Abstract
Reverse transcriptases, used in prime editing systems, exhibit lower fidelity, processivity and dNTP affinity than many DNA-dependent DNA polymerases. We report that a DNA-dependent DNA polymerase (phi29), untethered from Cas9, enables editing from a synthetic, end-stabilized DNA-containing template at up to 60% efficiency in human cells. Compared to prime editing, DNA polymerase editing avoids autoinhibitory intramolecular base pairing of the template, facilitates template synthesis and supports larger insertions (>100 nucleotides).
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Data availability
A Reporting Summary for this article is available as a supplementary information file. Plasmids for mRNA in vitro transcription, including MCP–RT, MCP–phi29 and nCas9 (H840A), have been deposited to Addgene for distribution. Illumina Sequencing data have been submitted to the Sequence Read Archive, and datasets are available under BioProject accession number PRJNA1004245 (ref. 37). Source data are provided with this paper.
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Acknowledgements
We thank S. Wolfe, B. Kelch, S. Liang, P. Liu and members of the laboratories of W.X. and E.J.S. for helpful discussions as well as T. Yu and Z. Weng for help with data analysis. We also thank Ben Kleinstiver for communicating results before publication. W.X. was supported by grants from the National Institutes of Health (DP2HL137167, P01HL158506 and UH3HL147367) and the Cystic Fibrosis Foundation. X.D. and E.J.S. acknowledge support from the Leducq Foundation Transatlantic Network of Excellence Program.
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Contributions
B.L., X.D., W.X. and E.J.S. conceptualized the project and designed experiments. B.L., X.D., C.Z. and Z.C. conducted molecular biological experiments. D.K. synthesized and purified LPETs and assisted in their design. B.L., X.D., C.Z. and H.C. conducted high-throughput sequencing and bioinformatic analyses. B.L., X.D., J.K.W., W.X. and E.J.S. interpreted the data and wrote the paper, and all authors edited the paper.
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E.J.S. is a co-founder and Scientific Advisory Board member of Intellia Therapeutics and a Scientific Advisory Board member at Tessera Therapeutics. The University of Massachusetts Chan Medical School has filed patent applications related to this work. All other authors have no competing interests.
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Extended data
Extended Data Fig. 1 LPET-mediated precision editing.
a, The levels of LPET and in vitro transcribed epegRNA were measured by qPCR after electroporation in 293T cells (n = 3). b, Diagram of LPET, MS2-less LPET, ssDNA, modified petRNA (LPET, −17), and unmodified petRNA. c, LPET and no-RT controls. HEK293T cells were electroporated with indicated mRNA (1 μg), sgRNA (100 pmol), nicking sgRNA (100 pmol), and FANCF LPET(+2), PRNP LPET (+0), IDS LPET (+0), RUNX1 LPET (+2), HBB LPET (+2). Editing was measured by deep sequencing (n = 3). Data and error bars indicate mean and s.d. of three independent biological replicates. d, Analysis of precise editing, imprecise editing, and scaffold incorporation events (FANCF) by CRISPResso2-prime editing mode (n = 3). 'MODIFIED’ represents reads containing unexpected insertions, deletions, or substitutions. Data and error bars indicate mean and s.d. of three independent biological replicates. e, Representative reads for FANCF and PRNP. Some deletions at FANCF are likely caused by short homology (underlined) near the nicking sites. Top five reads are shown.
Extended Data Fig. 2 MMLV RT-mediated precision editing with different LPET modifications.
a, Various modifications in the LPET. Precision editing by LPET with additional chemically modified residues [including Locked Nucleic Acid (LNA), 2′-F, 2′-O-methyl (OMe), 2′-fluoro (F) and 3′ phosphorothioate (PS)] as measured by deep sequencing in 293T cells (n = 3). * = 3′ PS linker. All sequences are written from 5′ to 3′. Data and error bars indicate the mean and s.d. of three independent biological replicates. b, RT mediates precise editing by LPETs with an all-DNA RTT and PBS with no modifications, or with the indicated 3′-terminal modifications, as measured by deep sequencing (n = 3). Blue and purple letters denote DNA, RNA and 2′-O-methyl RNA, respectively. Data and error bars indicate the mean and s.d. of three independent biological replicates.
Extended Data Fig. 3 Phi29 with unmodified and modified DPETs and additional controls.
a, Precise editing mediated by Phi29. HEK293T cells were electroporated with indicated mRNA (1 μg), sgRNA (100 pmol), nicking sgRNA (100 pmol), and indicated DPET(+0), MS2-less DPET, MS2-less ssDNA (RTT+PBS), modified linear DPET(−17)), or un-modified (all-RNA) DPET. b, Precise editing mediated by Phi29 and pegRNA. Editing efficiency was measured by deep sequencing (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates; two-tailed unpaired Student’s t-test; **P < 0.01.
