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.

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).

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.