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.
Source: Nature
Publication Date: (2019)
Issue: 10: 1038
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Species: human
Tissue Origin: cervix
Species: human
Tissue Origin: blood
U-2 OS
Species: human
Tissue Origin: bone
4D-Nucleofector® X-Unit

Nucleofection was used for transfection in all experiments using K562, HeLa, and U2OS cells. For PE conditions in these cell types, 800 ng
prime editor expression plasmid, 200 ng pegRNA expression plasmid, and 83 ng nicking sgRNA expression plasmid was nucleofected in a final volume of 20 µl in a 16-well nucleocuvette strip (Lonza). For HDR conditions in these three cell types, 350 ng Cas9 nuclease expression plasmid, 150 ng sgRNA expression plasmid and 200 pmol (6.6 µg) 100- nt ssDNA donor template (PAGE-purified; Integrated DNA Technologies) was nucleofected in a final volume of 20 µl per sample in a 16-well
Nucleocuvette strip (Lonza). K562 cells were nucleofected using the SF Cell Line 4D-Nucleofector X Kit (Lonza) with 5 × 105 cells per sample (program FF-120), according to the manufacturer’s protocol. U2OS cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 3–4 × 105 cells per sample (program DN-100), according to the manufacturer’s protocol. HeLa cells were nucleofected using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 2 × 105 cells per sample (program CN-114), according to the manufacturer’s protocol. Cells were harvested 72 h after nucleofection for genomic DNA extraction.


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.