Chapter 4 —— 69 —— S14). In line with this outcome, no exogenous DNA insertions were detected at CPNE5 (Figure 7C) in the absence of DSB formation at this locus (Figure 7D and Supplementary figure S14). Finally, additional AAV-HRS1 transduction experiments in cells exposed to AAVS1-targeting CRISPR-Cas9 complexes containing gRNA GS1 and either regular or high-specificity Cas9 nucleases or a Cas9D10A nickase (Figure 7E), confirmed the insertion of AAV donor DNA species at CPNE5 in cells receiving regular Cas9:GS1 nuclease complexes (Figure 7F). Taken together, these data stress the importance of selecting high-specificity CRISPR-Cas9 nucleases as opposed to parental Cas9 nucleases for achieving targeted and precise genome editing when using AAV donors. In this study, we introduce a dual viral vector genome-editing system based on the transfer of engineered CRISPR-Cas9 nucleases and donor DNA templates via AdVP and AAV particles, respectively. In common, these delivery agents lack the entire viral-gene complement of their parental viruses allowing for broader dose-ranges than those permitted when using viral genecontaining AdVs. Well-defined complementary attributes of AAV and AdVP systems (e.g. HDR-prone and strictly episomal genomes, respectively), were exploited for achieving selection-free and precise engineering of various human cell types. These experiments involved targeting commonly used safe harbor loci (i.e. AAVS1 and CCR5) and tagging DMD and LMNA alleles whose mutations cause notable human disorders. Specifically, mutations in former gene underpin DMD whilst in latter cause Emery–Dreifuss muscular dystrophy, limb girdle muscular dystrophy and Hutchinson–Gilford progeria syndrome, amongst others. We further exploited this dual viral vector platform to address the knowledge gap regarding the relationship between genome editing endpoints and different AAV donor designs (i.e. conventional HR versus ‘double-cut’ HMEJ templates) and structures (i.e. single-stranded versus double-stranded). These experiments revealed that, in contrast to HMEJ donors delivered in the context of circular plasmid DNA [7–9] or
RkJQdWJsaXNoZXIy MTk4NDMw