Chapter 4 —— 53 —— Figure 3. Assessing the role of chromosomal DNA breaks on AAV vector transduction. (A,B) Single-stranded AAV and double-stranded scAAV transduction kinetics in cells subjected to CRISPR-induced DSBs. HeLa cells exposed to Cas9:gRNACALM2 or Cas9:gRNAVEGFA complexes, inducing few or plenty off-target DSBs, respectively, were transduced with vectors AAV-HRS1 or scAAV-HRS1. AAV transgene expression was traced by real-time live-cell imaging for up to 66 h in an Incucyte apparatus. Each datapoint represents mean and SD values of a total of 16 fluorescence intensity measurements derived from 16 independent microscopy fields per well. Significant differences between datapoints corresponding to cells receiving gRNACALM2 versus gRNAVEGFA were calculated with Student’s t-tests; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; P > 0.05 was considered ns. (C) Representative microscopy fields corresponding to the indicated experimental settings at the 66-h timepoint. (D) Establishing faster transduction kinetics of scAAV over regular AAV vectors. HeLa cells were transduced with vectors AAV-HRS1 or scAAV-HRS1 and transgene expression levels were monitored through real-time live-cell imaging for up to 66 h in an Incucyte apparatus. Each datapoint represents mean and SD values of a total of 16 fluorescence intensity measurements derived from 16 independent microscopy fields per well. (E) Representative microscopy fields corresponding to HeLa cells transduced with AAV-HRS1 or scAAV-HRS1 at the 66-h timepoint.
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