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A split prime editor with untethered reverse transcriptase and circular RNA template


Delivery and optimization of prime editors (PEs) have been hampered by their large size and complexity. Although split versions of genome-editing tools can reduce construct size, they require special engineering to tether the binding and catalytic domains. Here we report a split PE (sPE) in which the Cas9 nickase (nCas9) remains untethered from the reverse transcriptase (RT). The sPE showed similar efficiencies in installing precise edits as the parental unsplit PE3 and no increase in insertion–deletion (indel) byproducts. Delivery of sPE to the mouse liver with hydrodynamic injection to modify β-catenin drove tumor formation with similar efficiency as PE3. Delivery with two adeno-associated virus (AAV) vectors corrected the disease-causing mutation in a mouse model of type I tyrosinemia. Similarly, prime editing guide RNAs (pegRNAs) can be split into a single guide RNA (sgRNA) and a circular RNA RT template to increase flexibility and stability. Compared to previous sPEs, ours lacks inteins, protein–protein affinity modules and nuclease-sensitive pegRNA extensions, which increase construct complexity and might reduce efficiency. Our modular system will facilitate the delivery and optimization of PEs.

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Fig. 1: sPE enables genome editing in cells and in adult mouse liver.
Fig. 2: sPE dual AAV rescues weight loss in Fah-mutant mice.
Fig. 3: Prime editing by modular RNA components.
Fig. 4: Effective prime editing by sPE through mRNA and RNP nucleofection.

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Data availability

A Reporting Summary for this article is available as a supplementary information file. The raw DNA sequencing data are available at the NCBI Sequence Read Archive database under project number PRJNA802843. Plasmids for mammalian expression of MS2-pegRNA, petRNA, alternative PEs and split RTs as well as for bacterial expression of recombinant NLS-M-MLV RT have been deposited to Addgene for distribution. Source data are provided with this paper.


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We thank S. Wolfe, P. Zamore and members of the Xue and Sontheimer labs for helpful discussions. We thank Y. Liu in the University of Massachusetts Chan Medical School Morphology Core and G. Gao, Q. Su and J. Xie in the University of Massachusetts Chan Medical School Viral Vector Core for support. W.X. was supported by grants from the National Institutes of Health (DP2HL137167, P01HL131471, P01HL158506 and UG3HL147367), American Cancer Society (129056-RSG-16-093) 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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Authors and Affiliations



B.L., X.D., W.X. and E.J.S. conceptualized the project and designed experiments. B.L., X.D., C.Z., S.-Q.L. and Z.C. conducted molecular biological experiments. B.L. performed mouse work. B.L., X.D., H.C. and T.C.R. conducted high-throughput sequencing and bioinformatic analyses. B.L., X.D., W.X. and E.J.S. interpreted the data and wrote the paper.

Corresponding authors

Correspondence to Wen Xue or Erik J. Sontheimer.

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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 a patent application on this work. The authors declare no competing interests.

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Nature Biotechnology thanks Jia Chen 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 MS2-PE2 and SunTag-PE2 design.

a, Schematic overview of MS2-PE2. The MS2 coat protein (MCP) was fused to the N terminus of M-MLV reverse transcriptase to enable recruitment by the MS2-pegRNAs. b, Sizes of Cas9 nickase and MCP-RT ORFs. c, Engineered MS2-pegRNAs with MS2 sequences appended into distinct sgRNA stem-loops, or onto the 3′ terminus. d, Schematic overview of SunTag-PE2. e, Schematics of scFv-RT and GCN4-Cas9 nickase. The scFv was fused to the N terminus of M-MLV RT (top). The 10xGCN4 epitope was fused to either the N terminus (SunTag-PE) or the C terminus of SpyCas9H840A (PE-SunTag).

Extended Data Fig. 2 Split-PE, SunTag-PE3 and MS2-PE3 tested in an mCherry reporter line and an endogenous locus.

a, A diagram of the mCherry reporter line that functions by converting a premature stop codon. b, Sequences of RTT and PBS, non-cognate (PBS + RTT), non-cognate PBS, and non-cognate RTT for the mCherry reporter line. c, Multiple MS2-pegRNAs tested in mCherry reporter cell lines. The pegRNA with MS2 on the repeat/anti-repeat stem-loop (pegRNA-1.1) has the highest editing efficiency (higher even than that of the original PE3) in this mCherry reporter line (n = 2). Therefore, the pegRNA1.1-Cas9H840A-MCP-RT system was designated as MS2-PE3. d, SunTag-PE3 and PE3-SunTag were tested in the mCherry reporter cell line. Two-tailed unpaired Student’s t-test: *P < 0.05 (n = 3). e, Sanger sequencing and EditR quantification of PE3, Split PE, SunTag-PE3 and MS2-PE3 by installing “CTT” at HEK3 sites in HEK293T cells. All plasmids were transfected at the same molar ratio. Genomic DNAs were isolated 72 h post transfection. f, Dose dependence of the RT-encoding plasmid. One microgram of H840A plasmid was co-transfected with plasmids encoding additional sPE components [pegRNA (0.3 µg), nicking sgRNA (0.1 µg), and RT (0.01-2 µg)] per well in a 12-well plate (n = 2). Data and error bars indicate the mean and standard deviation of two or three independent biological replicates, as indicated.

