DNA analysis

Genomic DNA was extracted with QIAamp DNA Micro Kit, AllPrep DNA/RNA 96 Kit (Qiagen, Hilden, Germany) or QuickExtract DNA Extraction Solution (Lucigen, Middleton, WI).

Fifty nanograms of genomic DNA were used to amplify the region that spans the cutting site of each gRNA using KAPA2G Fast ReadyMix (Kapa Biosystem, Wilmington, MA). After Sanger sequencing (Genewiz, Takeley, United Kingdom), the percentage of insertions and deletions (InDels) was calculated using TIDE³¹ and ICE (Synthego) softwares. See supplemental Table 2 for primer sequences.

Digital droplet PCR (ddPCR) was performed according to manufacturer’s instruction using ddPCR Supermix for Probes No dUTP (BioRad, Hercules, CA) and 1 to 50 ng of genomic DNA digested with HindIII (New England Biolabs, Ipswich, MA). Droplets were generated using AutoDG Droplet Generator and analyzed with a QX200 Droplet Reader; data analysis was performed with QuantaSoft (BioRad).

To quantify HBA2 copy number, primers and probes were designed on the 3′UTR of HBA2 gene because it differs significantly from HBA1.

To quantify on-target transgene integration events, primers and probes were designed spanning the donor DNA-genome 3′ junction. Human albumin (ALB) or ZNF843 genes were used as reference for copy number evaluation (assay ID: dHsaCP2506312, BioRad). See supplemental Table 2 for primer and probe sequences.