Fig 1.
A CRISPR/Cas9-based gene drive mechanism to spread prion disease resistance.
Diagram showing how Cas9/gRNA ribonucleoprotein complexes would induce a DSB within the Prnp ORF that is repaired by HDR in the presence of a donor DNA cassette, leading to the generation of a mobile null Prnp allele capable of gene drive. New abbreviation: GPI, glycophosphatidylinositol.
Fig 2.
Selected Prnp gRNAs induce Cas9-mediated cleavage within the Prnp ORF.
(A) Agarose gel image showing that recCas9 cleaves a half-genomic Prnp expression construct (MoPrP.Xho.wt) when Prnp gRNA–1 is present. A cleavage product of the expected size is indicated by the red arrow. (B) Agarose gel images showing the products of T7E1 mismatch cleavage assay reactions for DNA samples obtained from untransfected WT-5 RK13 cells (Cas9-negative) or cells transfected with eSpCas9(1.1) expression plasmids containing Prnp gRNA–1, –2 or –3, or no gRNA. In the absence of Prnp ORF disruptions, a PCR product of ~900 bp was expected. The faint bands appearing at ~400 and 500 bp only when Prnp gRNAs were expressed indicate that indels resulting from NHEJ events were present.
Fig 3.
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 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.
Fig 4.
Generation of CRISPR/Cas9-induced disruptions of the Prnp ORF in mice.
(A) Sanger sequencing chromatograms for mice derived from fertilized FVB/NJ oocytes that were electroporated with recCas9/Prnp gRNA–3 RNP complexes. The numbers below the automated base calls indicate the position in the sequencing read rather than the Prnp ORF. Changes to the PrPC amino acid sequence for the lines with disrupted Prnp are indicated in the bottom box. (B) Agarose gel image showing the products of T7E1 mismatch cleavage assay reactions for negative (homoduplex) and positive (heteroduplex) control DNA solutions and DNA obtained from the founder of line 34. The presence of the bands indicated by the red arrows confirms that line 34 has a disrupted Prnp allele. (C) Capillary western images confirming that PrPC expression is undetectable in brain homogenates from homozygous line 33 mice. Sha31 and 12B2 are two different PrPC antibodies. New abbreviation: KO, knockout.
Table 1.
Summary of gene editing events.
Fig 5.
Transgenic mice expressing Cas9 under control of the Prl3b1 promoter do not express Cas9 in the germline.
(A–C) Capillary western images showing that Cas9 expression was not detected in male urogenital tract tissues of Prl3b1-eCas9+/–Tg mice (n = 3) derived from founder 4 (A) or in testes and ovaries of mice derived from founders 5 and 19 (B) but was detectable in 15 dpc embryo-placenta homogenates of line 4 (C). The positive control (“ctrl”) was a lysate of RK13 cells transiently expressing Cas9. Caput, corpus and cauda are sections of the epididymis. The embryo-placenta tissues (n = 11) were obtained from two litters resulting from crosses between Prl3b1-eCas9+/–and WT mice (therefore, ~ half of the tissues would be expected to be positive for Cas9).