Extended Data Fig. 3 SunTag-PE3 and MS2-PE3 tested in reporter lines.

a, A diagram of the GFP reporter line that is activated by precise insertion of 18 bp (in place of a 39-bp non-functional sequence). Indels (+1) can restore mCherry expression. b, A diagram of the GFP reporter line that is activated by deletion of 47 bp; indels (+1) can restore mCherry expression. c, MS2-PE3 was tested in the GFP reporter line shown in panel a (n = 3). d, SunTag-PE3 was tested in the GFP reporter line shown in a (n = 3). e, MS2-PE3 was tested in the GFP reporter line shown in b (n = 3). Data and error bars indicate the mean and standard deviation of three independent biological replicates.

Extended Data Fig. 4 Amplicon sequencing of MS2-PE3 and SunTag-PE3 at multiple endogenous sites.

a, MS2-PE3 for editing by 1-bp substitution at multiple endogenous loci, including HEK3, RNF2, VEGFA, and FANCF in HEK293T cells. b, SunTag-PE3 for RNF2 and VEGFR editing to generate a 1-bp substitution in HEK293T cells. Two-tailed unpaired Student’s t-test: *P < 0.05, **P < 0.01. Data and error bars indicate the mean and standard deviation of three independent biological replicates.

Extended Data Fig. 5 Prime editing by alternative reverse transcriptases.

a, Illustration of prime editors with alternative RTs. Human codon-optimized E.r. maturase RT and GsI-IIC RT were cloned into the original PE2 in place of the M-MLV RT. b, Prime editing by alternative RT orthologs at the VEGFA site by 3-nt substitutions (+2 G to C and +4-5 GG to CT). Two-tailed unpaired Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001. Data and error bars indicate the mean and standard deviation of three independent biological replicates.

Extended Data Fig. 6 Prime editing using mutant PE2 and sPE components.

Representative Sanger sequencing traces from prime editing experiments using mutant PE2 and sPE components. HEK293T cells were transfected with indicated plasmids, along with others encoding pegRNA and nicking sgRNA. Prime editing introduces a 3-nt substitution at the FANCF locus (+2 C to T and +4-5 TG to AC). The experiment was repeated two times. Sanger sequencing traces were analyzed by EditR.

Extended Data Fig. 7 Split PE2 enables genome editing in adult mice.

a, Representative images of tumors in liver with PE3 or split PE. Control group was pegRNA-injected only. b, Amplicon sequencing from representative animals using genomic DNA isolated from tumors.

Extended Data Fig. 8 Prime editing by separate RNA modules.

a, Schematic of proof-of-concept experiment on delivering the RT template separately. The 3’ extension of the pegRNA (the RTT-PBS sequence) was removed from the 3’ of the tracrRNA scaffold and provided separately under the control of a U6 promoter. An sgRNA plasmid was co-transfected to carry out the nicking event in conjunction with the nCas9. b, Illustration of the circularization pathway to generate petRNAs. c, PE efficiency by modular RNA components at the FANCF locus introducing a 3-nt substitution (+2 C to T and +4-5 TG to AC). Plasmids expressing RNAs were co-transfected with Cas9H840A and the split RT, which lacks the MCP domain. Nicking sgRNAs were used for all prime editing. d, Validation of petRNA adaptability to an alternative nickase. The petRNA was designed to target a site at the FANCF locus where SpyCas9 and SauCas9 nickases share the same nick and thus a single petRNA guide/primer/template sequence. The petRNA and the MCP-RT were co-transfected with plasmids encoding SpyCas9H840A-sgRNA or SauCas9N580A. Nicking sgRNAs were used for all prime editing. Two-tailed unpaired Student’s t-test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data and error bars indicate the mean and standard deviation of three independent biological replicates.

Extended Data Fig. 9 Comparison of PE, sPE, and petRNA off-target effects at known Cas9 off-target sites of FANCF and HEK4 using deep sequencing.

On-target edits are shown in red and off-target edits are shown in green. Data and error bars indicate the mean and standard deviation of three independent biological replicates.

Extended Data Fig. 10 In vitro transcribed mRNA and purified RT protein used in the nucleofections, and FACS gating strategy.

a, Denaturing agarose gel analysis of the mRNAs produced in-house. The coding sequences of nCas9, MMLV-RT or PE2 were flanked by a capped 5’ UTR and a 3’ UTR, followed by a 110-nt poly(A) tract. b, SDS-PAGE analysis of the purified MMLV-RT protein. C, FACS gating examples for reporter cells.

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Supplementary information

Supplementary Information

Supplementary Note, Tables 1–4 and sequences.

Reporting Summary

Source data

Source Data Fig. 3

Unprocessed gel and blot images for Fig. 3e.

Source Data Extended Data Fig. 10

Unprocessed gel and blot images for Extended Data Fig. 10.

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Liu, B., Dong, X., Cheng, H. et al. A split prime editor with untethered reverse transcriptase and circular RNA template. Nat Biotechnol 40, 1388–1393 (2022).

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