Table 1.
Quantification of CRISPR-editing efficiency
| Samples | ICE (intron 5) |
ICE (intron 8) |
dPCR (genomic) |
dRT-PCR (transcript) |
||||||
|---|---|---|---|---|---|---|---|---|---|---|
| % indels | R2 | % indels | R2 | Δ6–8 (%) | CI (%) | Δ6–8 (%) | CI (%) | Δ6–9 (%) | CI (%) | |
| Untreated control | Ref | N/A | Ref | N/A | 0.006 | 0.003–0.044 | 0.002 | 0.0002–0.0091 | 0.002 | 0.0002–0.0093 |
| WT control | 0 | 1 | – | – | 0.012 | 0.011–0.036 | 0.002 | 0.0002–0.0086 | 0.005 | 0.0014–0.0140 |
| CRISPR (D1-S1) | 2 | 1 | 9 | 0.99 | 1.419 | 1.183–1.698 | 1.443 | 1.2950–1.5990 | 3.222 | 2.9630–3.4890 |
| CRISPR (D1-S2) | 1 | 1 | 6 | 1 | 0.981 | 0.796–1.204 | 0.654 | 0.5570–0.7620 | 2.320 | 2.0770–2.5710 |
| CRISPRa (D2-S1) | 1 | 1 | 1 | 1 | 0.089 | 0.051–0.153 | 0.067 | 0.0458–0.0974 | 0.139 | 0.1060–0.1810 |
| CRISPR (D2-S2) | 1 | 1 | 8 | 1 | 1.537 | 1.326–1.776 | 1.306 | 1.1650–1.4550 | 3.261 | 2.9930–3.5380 |
| μDys5 (D1-S1) | 0 | 1 | 0 | 1 | 0.006 | 0.001–0.033 | 0.002 | 0.0004–0.0134 | – | – |
| μDys5 (D1-S2) | – | – | – | – | 0.055 | 0.028–0.109 | 0 | – | – | – |
| μDys5 (D2-S1) | – | – | – | – | 0.016 | 0.004–0.057 | 0.006 | 0.0021–0.0164 | – | – |
| μDys5 (D2-S2) | – | – | – | – | 0.007 | 0.001–0.042 | 0 | – | – | – |
Quantification of editing frequency in CRISPR- versus μDys-treated muscle samples (S1-2) acquired from the 3-year-old dog (D1) and the 8-year-old dog (D2), as presented in Figure 4. ICE analysis reveals estimated cutting frequency at the individual genomic target sites within introns 5 and 8 and where the coefficient of determination (R2) represents the fit of the indel distribution to the Sanger sequences of the edited versus untreated negative control sample for ICE analyses. Note: ICE analysis of amplicons generated across the individual target sites is possible only for genomes where gene editing failed to remove the targeted genomic region. Digital PCR [dPCR (genomic)] and dRT-PCR (transcript) columns represent percent Δ6–8 deletion genomes per total genomes and percent Δ6–8 or Δ6–9 transcripts per total dystrophin transcripts, respectively. Confidence intervals (CI) for individual sample levels of corrected genomes and transcripts following dPCR and dRT-PCR are generated as part of the automated analysis performed using the Thermo Fisher Scientific Cloud-based dPCR analysis platform. CI, confidence interval; Ref, reference; N/A, not applicable.
CRISPR-treated muscle sample exhibiting near-undetectable gene editing in Figure 4.