Chapter 4 —— 55 —— challenges (e.g. reduced cell survival and tissue engraftment), it offers nonetheless a controlled gene correction setting and, simultaneously, minimizes immune responses directed against vector and gene-editing tool components [56–58]. Hence, to further assess the performance of the dual viral vector genome-editing system, we assembled the AAV donor construct AAV-HRDMDGIN4 for DMD gene knock-in using human muscle progenitors (myoblasts) as target cells (Figure 4A). This AAV donor encodes gRNA GIN4 for site-specific DNA cleavage within DMD intron 4 and it bears a matched HR template for directional HDR-mediated fusion of EGFP to the first four exons of DMD (Figure 4A). The gRNA GIN4 was selected via functional assays in HeLa cells and human myoblasts (Supplementary Figure S9). In addition to EGFP, the donor template in AAV-HRDMDGIN4 also has a constitutively active mCherry expression unit for tracing purposes (Figure 4A). DMD gene editing experiments using AdVP.C9KARA and AAVHRDMDGIN4 resulted in efficient AAV transduction of human myoblasts (Figure 4B, left graph), with the presence of AdVP.C9KARA having a significant enhancing effect on AAV transduction levels (Figure 4B, right graph), as previously observed in HeLa cells and hMSCs (Figure 2). Importantly, CRISPR-Cas9-dependent stable transduction frequencies of up to 52.6 ± 6.1% were reached in initially co-transduced myoblast populations (Figure 4C). A multiplex RT-PCR assay demonstrated the co-expression of EGFP-tagged and unmodified DMD transcripts in myotubes differentiated from myoblasts engineered via AAV-HRDMDGIN4 and AdVP.C9KARA cotransduction (Supplementary Figure S10A). Moreover, Sanger sequencing confirmed the accurate assembly of mRNA fusion transcripts in edited myotubes corresponding to the EGFP sequence linked to the first four DMD exons (Supplementary Figure S10B). In addition to the efficiency, the specificity and precision of donor DNA insertion are other parameters of paramount importance associated with programmable nuclease-assisted gene knock-in procedures. The specificity is established by detecting donor DNA sequences at the target site whilst the
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