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.
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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
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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.
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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.
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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.
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Nature Biotechnology thanks Daesik Kim and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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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.
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Liang, R., He, Z., Zhao, K.T. et al. Prime editing using CRISPR-Cas12a and circular RNAs in human cells. Nat Biotechnol 42, 1867–1875 (2024). https://doi.org/10.1038/s41587-023-02095-x
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DOI: https://doi.org/10.1038/s41587-023-02095-x
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