RNA Sequencing Reveals Transcriptomic Changes in HEK293 Cells Following Introduction of rs16851030 DNA Variant

rs16851030, a single-nucleotide variant located in the 3’-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity, aspirin-induced asthma, and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce physiological changes associated with the DNA variant. rs16851030 was introduced into HEK293 cells through homology-directed repair. Edited cells were then fluorescence-enriched, sorted, isolated, and grown into single cell-derived clones. The single-base edit was confirmed by Sanger sequencing. Finally, RNA sequencing was performed to elucidate the pathways affected by rs16851030. Our study provides valuable information about key pathways associated with rs16851030 DNA variant. Transcriptome analysis was used to detect the mRNA expression profiles of rs16851030-mutant HEK293 cells and wild-type cells. A total of six RNA libraries were constructed from the total RNA isolated from the following two groups of cells: rs16851030-mutant group and wild-type group (three biological replicates in each group). The total RNA of each sample was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions, and the residual DNA was removed using the RapidOut DNA Removal Kit (Thermo Fisher Scientific, USA). After RNA quantification and purity checks, the samples were shipped to GENEWIZ (Suzhou, China) for RNA sequencing and pre-processing of raw sequencing data.