Construction and characterization of ribonuclease H2 knockout NIH3T3 cells

Ribonuclease H (RNase H) specifically hydrolyzes the 5’-phosphodiester bonds of the RNA of RNA/DNA hybrid. Both types 1 and 2 RNases H act on the RNA strand of the hybrid, while only type 2 acts on the single ribonucleotide embedded in DNA duplex. In this study, to explore the role of mammalian type 2 RNase H (RNase H2) in cells, we constructed the RNase H2 knockout NIH3T3 cells (KO cells) by CRISPR/Cas9 system. KO cells hydrolyzed RNA strands in RNA/DNA hybrid, but not single ribonucleotides in DNA duplex, while wild-type NIH3T3 cells (WT cells) hydrolyzed both. Genomic DNA in the KO cells was more heavily hydrolyzed than in the WT cells by the alkaline or RNase H2 treatment, suggesting that the KO cells contained more ribonucleotides in genomic DNA than the WT cells. The growth rate of the KO cells was 60% of that of the WT cells. Expression of interferon-stimulated genes (ISGs) in the KO cells was not markedly elevated compared with the WT cells. These results suggest that in NIH3T3 cells, RNase H2 is crucial for suppressing the accumulation of ribonucleotides in genomic DNA but not for the expression of ISGs.