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Nature. 2019 Dec;576(7785):149-157. doi: 10.1038/s41586-019-1711-4. Epub 2019 Oct 21.

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

Anzalone AV1,2,3, Randolph PB1,2,3, Davis JR1,2,3, Sousa AA1,2,3, Koblan LW1,2,3, Levy JM1,2,3, Chen PJ1,2,3, Wilson C1,2,3, Newby GA1,2,3, Raguram A1,2,3, Liu DR4,5,6.

Author information

1
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA.
2
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
3
Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
4
Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA, USA. drliu@fas.harvard.edu.
5
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
6
Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.

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 endonuclease 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 used 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 shows higher or similar efficiency and fewer byproducts than homology-directed repair, has complementary strengths and weaknesses compared to base editing, and induces 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 up to 89% of known genetic variants associated with human diseases.

PMID:
31634902
PMCID:
PMC6907074
[Available on 2020-04-21]
DOI:
10.1038/s41586-019-1711-4
[Indexed for MEDLINE]

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