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

By Andrew V. Anzalone, Peyton B. Randolph, Jessie R. Davis, Alexander Sousa, Luke W. Koblan, Jonathan M. Levy, Peter J. Chen, Christopher Wilson, Gregory A. Newby, Aditya Raguram and 1 more. Nature, 2019

3,884 citations recorded by the source corpus.

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. A new DNA-editing technique called prime editing offers improved versatility and efficiency with reduced byproducts compared with existing techniques, and shows potential for correcting disease-associated mutations.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/3af5518cd1a523532cb2f2b43142843980af15a2

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

Citations in the corpus

CRISPR technology: A decade of genome editing is only the beginning (2023, background): “Prime editing represents one such alternative that can insert and delete DNA sequences without introducing DSBs (12), though this technology still needs further refinement (Fig.”
Genome engineering for crop improvement and future agriculture. (2021, positive): “However, a recent technological breakthrough, prime editing, allows for the creation of all 12 types of base substitutions and small DNA insertions and deletions in human cells (Anzalone et al., 2019; Figure 3C).”
Adeno-associated virus as a delivery vector for gene therapy of human diseases (2024, background): “146,147 Various strategies have emerged to overcome this hurdle by dividing a large transgene into two parts, with each encapsulated in an rAAV capsid.”
CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future (2020, background): “CRISPR-induced DSBs often trigger apoptosis rather than the intended gene edit (68).”
Enhanced prime editing systems by manipulating cellular determinants of editing outcomes (2021, methodology): “Toward the goal of enabling a wide range of sequence changes, we developed prime editing, a versatile gene editing approach that can install all types of targeted DNA base pair substitutions, small insertions, small deletions, and combinations thereof without requiring double-strand DNA breaks (DSBs”