Chapter 4 —— 66 —— disclosed that most genome-modifying events consisted of on-target exogenous DNA insertions resulting from HDR at both termini, with such outcomes being especially prevalent when implementing longer ‘homology arms’ in the vector design (Figure 6D and E). Hence, we set out to further explore the genome-editing specificity and fidelity achieved with the dual viral vector system and, in parallel, probe the role that high-specificity CRISPR-Cas9 complexes have on these parameters. To this end, after exposing hMSCs to AAV-HRS1 and high-specificity or regular CRISPR-Cas9 complexes (i.e. eC94NLS:GS1 or Cas9:GS1, respectively), stably transduced cell populations were sorted by EGFP-directed flow cytometry and subjected to junction PCR analyses diagnostic for on-target and off-target insertions at AAVS1 and CPNE5, respectively (Figure 7A). Regarding the latter locus, previous work from our laboratory tracing off-target DNA cleavage in HEK293T cells through orthogonal high-throughput genome-wide translocation sequencing validated CPNE5 as the top-ranked off-target site for gRNA GS1 [34,35]. Junction PCR analysis detected HDR-mediated AAVS1 gene targeting in hMSCs exposed to AAV-HRS1 together with either eC94NLS:GS1 or regular Cas9:GS1 complexes (Figure 7B). Yet, CPNE5 offtarget insertions were detected exclusively in hMSCs initially exposed to regular Cas9:GS1 complexes (Figure 7C). Moreover, these insertions displayed heterogenous sizes consistent with end-ligation of truncated and/or rearranged AAV vector DNA. Related to the emergence of these byproduct species, as forementioned, it is known that AAV capsids package heterogeneously sized vector DNA that can integrate as such or as multimers. In fact, recent experiments have demonstrated that fragments encompassing AAV ITR sequences contribute to a significant fraction of off-target insertions [63]. Besides insertional mutagenesis, AAV ITR insertions create additional safety concerns in the form of potential deregulation of cellular
RkJQdWJsaXNoZXIy MTk4NDMw