Format

Send to

Choose Destination
Nat Commun. 2017 Jul 7;8(1):66. doi: 10.1038/s41467-017-00124-3.

Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA.

Author information

1
Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus University Medical Centre, Rotterdam,, 3000 CA, The Netherlands.
2
Department of Human Genetics, Leiden University Medical Centre, PO Box 9600, Leiden,, 2300 RC, The Netherlands.
3
Department of Molecular Genetics, Cancer Genomics Netherlands, Erasmus University Medical Centre, Rotterdam,, 3000 CA, The Netherlands. R.Kanaar@erasmusmc.nl.
4
Department of Human Genetics, Leiden University Medical Centre, PO Box 9600, Leiden,, 2300 RC, The Netherlands. M.Tijsterman@lumc.nl.

Abstract

Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

PMID:
28687761
PMCID:
PMC5501794
DOI:
10.1038/s41467-017-00124-3
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center