Live-cell quantification of phenotypic changes induced by AdVP-based prime editing

Assessing the phenotypic changes induced by AdVP-based prime editing
Conclusion:
Transduction experiments using a broad range of AdVP.PE2 MOIs (i.e. 500, 1000, 5000 and 10,000 GC/cell) all lead to the acquisition of the EBFP+ phenotype by fractions of reporter cell populations that were quantified by live-cell flow cytometry.
Notes:
Reporter HEK293T.EGFP+ cells stably expressing different amounts of EGFP-targeting pegRNA/gRNA pairs were generated by lentiviral vector transduction at 0.1 and 5.0 TU cell-1 and puromycin selection. The resulting cell populations were transduced with AdVP.PE2 at the indicated MOI and flow cytometry quantified target protein spectral conversions on a per cell basis. Acquisition of the EBFP-positive phenotype (left graph) and loss of the EGFP-positive phenotype (right graph) was determined at 7 days after AdVP.PE2 transductions by flow cytometry. A minimum of 10000 live single-cell events were acquired per sample. Mock cells were used to set fluorescence background thresholds.