Identification of human endogenous retroviruses working as enhancers in cancer cells

Human endogenous retroviruses (HERVs) are a sort of transposable elements. HERVs harbor abundant regulatory elements in their sequences and have a potential to work as enhancers modulating the expressions of the adjacent genes. Some HERV-enhancers are particularly activated in cancer cells and suspected to be associated with cancer progression. To verify the enhancer activity of HERVs on the gene expressions in lung adenocarcinoma cells, we established the HERV-excised A549 cell lines using a CRISPR-Cas9 system and performed RNA sequencing (RNA-Seq) analysis of these cells. First, an A549 cell line stably expressing Cas9 (referred to as A549/Cas9 cell) was established. Second, a pair of guide RNAs that are specific to the 5´- and 3´-flanking regions of the target HERV were co-transfected into A549/Cas9 cells. Through limiting dilution, single cell clones were picked up and screened by genotyping PCR. The resultant clones in which the targeted HERV was excised homozygously were obtained. As a control, the A549/Cas9 cells that were transfected with non-target control guide RNA were prepared and cloned as described above. Total RNA was extracted from these cells by QIAamp RNA Blood Mini Kit (QIAGEN #52304) and treated with RNase-Free DNase Set (QIAGEN #79254). Quality check of the purified RNA, library construction, and sequencing were performed by Novogene (https://en.novogene.com). Pair-ended and 150bp read length sequencing was performed by Illumina Novaseq6000.