Chapter 4 —— 83 —— Aldrich). The procedure for isolating individual mCherry-positive human myoblast clones was similar to that applied to HeLa cells except that heatinactivated FBS was used instead. After 2–3 weeks, single cell-derived clones expressing reporter proteins were randomly collected for genomic DNA analysis via junction PCR assays using the Phire Tissue Direct PCR Master mix following the manufacturer’s recommendations (Thermo Fisher Scientific; cat. no.: F170L). The compositions of the PCR mixtures and thermocycling parameters used for the clonal screening of HeLa cells are described in the Supplementary Tables S2 and S3; the compositions of the PCR mixtures and thermocycling parameters applied for the clonal screening of human myoblasts are indicated in Supplementary Tables S4 and S5. T7EI genotyping assays Targeted DSB formation in HeLa cells and human myoblasts driven by CRISPR-Cas9 complexes containing DMD-targeting gRNAs was assessed by T7EI assays. In brief, 10 μl of PCR mixtures containing amplicons spanning the DMD target sites were incubated in 15-μl reactions consisting of 5 U of the mismatch-sensing T7EI enzyme (New England Biolabs; cat. no.: M0302) and 1 × NEBuffer 2 (New England Biolabs; cat. no.: B7002S). After incubation at 37℃ for 17 min, the DNA samples underwent electrophoresis through 2% (w/v) agarose gels in 1 × Tris-acetate-EDTA buffer. Ethidium bromide-stained DNA species were subsequently imaged by using a GelDocTM XR + apparatus (Bio-Rad) and the Image Lab 6.0.1 software (BioRad). Amplicon deep sequencing To quantify and characterize DNA cleavage at AAVS1 target sequences and at the CPNE5 and BBOX1 off-target sites of gRNA GS1 [34], hMSCs were transduced with different MOIs of adenovectors encoding high-specificity eC94NLS:GS1 or regular Cas9:GS1 complexes. At 3 days post-transduction, genomic DNA was extracted by using the protocol and reagents from the DNeasy Blood & Tissue Kit (Qiagen; cat. no.: 69506). The resulting DNA
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