Journal: Nucleic Acids Research
Article Title: Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants
doi: 10.1093/nar/gkaf037
Figure Lengend Snippet: Repair of 8-oxoG•A using a MUTYH lesion-specific plasmid reporter. ( A ) Schematic diagram of the fluorescent reporter for 8-oxoG•A repair. ( B ) Representative flow cytometry plots and gating schemes to quantify 8-oxoG•A repair in live cells using matching L111P MUTYH mutant cell lines as an example. The plots show compensated red fluorescence intensity ( y -axis) versus compensated EGFP fluorescence intensity ( x -axis) for four representative samples, left to right: unedited HEK293T cells, HEK293T cells that were transfected with the L111P gRNA, but produced no editing at the target site (null clone), heterozygous L111P MUTYH clone 1, and homozygous L111P MUTYH clone 1. 8-oxoG•A repair is quantified by calculating the percent of EGFP+ cells divided by the transfected, or mCherry+, cells. Scatter gates were applied to remove nonviable cells and doublets as shown in . Quadrant boundaries for analysis were set by using unedited HEK293T cells that were transfected with mCherry only or EGFP only plasmids. The numbers in each quadrant represent the percentage of cells within that population. “+’’s in the quadrants indicate the median EGFP fluorescence intensity of EGFP-positive cells. ( C ) Percentage of 8-oxoG•A repair in living cells harboring various MUTYH mutants. Values calculated as described in (B). Bars represent the average of n = 3 biological replicates (circles show clone 1, triangles show clone 2, and squares show clone 3). Error bars represent the standard deviation of the three biological replicates. Data were analyzed with unpaired, one-tailed, parametric t -tests (in which the heterozygous or homozygous lines were compared to their wild-type counterparts), and P- values are marked as follows: ns = P ≥ 0.05, not significant: * P ≤ .05, ** P ≤ .01, and **** P ≤ .0001 are significant.
Article Snippet: Guide RNA (gRNA) plasmids were cloned by site-directed mutagenesis using a 5′ tail in the forward primer to replace the 20 nt spacer region (Basic Protocol 1 [ ]; Streptococcus pyogenes Cas9 gRNA vector Addgene plasmid #47511).
Techniques: Plasmid Preparation, Flow Cytometry, Mutagenesis, Fluorescence, Transfection, Produced, Standard Deviation, One-tailed Test
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