Transcriptomic analysis of KMT5A-knockout healthy volunteer peripheral blood CD8+ T cells under activated conditions

This study aims to investigate the role of Lysine Methyltransferase 5A (KMT5A, also known as SETD8) in CD8+ T cell biology. CD8+ T cells were isolated from healthy volunteer PBMCs using magnetic bead-based negative selection. Isolated cells were subjected to CRISPR-Cas9 mediated gene editing targeting KMT5A (KMT5A-KO group) or a non-targeting control guide RNA (control group, CTRL) via ribonucleoprotein (RNP) complex electroporation. Cells were harvested at 72 hours post-electroporation for RNA extraction. RNA sequencing (RNA-seq) libraries were constructed using the Illumina TruSeq Stranded mRNA kit and sequenced on an Illumina platform to generate paired-end reads. Transcriptome profiles of KMT5A-KO CD8+ T cells were compared to those of CTRL cells to identify differentially expressed genes (DEGs) and elucidate potential pathways regulated by KMT5A in this cell type. Overall design: This study employed a comparative transcriptomic approach to assess the effects of KMT5A gene knockout on CD8+ T cells. The primary comparison is between CD8+ T cells edited with KMT5A-targeting sgRNA RNP (KMT5A-KO group) and CD8+ T cells edited with non-targeting control sgRNA RNP (CTRL group). All samples were derived from CD8+ T cells isolated from healthy volunteer PBMC and processed in parallel.