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Template plasmid integration in germline genome-edited cattle

Nature Biotechnology volume 38, pages 163–164 (2020)Cite this article

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An Author Correction to this article was published on 05 March 2020

The Original Article was published on 06 May 2016

This article has been updated

arising from D. F. Carlson et al. Nature Biotechnology https://doi.org/10.1038/nbt.3560 (2016)

As genome-editing technology evolves, so does our understanding of the unintended alterations it produces, both in form and frequency. Several sequencing-based methods have been developed to screen for off-target errors (GUIDE-Seq1, SITE-Seq2, CIRCLE-Seq3, DISCOVER-Seq4), while long-read sequencing of the target site can be used to detect on-target errors5. Each screening approach carries assumptions and biases that may allow alterations of unexpected types to go undetected. Recent examples of previously unexpected alterations include complex genomic rearrangements at or near the target site in mammalian genome-editing experiments5,6. The complex rearrangements included insertions, deletions, inversions and translocations that were difficult to detect by standard PCR and DNA sequencing methods.

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Fig. 1: Template plasmid integration at the target site of genome-edited calves.

Data availability

The Carlson et al. whole-genome sequencing data are available from NCBI SRA: https://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP072240.

Change history

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Acknowledgements

We acknowledge Recombinetics, Inc. for generating the animals and sharing the sequencing data through NCBI SRA. We appreciate A. Van Eenennaam, C. T. Brown and T. Mansour for helpful discussions concerning our discovery of the template plasmid integration. We thank M. Mikhailov and F.-J. Luo for technical expertise, as well as H. Howard, A. Fidler and K. Underwood for veterinary expertise.

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Author notes

  1. These authors contributed equally: Alexis L. Norris, Stella S. Lee.

Authors and Affiliations

  1. Office of New Animal Drug Evaluation, US Food and Drug Administration, Center for Veterinary Medicine, Rockville, MD, USA

    Alexis L. Norris, Stella S. Lee, Kevin J. Greenlees & Heather A. Lombardi

  2. Office of Research, Center for Veterinary Medicine, US Food and Drug Administration, Laurel, MD, USA

    Daniel A. Tadesse & Mayumi F. Miller

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  1. Alexis L. Norris
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Contributions

A.L.N. and S.S.L. developed the strategy. A.L.N. conducted the experiments. A.L.N., S.S.L. and D.A.T. analyzed the data. A.L.N, S.S.L, K.J.G., D.A.T., M.F.M. and H.A.L. wrote the manuscript.

Corresponding author

Correspondence to Heather A. Lombardi.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Plasmid DNA is present in genome-edited calves.

Shown are all sequencing reads that aligned to the linearized plasmid backbone, with origins, genes, and gene promoters listed. For each sample, the coverage track is shown above the individual sequencing read pairs, for which lines connect the mate pairs. The template-plasmid junctions are indicated by discordantly mapped pairs and split reads. In discordant pairs, the read’s mate is aligned to the template, not the plasmid. In split reads, the mismatched bases at an end of the read are the template sequence, because the read spans the template-plasmid junction. In both genome-edited calves, the reads span the entire length of the plasmid, and at a coverage consistent with heterozygosity (~10X coverage). The unedited parental cell lines have sparse, low-level coverage of the plasmid, consistent with contamination. The coverage track scale is uniform across samples (0-19 reads). Reads at the start and end of the plasmid sequence are artifacts of linearization of the circular plasmid.

Supplementary Figure 2 Template plasmid is integrated at the target site in genome-edited calves.

Compared to the naturally occurring Pc sequence (a), the chromosome with the template plasmid integration (b) adds two novel junctions created by the plasmid and flanking template sequences. The junctions are evidenced by the aligned sequencing reads, shown here for a representative sample (SRR3290615) in IGV (c). The plasmid sequence is inverted as a result of the antisense insertion of the repair template at the TOPO site during cloning. For each template-plasmid junction, the left (L) and right (R) breakpoints are indicated. The first novel junction’s breakpoints are the end of the first template copy (bt9 chr1:2,429,911; 1L) with the start of the plasmid (pCR2.1-TOPO:294-bp(-); 1R). The second novel junction’s breakpoints are the end of the plasmid (pCR2.1-TOPO:295-bp(-); 2L) and the start of the second template copy (bt9 chr1:2,428,500; 2R).

Supplementary Figure 3 Template plasmid integration was not detected by PCR genotyping.

In the Carlson, et al. study, two primer sets, btHP and HP1748 (Supplemental Table 1 and Supplemental Methods), were used for confirming Pc homozygosity of the genome-edited calves (Nature Biotechnology 34, 479–481, 2016). Neither primer set detected the template plasmid integration by gel electrophoresis or Sanger sequencing of the TOPO-cloned amplicon. The btHP primers overlapped Pc, while the HP1748 primers flanked Pc. In HP1748, the forward primer was 154-bp upstream of the start of the repair template, and the reverse primer at the end of the repair template. For both primer sets, amplicons containing the plasmid would be prohibitively large (6,098-bp and 7,255-bp, respectively). With regards to the second copy of the repair template, the doubled amount of Pc amplicons for primer set btHP (591-bp) would be difficult to detect without a quantitative method, and the HP1748 primer set does not amplify the second copy because the HP1748 forward primer was upstream of the repair template.

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Supplementary Figs. 1–3 and Data 1.

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Norris, A.L., Lee, S.S., Greenlees, K.J. et al. Template plasmid integration in germline genome-edited cattle. Nat Biotechnol 38, 163–164 (2020). https://doi.org/10.1038/s41587-019-0394-6

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