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A genome-wide map of adeno-associated virus–mediated human gene targeting

Nature Structural & Molecular Biology volume 21, pages 969–975 (2014)Cite this article

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Abstract

To determine which genomic features promote homologous recombination, we created a genome-wide map of gene targeting sites. We used an adeno-associated virus vector to target identical loci introduced as transcriptionally active retroviral vectors. A comparison of ~2,000 targeted and untargeted sites showed that targeting occurred throughout the human genome and was not influenced by the presence of nearby CpG islands, sequence repeats or DNase I–hypersensitive sites. Targeted sites were preferentially located within transcription units, especially when the target loci were transcribed in the opposite orientation to their surrounding chromosomal genes. We determined the impact of DNA replication by mapping replication forks, which revealed a preference for recombination at target loci transcribed toward an incoming fork. Our results constitute the first genome-wide screen of gene targeting in mammalian cells and demonstrate a strong recombinogenic effect of colliding polymerases.

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Figure 1: Genome-wide gene targeting.
Figure 2: Transcriptional effects on targeting.
Figure 3: Genome-wide replication-fork mapping.
Figure 4: DNA-replication effects on targeting.
Figure 5: Targeting frequencies in subclones with specific, mapped integration sites.
Figure 6: Stalled replication forks may promote vector pairing at target loci.

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Acknowledgements

We thank J. Delrow, A. Dawson and R. Basom for microarray analysis, P. Hendrie for MVM data, T. Canfield for Repli-Seq processing and R. Hirata and R. Stolitenko for technical assistance. This work was supported by grants from the US National Institutes of Health (R01DK55759, P01HL53750 and R01AR48328) to D.W.R., (K08AR053917) to D.R.D. and (U54HG007010 and P01HL53750) to R.S.H. and J.A.S. This work was also supported by grants from the Australian Department of Innovation, Industry, Science and Research (CG130052) to D.W.R., I.E.A. and C.-L.W., and the Genome Institute of Singapore (GIS) funded by the Agency for Science, Technology and Research (A*STAR), Singapore, to C.-L.W.

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Author notes

  1. David R Deyle

    Present address: Present address: Department of Medical Genetics, Mayo Clinic, Rochester, Minnesota, USA.,

  2. David R Deyle and R Scott Hansen: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine, University of Washington, Seattle, Washington, USA

    David R Deyle, R Scott Hansen, Li B Li, Amber A Burt & David W Russell

  2. Department of Molecular and Cellular Biology, University of Washington, Seattle, Washington, USA

    Anda M Cornea

  3. Gene Therapy Research Unit, Children's Medical Research Institute, Westmead, New South Wales, Australia

    Ian E Alexander

  4. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Richard S Sandstrom & John A Stamatoyannopoulos

  5. Genomic Technologies Department, Joint Genome Institute, Walnut Creek, California, USA

    Chia-Lin Wei

  6. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    David W Russell

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Contributions

D.R.D., R.S.H., A.M.C., C.-L.W., I.E.A. and D.W.R. designed experiments. A.M.C. performed gene targeting and plasmid rescue experiments. C.-L.W. performed high-throughput sequencing. R.S.H. and R.S.S. conducted the Repli-Seq experiments and processed the data. A.A.B. provided bioinformatics support. D.R.D. performed target-site mapping, bioinformatics processing, microarray analysis and data collection. L.B.L. mapped the MVM vector target site. J.A.S. and I.E.A. provided support for the project. D.R.D., R.S.H., A.M.C. and D.W.R. analyzed the data and wrote the manuscript. All authors commented on the manuscript. D.W.R. supervised the project.

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Correspondence to David W Russell.

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Integrated supplementary information

Supplementary Figure 1 Lack of influence of neighboring genetic elements on targeting.

The percentage of targeted, control, and random sites found within each interval (per kb) is shown in relation to short interspersed nuclear elements (SINE), long terminal repeats, long interspersed nuclear elements (LINE), DNA repeats, simple repeats, CpG islands, microsatellite repeats, and DNase I hypersensitive sites. The x-axis displays the binned distances up to 10 kb upstream and downstream from elements, with absolute values of distances used for elements that lack a sequence orientation. There were no significant differences between targeted and untargeted sites (P > 0.05, Chi-square test).

Supplementary Figure 2 Transcription and replication effects on targeting in very long genes.

(a) The number of very long chromosomal genes (>500 kb) containing the random, targeted or untargeted sites analyzed in this study is shown with their relative transcription and replication fork directions. (b) The number of very long chromosomal genes is shown with their transcription direction relative to that of the neo gene. (c) The number of targeted, untargeted or random sites located in very long genes is shown with their neo transcript direction compared to replication fork direction. Transcription directions of random sites were assigned randomly in Excel. *Statistically significant comparisons (P<0.05, Chi-square test) are indicated by asterisks.

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Supplementary Text and Figures

Supplementary Figures 1 and 2, and Supplementary Tables 2 and 3 (PDF 98 kb)

Supplementary Table 1

Genomic positions of uniquely mapped targeted and untargeted sites (XLSX 154 kb)

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Deyle, D., Hansen, R., Cornea, A. et al. A genome-wide map of adeno-associated virus–mediated human gene targeting. Nat Struct Mol Biol 21, 969–975 (2014). https://doi.org/10.1038/nsmb.2895

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