Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining

The efficiency of homologous recombination-based Cas9 genome editing is increased by inhibiting non-homologous end joining. Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorpora...

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Bibliographic Details
Published in:Nature biotechnology 2015-05, Vol.33 (5), p.538-542
Main Authors: Maruyama, Takeshi, Dougan, Stephanie K, Truttmann, Matthias C, Bilate, Angelina M, Ingram, Jessica R, Ploegh, Hidde L
Format: Article
Language:English
Subjects:
45/41
631/1647/1513/1967
631/337/1427/2122
631/61/17/1511
64/60
Agriculture
Animals
Bioinformatics
Biomedical Engineering/Biotechnology
Biomedicine
Biotechnology
Cell Line
CRISPR-Cas Systems - genetics
Deoxyribonucleic acid
DNA
DNA Breaks, Double-Stranded - drug effects
DNA End-Joining Repair - genetics
DNA Ligase ATP
DNA Ligases - genetics
DNA repair
DNA Repair - genetics
Forecasts and trends
Gene mutations
Genetic engineering
Genetic Engineering - methods
Genetic research
Genome
Homologous Recombination - genetics
Identification and classification
letter
Life Sciences
Mice
Pyrimidines - administration & dosage
Schiff Bases - administration & dosage
Zinc Fingers - genetics
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Summary:The efficiency of homologous recombination-based Cas9 genome editing is increased by inhibiting non-homologous end joining. Methods to introduce targeted double-strand breaks (DSBs) into DNA enable precise genome editing by increasing the rate at which externally supplied DNA fragments are incorporated into the genome through homologous recombination. The efficiency of these methods is limited by nonhomologous end joining (NHEJ), an alternative DNA repair pathway that competes with homology-directed repair (HDR). To promote HDR at the expense of NHEJ, we targeted DNA ligase IV, a key enzyme in the NHEJ pathway, using the inhibitor Scr7. Scr7 treatment increased the efficiency of HDR-mediated genome editing, using Cas9 in mammalian cell lines and in mice for all four genes examined, up to 19-fold. This approach should be applicable to other customizable endonucleases, such as zinc finger nucleases and transcription activator–like effector nucleases, and to nonmammalian cells with sufficiently conserved mechanisms of NHEJ and HDR.
ISSN:1087-0156
1546-1696
DOI:10.1038/nbt.3190