Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage.

Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR
Nature. 2017 551 (7681): 464-471

PMID: 29160308 · PMCID: PMC5726555 · DOI:10.1038/nature24644

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

MeSH Terms (12)

Adenosine Deaminase Base Pairing Cell Line, Tumor CRISPR-Associated Proteins DNA DNA Cleavage Gene Editing Genome, Human HEK293 Cells Humans Models, Molecular Polymorphism, Single Nucleotide

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