Chapter 4 —— 59 —— arms’); black boxes, EF1α and U6 gene promoters driving mCherry and gRNA GIN4 expression, respectively. The elements of the junction PCR assays diagnostic for HDRmediated gene tagging are also depicted. (B, C) Testing DMD gene tagging in human myoblasts. Human myoblasts were co-transduced with AdVP.C9KARA and AAVHRDMDGIN4 at the indicated MOI. The initial and stable transduction levels, determined by flow cytometry at 3 days and 20 days post-transduction, are plotted in panel (B) and (C), respectively. Mock-transduced myoblasts and myoblasts transduced with AAVHRDMDGIN4 alone served as controls. Significant differences between the marked datasets were assessed with one-way ANOVA followed by Tukey’s test for multiple comparisons; **P < 0.01; ***P < 0.001. Bars and error bars represent mean and SD values, respectively, of three biological replicates. Numerals above the graph bars correspond to AAV stable transduction frequencies (mean ± SD) normalized to the initial transduction levels. (D) Diagrammatic representation of the LMNA gene tagging strategy. (E) Assessing LMNA gene tagging in HeLa cells. HeLa cells were co-transduced with AdVP.C9KARA and AAVHRLMNAGEX1 at the indicated MOI. The gene tagging frequencies were determined by flow cytometry at 3- and 14-days post-transduction. Bars and error bars correspond to mean and SD values, respectively, of three biological replicates. (F) Direct fluorescence microscopy analysis of LMNA-tagged cells. Representative fluorescence microscopy images of HeLa cells exposed only to AAV-HRLMNAGEX1 at 4000 GC cell−1 or to this donor and AdVP.C9KARA at 4000 GC cell−1 and 4 GC cell−1, respectively. HeLa cell nuclei containing reporter-tagged LMNA proteins were visualized via direct fluorescence microscopy for mScarlet-I and the DNA dye Hoechst 33342. We next sought to exploit the dual viral vector genome-editing platform to investigate the performance of different AAV donor designs (i.e. HR- versus HMEJ-prone) and structures (i.e. single-stranded versus double-stranded). To this end, AAV-HRS1 and AAV-HMEJS1 were first combined with AdVP.eC94NLSGS1 for introducing AAVS1-tailored HR and HMEJ donors into hMSCs, respectively. CRISPR-Cas9-dependent stable transduction frequencies measured upon sub-culturing of co-transduced hMSCs were similar in cultures initially exposed to AAV-HRS1 or AAV-HMEJS1 (Figure 5A). In contrast, it is well-established that amongst HR and HMEJ substrates placed in plasmid or adenovector double-stranded DNA, genome editing frequencies are typically higher when using the latter ‘double-cut’ donor
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