector construction. Supplementary Table 3. MiSeq 1st PCR primers used for target sites in this study. Supplementary Table 4. MiSeq 2nd PCR primers used in this study. Supplementary Table 5. Summary of crRNA spacer sequence-like off-target sites identified by Cas-OFFinder. Supplementary Table 6. MiSeq 1st PCR primers used for off-target sites in this study.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Liang, R., He, Z., Zhao, K.T. et al. Prime editing using CRISPR-Cas12a and circular RNAs in human cells. Nat Biotechnol (2024). https://doi.org/10.1038/s41587-023-02095-x
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41587-023-02095-x
This article is cited by
-
Harnessing noncanonical crRNA for highly efficient genome editing
Nature Communications (2024)
-
Prime editing using CRISPR-Cas12a and circular RNAs in human cells
Nature Biotechnology (2024)
