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Template plasmid integration in germline genome-edited cattle

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

The Original Article was published on 06 May 2016

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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:

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  1. Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  Google Scholar 

  2. Cameron, P. et al. Mapping the genomic landscape of CRISPR–Cas9 cleavage. Nat. Methods 14, 600–606 (2017).

    Article  CAS  Google Scholar 

  3. Bolger, A.M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

    Article  CAS  Google Scholar 

  4. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    Article  CAS  Google Scholar 

  5. Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).

    Article  CAS  Google Scholar 

  6. Robinson, J. T. et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

    Article  CAS  Google Scholar 

  7. Carlson, D. F. et al. Production of hornless dairy cattle from genome-edited cell lines. Nat. Biotechnol. 34, 479–481 (2016).

    Article  CAS  Google Scholar 

  8. Tan, W. et al. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proc. Natl Acad. Sci. USA 110, 16526–16531 (2013).

    Article  CAS  Google Scholar 

  9. Medugorac, I. et al. Bovine polledness—an autosomal dominant trait with allelic heterogeneity. PLoS ONE 7, e39477 (2012).

    Article  CAS  Google Scholar 

  10. Olsen, P. A., Gelazauskaite, M., Randol, M. & Krauss, S. Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction. BMC Mol. Bi.ol 11, 35 (2010).

    Article  Google Scholar 

  11. Radecke, S., Radecke, F., Cathomen, T. & Schwarz, K. Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications. Mol. Ther. 18, 743–753 (2010).

    Article  CAS  Google Scholar 

  12. Dickinson, D. J., Ward, J. D., Reiner, D. J. & Goldstein, B. Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination. Nat. Methods 10, 1028–1034 (2013).

    Article  CAS  Google Scholar 

  13. Gutierrez-Triana, J.A. et al. Efficient single-copy HDR by 5′ modified long dsDNA donors. Elife 7, e39468 (2018).

    Article  Google Scholar 

  14. Skryabin, B.V. et al. Pervasive head-to-tail insertions of DNA templates mask desired CRISPR/Cas9-mediated genome editing events. Preprint at bioRxiv (2019).

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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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Authors and Affiliations



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).

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