Chapter 4 —— 65 —— depicted (jT and jC, respectively). (B) Cumulative characterization of genome editing events resulting from AAV donors with different structures and designs. Individual EGFPpositive HeLa cell clones genetically modified via transduction with AdVP.eCas94NLSGS1 and AAV-HRS1, scAAV-HRS1 or scAAV-HMEJS1 were subjected to junction PCR screens targeting HDR-derived telomeric and centromeric junctions (jT and jC, respectively). The frequencies of gene targeting events resulting from HDR at both termini (jT+/jC+) or involving imprecise HDR-independent processes (non-HDR and jT+/jC-), are plotted. The frequencies of clones with off-target insertions (jT-/jC-) are equally plotted. The respective PCR screening data are presented in Supplementary Figure S13. (C) Transient and stable transduction levels as a function of AAV donor input. HeLa cells transduced with a fixed MOI of AdVP.eC94NLSGS1 together with different MOI of AAV-HRL.S1 were monitored through flow cytometry at early and late timepoints post-transduction to quantify initial and stable transduction levels corresponding to DSB-dependent genome editing frequencies. Controls consisted of HeLa cells exposed exclusively to the AAVHRL.S1 dose-range. (D) Characterization of genome editing outcomes using AAV-HRL.S1. Screening of individual EGFP-positive HeLa cell clones genetically modified via transduction with AdVP.eCas94NLSGS1 and AAV-HRL.S1 at 2 TU cell−1 (top panel) or 0.1 TU cell−1 (bottom panel). All randomly selected clones yielded amplicons diagnostic for HDR-derived telomeric and centromeric junctions (jT and jC, respectively). One clone yielded, in addition, an amplicon consistent with imprecise HDR-independent targeted insertion (yellow arrowhead). PCR mixtures with DNA from EGFP-positive cell populations and water provided for positive and negative controls, respectively. EGFP served as an internal control template. Lanes M, GeneRuler DNA Ladder Mix molecular weight marker. (E) Plots summarizing the PCR screening data presented in panel (D). Earlier and recent research has established that AAV vectors yield imprecise genome-editing events at appreciable levels in vitro and in vivo whose origins are consistent with end-joining of vector DNA at on-target genomic positions (‘capture’), including fragment derivatives containing ITR sequences [61–63]. Related to this, it is well-established that, in addition to full-length genomes, AAV particles package heterogeneous species, such as, truncated genomes with ITR sequences [64,65]. Notably, the previous clonal analyses revealed that combining high-specificity CRISPR-Cas9 complexes with singlestranded AAV HR donor templates mostly yields precise HDR-mediated gene targeting at AAVS1 (Figure 6B, D and E). Indeed, these analyses
RkJQdWJsaXNoZXIy MTk4NDMw