Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
Top Cited Papers
Open Access
- 20 April 2016
- journal article
- research article
- Published by Springer Science and Business Media LLC in Nature
- Vol. 533 (7603), 420-424
- https://doi.org/10.1038/nature17946
Abstract
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction.1,2 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 from the cellular response to dsDNA breaks.1,2 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 (nt), 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 (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor 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.This publication has 33 references indexed in Scilit:
- Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavageScience, 2016
- A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable mannerNature Biotechnology, 2009
- The Role of Apolipoprotein E in Alzheimer's DiseaseNeuron, 2009
- DNA Repair in Mammalian CellsCellular and Molecular Life Sciences, 2009
- The AID/APOBEC family of nucleic acid mutatorsGenome Biology, 2008
- The Missing ApoE AlleleAnnals of Human Genetics, 2007
- Replisome assembly and the direct restart of stalled replication forksNature Reviews Molecular Cell Biology, 2006
- Comparison of the Differential Context-dependence of DNA Deamination by APOBEC Enzymes: Correlation with Mutation Spectra in VivoJournal of Molecular Biology, 2004
- RNA Editing Enzyme APOBEC1 and Some of Its Homologs Can Act as DNA MutatorsMolecular Cell, 2002
- Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: Protein mimicry of DNACell, 1995