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

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 ? (Pol ?) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase ? 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 ? and classical non-homologous recombination eliminates random integration.

SUBMITTER: Zelensky AN

PROVIDER: S-EPMC5501794 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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

Zelensky Alex N AN Schimmel Joost J Kool Hanneke H Kanaar Roland R Tijsterman Marcel M

Nature communications 20170707 1

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, ...[more]

PMID: 28687761

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Project description:Background:The comparatively small genome, well elucidated functional genomics and rapid life cycle confer T7 bacteriophage with great advantages for bio-application. Genetic manipulation of T7 genome plays a key role in T7 related applications. As one of the important aspects in T7 phage genetic modification, gene knock-in refers to two main approaches including direct genetic manipulation in vitro and recombineering. Neither of these available methods are efficient enough to support the development of innovative applications capitalizing on T7 bio-system and thus there is room for novel strategies that address this issue. Integration mediated by the ?C31 integrase is one of the most robust site-specific recombination systems. ?C31 integrases with enhanced activity and specificity have been developed such that it is ideal to effectuate exogenous DNA knock-in of T7 phage with advanced ?C31 integrase. Methods:Plasmid construction was conducted by routine molecular cloning technology. The engineered T7 bacteriophages were constructed through homologous recombination with corresponding plasmids and the functional T7 phage was designated as T7?G10G11-attB. In the integration reaction, hosts with both executive plasmids (pEXM4) and donor plasmids (pMCBK) were lysed by T7?G10G11-attB. Progenies of T7 phages that integrated with pMCBK were isolated in restrict hosts and validated by sequencing. T7?G10G11-attB capacity limit was explored by another integration reactions with donor plasmids that contain exogenous DNA of various lengths. Results:T7?G10G11-attB exhibits abortive growth in restrictive hosts, and a bacterial attachment site recognized by ?C31 integrase (attB) was confirmed to be present in the T7?G10G11-attB genome via sequencing. The integration reaction demonstrated that plasmids containing the corresponding phage attachment site (attP) could be integrated into the T7?G10G11-attB genome. The candidate recombinant phage was isolated and validated to have integrated exogenous DNA. The maximum capacity of T7?G10G11-attB was explored, and it's found that insertion of exogenous DNA sequences longer than 2 kbp long can be accommodated stably. Conclusion:We advanced and established a novel approach for gene knock-in into the T7 genome using ?C31 integrase.

Dataset Information

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

Publications

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

Similar Datasets

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