Extended Data Fig. 4 Phi29 DPE with different DPET modifications.
a, Phi29-mediated precision editing with various modifications in the DPET (including locked nucleic acid, 2′-fluoro, 2′-O-methyl, and 3′ phosphorothioate) as measured by deep sequencing (n = 3). * = 3′ PS linker. All sequences are written from 5′ to 3′. Data and error bars indicate the mean and s.d. of three independent biological replicates. b, Phi29 mediates precise editing by DPETs with an all-DNA RTT and PBS with no modifications, or with the indicated 3′-terminal modifications, as measured by deep sequencing (n = 3). Blue and purple letters denote DNA, RNA and 2′-O-methyl RNA residues, respectively. Data and error bars indicate the mean and s.d. of three independent biological replicates.
Extended Data Fig. 5 Incremental DNA replacement in the PBS/RTT at the PRNP site.
Precision editing with DNA replacement in the PBS/RTT with LPET/RT or DPET/Phi29. Data and error bars indicate the mean and s.d. of three independent biological replicates.
Extended Data Fig. 6 LPETs and DPETs enable precise genome editing without an additional nicking sgRNA in multiple endogenous sites.
a–c, HEK293T cells were electroporated with indicated mRNAs (1 μg), pegRNA (100 pmol, PE2) and nicking sgRNA (100 pmol, PE3). LPET and DPET groups include mRNAs, sgRNA and nicking sgRNA (100 pmol), and LPET/DPET (FANCF +2 (a), PRNP +0 (b), RUNX1 +2 (c)). Data and error bars indicate the mean and s.d. of three independent biological replicates.
Extended Data Fig. 7 LPETs and DPETs enable precise genome editing.
a–c, LPET/RT and DPET/Phi29 editing in multiple cell lines, as measured by deep sequencing (n = 3). ND, not determined. ‘SNP’ indicates the existence of a SNP in the HBB target region in the U2OS cell line, precluding comparative testing. d,e, Representative FACS plots in Fig. 2f. f, Diagram depicting mSA-RT and Phi29. The 5′-biotinylated LPET or DPET is recruited by mSA and anneals to the nicked DNA. g, Tests of biotinylated-LPETs and biotinylated-DPETs in an mCherry reporter line and at endogenous sites (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates.
Extended Data Fig. 8 PoII5M DNA polymerase, MarathonRT and TGIRT editing with synthetic templates.
a, Diagram depicting PoII5M/DPET editing (left). MCP-tethered PoII5M binds to the DPET via MS2. The DPET anneals to the nicked DNA strand and serves as the template for PoII5M DNA polymerase. Data indicate precision editing (3-nt substitution) at the FANCF locus with DPET (+2) and fully 2′-O-methyl (OMe)-substituted PBS (right). b, Precise editing at the FANCF locus with MarathonRT and TGIRT. Editing efficiency was measured by deep sequencing on day 3 (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates.
Extended Data Fig. 9 Off-target and cytotoxicity evaluation.
a, On-target and off-target editing efficiencies for FANCF. Data indicate the mean and standard deviation of three independent biological replicates. b, HEK293T cells were electroporated with indicated mRNA reagents or buffers. Cell viability was measured by the CellTiter-Glo assay (n = 3) on the indicated time points. Data and error bars indicate mean and s.d. of three independent biological replicates. Two-tailed unpaired Student’s t-test: *, P < 0.05.
Extended Data Fig. 10 Precise editing for a 40-bp replacement and a 132-bp insertion.
a, Precise editing for a 40-bp insertion and 90-bp deletion at two AAVS sites (1615 and 1705) using LPET/DPET through mRNA nucleofection, followed by PCR amplification and agarose gel electrophoresis. b, Quantification of 40-bp insertion in a by deep sequencing (n = 3). Data and error bars indicate the mean and s.d. of three independent biological replicates, unless otherwise indicated. c, Diagram of the TwinPE by LPETs/DPETs for long insertion. d, Precise editing for a 132-bp insertion at the HEK3 site. Editing efficiency was measured by ddPCR (n = 2).
Supplementary information
Supplementary Information
Supplementary Table 1. Synthetic sgRNAs, nicking sgRNAs and pegRNAs. Supplementary Table 2. Synthetic LPETs and DPETs. Supplementary Table 3. Primers used for high-throughput sequencing. Supplementary Table 4. Primers used for ddPCR. Supplementary Table 5. Primers used for qPCR. Supplementary sequences.
Source Data Extended Data Fig. 10
Unmodified gel
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Liu, B., Dong, X., Zheng, C. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01947-w
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DOI: https://doi.org/10.1038/s41587-023-01947-w
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