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

Bin Liu^#¹, Xiaolong Dong^#¹, Haoyang Cheng¹, Chunwei Zheng¹, Zexiang Chen¹, Tomás C Rodríguez¹, Shun-Qing Liang¹, Wen Xue^{2

3

4

5}, Erik J Sontheimer^{6

7

8}

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
² RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
³ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁴ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁵ Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁶ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
⁷ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
⁸ Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.

^# Contributed equally.

PMID: 35379962
DOI: 10.1038/s41587-022-01255-9

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

Bin Liu et al. Nat Biotechnol. 2022 Sep.

. 2022 Sep;40(9):1388-1393.

doi: 10.1038/s41587-022-01255-9. Epub 2022 Apr 4.

Authors

Bin Liu^#¹, Xiaolong Dong^#¹, Haoyang Cheng¹, Chunwei Zheng¹, Zexiang Chen¹, Tomás C Rodríguez¹, Shun-Qing Liang¹, Wen Xue^{2

3

4

5}, Erik J Sontheimer^{6

7

8}

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA.
² RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
³ Department of Molecular, Cell and Cancer Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁴ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁵ Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. wen.xue@umassmed.edu.
⁶ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
⁷ Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.
⁸ Department of Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA. erik.sontheimer@umassmed.edu.

^# Contributed equally.

PMID: 35379962
DOI: 10.1038/s41587-022-01255-9

Abstract

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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References

1. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). - DOI
1. Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021). - DOI
1. Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021). - DOI
1. Wang, D., Zhang, F. & Gao, G. CRISPR-based therapeutic genome editing: strategies and in vivo delivery by AAV vectors. Cell 181, 136–150 (2020). - DOI
1. Truong, D. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015). - DOI

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

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

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