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.
Author information
Author notes
Xiaolong Dong
Present address: Tessera Therapeutics, Somerville, MA, USA
These authors contributed equally: Bin Liu, Xiaolong Dong, Chunwei Zheng.
Authors and Affiliations
RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA
Bin Liu, Xiaolong Dong, Chunwei Zheng, David Keener, Zexiang Chen, Haoyang Cheng, Jonathan K. Watts, Wen Xue & Erik J. Sontheimer
Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA
Jonathan K. Watts, Wen Xue & Erik J. Sontheimer
Department of Biochemistry and Molecular Biotechnology, University of Massachusetts Chan Medical School, Worcester, MA, USA
Jonathan K. Watts & Erik J. Sontheimer
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA
Wen Xue
Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
Wen Xue & Erik J. Sontheimer
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.
Corresponding authors
Ethics declarations
Competing interests
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
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