Fig. 1.

Enrichment of cells targeted by genome editing tools using the pBSR selection plasmid. a The selection plasmid contains a cassette in which the coding sequences for mCherry and the Blasticidin S resistance gene (bsr) are connected by a linker containing the target sequence (blue) for sgRNA/Cas9. The linker (blue) places the bsr out-of-frame (grey) with the mCherry. Upon cotransfection, the sgRNA/Cas9 targets the plasmid, and the frame shift resulting from the resolution of the double-strand break (red) allows expression of the bsr protein (purple). The acquired transient resistance to Blasticidin S will provide a proxy for the activity of the sgRNA/Cas9 in the cells, thereby allowing the selection of potentially edited cells. Removal of the antibiotic from the culture medium and limiting dilutions will enable to obtain clones targeted by the genome editing tool and devoid of the selection plasmid. b Representative FACS analysis of class switch recombination (CSR) in CH12F3 clones targeted for inactivation of the Aicda gene. The population of cells expressing IgA is gated in the red box. CSR levels could vary among experiments. Normal levels are indicative of those obtained from wild-type CH12F3 clones (typically an IgA(+) population of 40–60%); a CSR efficiency lower than 50% of wild-type CH12F3 was considered reduced; CSR levels lower than 0.1% were considered indicative of lack of CSR (absent). c Comparison of the efficiency of Aicda targeting by pBSR enrichment to other approaches. Samples were transfected with the sgRNA/Cas9 plasmid alone or in combination with a control plasmid or the pBSR. Cells were left untreated, or treated with puromycin (Puro) or Blasticidin S (BlsS). The bar diagram shows the percentage of clones obtained in each CSR efficiency group. The error bars indicate the SEM of at least three independent experiments. There is no statistical difference among the various groups when overall clones with impaired CSR are considered. Enrichment of CSR-absent clones using the pBSR is significantly different from all treatments (vs Cas9, p = 0.0006; vs Cas9(Puro), p = 0.001; vs Cas9 + ctrl(BlsS), p = 0.01). d The status of the Aicda alleles in selected clones from the pBSR sample has been assessed by sequencing of the targeted region. The plot depicts the allele composition of a number of independent clones from each CSR efficiency group. Wild-type (WT) and mutated (Mut) alleles are indicated on the x-axis, while the zygosity of the alleles is indicated by the colour