Figure 6. CRISPR/Cas-mediated RNASEH2A knockout induces DNA hypomethylation.
(A) Western blot using an anti-RNase H2A antibody shows successful gene knockout in HeLa-GFP cells. Scramble cells were generated with a CRISPR vector carrying a scrambled guide RNA sequence. Tubulin was used as a loading control. (B) Immunocytochemistry confirms elevated DNA damage response in RNASEH2A KO cells; DAPI (blue), gamma H2AX antibody (green). (C) Bright field and green fluorescent protein (GFP) microscopy images of RNASEH2A KO and scramble control cells. Knockout of the RNASEH2A gene triggers the reactivation of the silent GFP reporter in HeLa-GFP cells. (D) (Top) Western blot showing GFP expression in HeLa-GFP cells (RNASEH2A KO and scramble); (bottom) bar graph quantification of Western blot. (E) Bisulfite methylation sequencing for two different LINE-1 loci in RNASEH2A KO and scramble control cells. Black and white circles represent methylated and unmethylated CpG sites, respectively. Missing bubbles indicate that a CpG site is absent from the sequence of that particular molecule. The coordinates for each fragment analyzed are indicated at top. (F) Quantification of percent methylation at each CpG sites surveyed in panel E. p-value was calculated using a paired Wilcoxon test with the alternative hypothesis that RNASEH2A KO is less methylated than scramble.