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Nature DOI:10.1038/nature17946

Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

Publication TypeJournal Article
Year of Publication2016
AuthorsKomor, AC, Kim, YB, Packer, MS, Zuris, JA, Liu, DR
JournalNature
Volume533
Issue7603
Pages420-4
Date Published2016 05 19
ISSN1476-4687
KeywordsAnimals, APOBEC-1 Deaminase, Apolipoprotein E4, Base Sequence, Cell Line, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Proteins, CRISPR-Cas Systems, Cytidine, Cytidine Deaminase, Deoxyribonuclease I, DNA, DNA Cleavage, DNA Repair, Genes, p53, Genetic Engineering, Genome, Humans, INDEL Mutation, Mice, Point Mutation, RNA, Guide, Templates, Genetic, Uracil-DNA Glycosidase, Uridine
Abstract

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.

DOI10.1038/nature17946
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/27096365?dopt=Abstract

Alternate JournalNature
PubMed ID27096365
PubMed Central IDPMC4873371
Grant ListF32 GM 112366-2 / GM / NIGMS NIH HHS / United States
F32 GM 106601-2 / GM / NIGMS NIH HHS / United States
/ / Howard Hughes Medical Institute / United States
F32 GM106601 / GM / NIGMS NIH HHS / United States
R01 GM065400 / GM / NIGMS NIH HHS / United States
F32 GM112366 / GM / NIGMS NIH HHS / United States
T32 GM008313 / GM / NIGMS NIH HHS / United States
R01 EB022376 / EB / NIBIB NIH HHS / United States