Chapter 4 —— 50 —— Figure 2. Genome editing combining AAV delivery of donor and gRNA components with high-capacity AdVP transfer of Cas9. (A) AAV donor transductions targeting AAVS1 safe harbor loci in hMSCs. The structure of AAV-HRL.S1GS1 donor is shown. Horizontal gray bars, DNA homologous to sequences flanking the AAVS1 target site of gRNA GS1 (‘homology arms’). hMSCs were transduced with AAV-HRL.S1GS1 and AdVP.C9KARA at the specified MOI. The former vector encodes the AAVS1-targeting gRNA GS1; the latter vector encodes the high-specificity Cas9 nuclease SpCas9KARA. AAV donor transduction was assessed by measuring EGFP-positive cells and respective MFI values by flow cytometry at 3 days post-transduction (top and bottom graphs, respectively). Mock-transduced hMSCs and hMSCs transduced solely with the AAV donor provided for controls. Data are shown as mean ± SD of three biological replicates. Significant differences amongst the marked datasets were calculated with one-way ANOVA followed by Tukey’s test for multiple comparisons; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (B) Monitoring transient and stable transduction of hMSCs upon AAV-HRL.S1GS1 delivery. Representative live-cell fluorescence microscopy images of hMSCs co-transduced with AAV-HRL.S1GS1 and AdVP.C9KARA at the indicated MOI. Images showing transiently and stably transduced cells were acquired at 2- and 19-days post-transduction, respectively. (C) Quantification of hMSCs stably transduced with AAV-HRL.S1GS1. Stable transduction frequencies were measured via EGFP-directed flow
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