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Prime editing for precise and highly versatile genome manipulation

Abstract

Programmable gene-editing tools have transformed the life sciences and have shown potential for the treatment of genetic disease. Among the CRISPR–Cas technologies that can currently make targeted DNA changes in mammalian cells, prime editors offer an unusual combination of versatility, specificity and precision. Prime editors do not require double-strand DNA breaks and can make virtually any substitution, small insertion and small deletion within the DNA of living cells. Prime editing minimally requires a programmable nickase fused to a polymerase enzyme, and an extended guide RNA that both specifies the target site and templates the desired genome edit. In this Review, we summarize prime editing strategies to generate programmed genomic changes, highlight their limitations and recent developments that circumvent some of these bottlenecks, and discuss applications and future directions.

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Fig. 1: Precision genome editing in mammalian cells.
Fig. 2: Original prime editing systems.
Fig. 3: Advancements in prime editing systems.
Fig. 4: Prime editing variants.

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Acknowledgements

D.R.L. acknowledges support from US National Institutes of Health awards U01 AI142756, RM1 HG009490 and R35 GM118062, from the Bill and Melinda Gates Foundation, and from the Howard Hughes Medical Institute. P.J.C. acknowledges support from a US National Science Foundation (NSF) graduate research fellowship. The authors thank A. Anzalone for helpful comments.

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P.J.C. and D.R.L. contributed to all aspects of the manuscript.

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Correspondence to David R. Liu.

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Competing interests

The authors have filed patent applications on gene-editing technologies through the Broad Institute of MIT and Harvard. P.J.C. is currently an employee of Prime Medicine. D.R.L. is a consultant and equity owner of Beam Therapeutics, Pairwise Plants, Prime Medicine, Chroma Medicine, and Nvelop Therapeutics, companies that use or deliver genome-editing or genome-engineering technologies.

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Glossary

Chromothripsis

A process in which tens to thousands of chromosomal rearrangements occur in a single event.

Heteroduplex

Double-stranded DNA in which the sequences of the strands are not perfectly complementary.

Mosaicism

A condition in which an animal contains multiple cell lineages with different genotypes.

Nickase Cas9

(nCas9). Cas9 that has either its HNH or RuvC nuclease domain catalytically inactivated, resulting in a Cas9 enzyme that can only cut one strand of targeted double-stranded DNA.

Polygenic diseases

Diseases that are mediated by numerous genetic variants that each individually contribute small effects.

R loop

A three-stranded nucleic acid structure that contains a DNA:RNA hybrid and a displaced strand of DNA.

Single-guide RNA

(sgRNA). A single-guide RNA molecule, composed of a CRISPR RNA (crRNA) fused to its corresponding trans-activating CRISPR RNA (tracrRNA) scaffold sequence, that directs the binding and nuclease activity of Cas9 enzymes.

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Chen, P.J., Liu, D.R. Prime editing for precise and highly versatile genome manipulation. Nat Rev Genet (2022). https://doi.org/10.1038/s41576-022-00541-1

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