Fig. 4: Improvements in gene knock-in with dsDNA donor templates using the double tap method. | Nature Communications

Fig. 4: Improvements in gene knock-in with dsDNA donor templates using the double tap method.

From: Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies

Fig. 4

Selected scatter plots of GFP fluorescence (y-axis) and cell forward scatter (x-axis), showing gating for GFP fluorescence for HEK293T cells transfected with plasmids encoding dsDNA donor template, Cas9, and non-targeting gRNA only (top), primary and non-targeting gRNAs (middle), or primary and secondary gRNAs (bottom) for the ACTB gene (a) and the LMNA gene (b). c Quantification of the percent of cells with GFP fluorescence in the GFP knock-in experiment for the ACTB (top) and LMNA (bottom) genes. NT stands for non-targeting, OG + NT stands for primary with non-targeting, and OG + DT stands for primary and gRNAs. One secondary gRNA was used at both sites. Values on the whisker plots represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs.

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