Figure 2.

Simultaneous CRISPR-Cas9 application induces multi-copy deletion of Pmp22-SE. (A) Schematic of the Pmp22 locus in the cell line used for this approach. Pmp22 constitutes six exons, including non-coding alternative first exons 1A and 1B. White regions indicate non-translated portions of exons; black regions indicate translated portions of exons. A modified derivative of the S16 rat Schwann cell line was developed in which one allele of Pmp22 includes a reporter cassette inserted at the end of the last exon of Pmp22 on one of the three copies of the Pmp22 gene. (B) Cells transfected with the CRISPR-Cas9 expression constructs are tested for successful deletion by pairing primers that flank each individual target site (indicated by arrows at positions 1 and 4). The number of copies of chromosome 10 possessing the deletion is evaluated by quantitative PCR of genomic DNA using primers within the deletion (e.g. those at positions 5 and 6) as well as a region outside of the deletion, found 74 kb upstream of the Pmp22 translation start site. (C) PCR amplification of the repair junction was used to detect the deletion in CRISPR-Cas9-transfected unsorted cells and in clones C6, C7 and C8. (D) Evaluation of copy number of the deleted region relative to a nearby segment outside of the deleted region using the Comparative Ct method. Error bars represent the standard deviation of four technical replicates (*P < 0.05).