Chapter 5 —— 110 —— therapeutic relevance, e.g., stem cells. Further consistent with precise gene editing, direct fluorescence microscopy revealed that mScarlert::LMNA fusion products were present and properly located in cell nuclei exclusively in cultures exposed simultaneously to CRISPR-Cas9 and donor DNA reagents (Figure 3D and Supplementary Figure S3B). Finally, RFLP assays on unsorted cells and on mScarlet-positive and mScarlet-negative cells, further confirmed the strict ouabain-dependent selection of the ATP1A1 RD variant. The strong positive selection for this endogenous marker gene is in fact particularly evident in cultures of mScarlet::LMNA-negative cells treated with ouabain (Figure 3E). Taken together, the above-described experiments demonstrate that combining ouabain with tailored selector AAV vectors achieves a strong elimination of cells with imprecise on-target edits and off-target exogenous DNA insertions from CRISPR-edited cell populations. However, although the co-delivery of selectable ATP1A1 and target donor sequences in single AAV vectors guarantees the presence of both donor templates in individual cells, at low vector doses, bipartite donor availability at primary and secondary loci is expected to become limiting. This consideration is supported by the decreasing ouabain-dependent genome editing frequencies at primary target loci as a function of diminishing AAV vector amounts, i.e., at AAVS1 safe harbour and LMNA loci (Figure 2C and Figure 3B).
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