Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection
Fig 1
Targeting the locus of the cell surface protein CD46 in primary human B cells.
(A) Schematic overview of the delivery of CD46-Cas9 RNP complexes to primary human B cells by nucleofection. 2×106 primary human B cells were nucleofected with RNP complexes assembled with a two-component guide RNA (gRNA) protocol using tracrRNA, crRNA and recombinant Streptococcus pyogenes Cas9 nuclease. The cells recovered for 1 h after nucleofection before they were infected with wild-type (WT) EBV. The efficiency of gene editing was analyzed at the level of nucleotide sequence and surface protein. (B) Indel frequencies derived from next generation sequencing analysis of the second exon of the CD46 gene are shown together with flow cytometry analysis of CD46 protein expression from all investigated samples. Cells were nucleofected with individual Cas9 RNP complexes (gRNA1 or 2) or concomitantly with both complexes and were analyzed 1 week later. Mean and standard deviation from four independent biological replicates are shown. (C) Flow cytometry analysis of CD46 surface staining of living cells treated with two RNP complexes 1 week post infection.