Integration of a DNA cassette into the Prnp locus of murine neuroblastoma cells by HDR of Cas9-induced DSBs.

(A) Diagram of the donor vector generated for HDR experiments. Prnp homology arms flank a promoter-less reporter gene consisting of the Gfp ORF fused to the 3’ end of the Prnp ORF that would normally encode residues 230–254 of PrP^C. (B–D) Images from agarose gel electrophoresis of junction PCR products showing that co-transfection of N2a cells with the donor vector and eSpCas9(1.1) expression plasmids containing Prnp gRNAs results in integration of the Gfp-GPI transgene into the Prnp locus, at least in some cells. DNA was isolated 3 days (B, C) or 6 days (D) after transfection. “L” and “H” denote low (1:1) and high (5:1) ratios of donor vector to eSpCas9(1.1), respectively. Red arrows indicate the presence of PCR products of the expected sizes. The white line on the bottom part of panel C indicates that irrelevant lanes have been omitted from the image.