Search-and-replace genome editing without double-strand breaks or donor DNA

This is an unedited manuscript that has been accepted for publication. Nature Research are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be…

This is an unedited manuscript that has been accepted for publication. Nature Research are providing this early
version of the manuscript as a service to our customers. The manuscript will undergo copyediting,
typesetting and a proof review before it is published in its final form. Please note that during
the production process errors may be discovered which could affect the content, and all legal
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Nature

(2019)
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Abstract

Most genetic variants that contribute to disease1 are challenging to correct efficiently and without excess byproducts2–5. Here we describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed more than 175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutation without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to insert various tags and epitopes precisely into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over homology-directed repair, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle could correct about 89% of known pathogenic human genetic variants.

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Author information

Affiliations

  1. Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA

    • Andrew V. Anzalone
    • , Peyton B. Randolph
    • , Jessie R. Davis
    • , Alexander A. Sousa
    • , Luke W. Koblan
    • , Jonathan M. Levy
    • , Peter J. Chen
    • , Christopher Wilson
    • , Gregory A. Newby
    • , Aditya Raguram
    •  & David R. Liu
  2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA

    • Andrew V. Anzalone
    • , Peyton B. Randolph
    • , Jessie R. Davis
    • , Alexander A. Sousa
    • , Luke W. Koblan
    • , Jonathan M. Levy
    • , Peter J. Chen
    • , Christopher Wilson
    • , Gregory A. Newby
    • , Aditya Raguram
    •  & David R. Liu
  3. Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA

    • Andrew V. Anzalone
    • , Peyton B. Randolph
    • , Jessie R. Davis
    • , Alexander A. Sousa
    • , Luke W. Koblan
    • , Jonathan M. Levy
    • , Peter J. Chen
    • , Christopher Wilson
    • , Gregory A. Newby
    • , Aditya Raguram
    •  & David R. Liu

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Corresponding author

Correspondence to
David R. Liu.

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