pone.0269342.g003.tif (245.73 kB)

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

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posted on 2022-06-07, 17:24 authored by Andrew R. Castle, Serene Wohlgemuth, Luis Arce, David Westaway

(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 PrPC. (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.