CRISPR-Cas9 mediated editing of the CD46 gene in primary human B cells prior to their infection with Epstein-Barr virus (EBV) – analysis of CD46 transcripts after 24 hours

Adenoid B cells from anonymous human donors were isolated and nucleofected with RNP complexes targeting two loci within exon 2 of the CD46 gene, which encodes a cell surface protein. One hour after nucleofection, the same number of control cells (w/o-RNP) and CD46-RNP complex treated cells were infected with Epstein-Barr virus (strain 6008_B95-8) with a multiplicity of infection of 0.1. Newly transcribed RNAs were metabolically labeled with 4-thiouridine (4sU) for 60 min 23 hours after EBV infection. Twenty-four hours after the start of the experiment, 4sU-biotin-labeled, newly transcribed RNAs were thiol-specifically biotinylated and separated from unlabeled RNA using Streptavidin beads. RNA-seq experiments with the enriched RNA samples were performed to analyze the edited CD46 locus encompassing the RNP complex target sites. The experiment revealed that 24 hours after CRISPR-Cas9 mediated gene editing a substantial fraction of CD46 transcript contained indels and a larger deletion in between the two targets sites within exon 2 of CD46.