Chapter 4 —— 78 —— and a primer pair specific for the mScarlet-I reporter (i.e. 5′- CTACCTGGCGGACTTCAAGA-3′ and 5′-ACGGTGTAGTCCTCGTTGT G-3′). In brief, HeLa cells seeded one day before at a density of 8.5 × 104 cells per well of 24-well plates (Greinder Bio-One), were incubated with seven 3fold serial dilutions of the purified AAV stock. Genomic DNA isolated at 24 h post-transduction with the DNeasy Blood & Tissue kit was subsequently used for the qPCR analysis. In parallel, eight serial 10-fold dilutions of the linearized parental AAV construct containing 1 × 107 GC ml-1 were used for qPCR standard curve generation. Data analysis was performed with the BioRad CFX Manager 3.1 software (Bio-Rad Laboratories) and the titer determined based on the AAV DNA and plasmid standard curve Ct values. Selection of DMD-targeting gRNA The screens to identify a gRNA for AAV donor DNA targeting DMD in human myoblasts was done via transient transfections and viral vector transductions on HeLa cells and human myoblasts, respectively. The transient transfections started by seeding 4 × 104 HeLa cells per well of 24-well plates and, the next day, the cells were exposed to complexes formed by incubating 150 mM NaCl solutions containing 1.1 μl of PEI (1 mg ml-1) and 300 ng of plasmid mixtures at 1:1 ratio. The later mixtures consisted of 218.5 ng of AV50_pCAG.eSpCas9.bGHpA encoding the high-specificity eSpCas9(1.1) nuclease [29] and 81.5 ng of each of the test gRNA constructs, i.e. BC52_pgRNADMD.IN3.1, BC53_pgRNADMD.IN3.2, BC54_pgRNADMD.IN3.3, BC55_pgRNADMD.IN4.1 and BC56_pgRNADMD.IN4.2 (Supplementary information). As controls, previously validated and non-targeting gRNA constructs, i.e. AY60_pgRNAAAVS1-T2 [30] and AM51_pgNT [31], respectively, provided for positive and negative controls, respectively. All gRNAs contain an optimized scaffold [32] derived from the gRNA acceptor construct AY56_pUC.U6.opt-sgRNA.BveI-stuffer [30]. At 2 days posttransfection, genomic DNA was isolated with the DNeasy Blood & Tissue Kit (Qiagen; cat. no.: 69506) and targeted DSB formation was assessed via
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