CRISPR-mediated deletion of Prdm12 alters H3K9me3 landscape in CD8+ T cells: a CUT&Tag study

In this study, we used magnetic bead-based separation to isolate CD8+ T cells from OTI:Cas9 transgenic mice. These cells were then transfected with Cas9 ribonucleoprotein (RNP) targeting the Prdm12 gene or non-targeting control RNP via electroporation. After 72 hours, both edited (Prdm12 knockout) and control cells were collected for chromatin profiling of histone modification H3K9me3 using CUT&Tag sequencing. The aim of this study is to investigate how Prdm12 regulates the epigenetic landscape, particularly H3K9me3 distribution, in CD8+ T cells. Overall design: CD8+ T cells were isolated from OTI:Cas9 transgenic mice using magnetic bead separation. Cells were divided into two groups: one group was transfected with Cas9 RNP targeting Prdm12, while the other received non-targeting control RNP via electroporation. Following 72 hours of recovery, genomic editing efficiency was confirmed by T7E1 assay and/or Sanger sequencing. Both Prdm12-edited and control cells were subjected to CUT&Tag analysis using an antibody specific for H3K9me3. Libraries were prepared according to standard protocols and sequenced on an Illumina platform (e.g., NovaSeq 6000). Sequencing data were analyzed to identify genome-wide changes in H3K9me3 occupancy associated with Prdm12 loss.