4.3. Droplet Digital PCR (ddPCR)

The droplet digital PCR was carried out following published protocols [55]. Briefly, sequence-specific PCR primers and probes were designed (Table S2). The extracted genomic DNA was used for ddPCR. The assay consisted of the following components (final concentrations in 20 µL total reaction volume): ddPCR SuperMix for Probes (no dUTP) (1×), forward primer (900 nM), reverse primer (900 nM), reference probe (HDR-insensitive probe, Hex, 250 nM), HDR-sensitive probe (different fluorophore than reference; FAM, 250 nM), nuclease-free water, and ~40 ng gDNA was used as template. All primers and probes were designed using Primer3 plus (http://primer3plus.com, accessed on 1 January 2021) from Eurofns (Eurofns Genomics, Louisville, KY, USA). All ddPCR assays were analyzed using the QX100 droplet reader and Quantasoft software version 1.7.4 (Bio-Rad). Genome editing was calculated according to the ratio based on the concentrations of events per µL, and it was used for the calculation of gene-editing frequency.