CRISPR/Cas9-mediated ApoE-/- and LDLR-/- double gene knockout in pigs elevates serum LDL-C and TC levels

Figures

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  Figure 1. Design and CRISPR/Cas9 assay (A, B) Schematic and sequencing chromatography of genomic DNA of the CRISPR/Cas9-targeting sites in pig APOE (A) and LDLR (B) genes. Exons are shown as boxes. The black arrow indicates the direction of transcription. The gRNA-targeting sequence is labeled in red, and the protospacer-adjacent motif (PAM) sequence is labeled in green. The different gRNAs are named as gRNA 1-4, respectively. Sequencing chromatography of genomic DNA of ApoE (A) and LDLR (B) genes showd a double curve after the guided double strand break induced by the CRISPR/Cas9. (C) Agarose gel electrophoresis showing the PCR product of the target region derived from PEF cell lines digested with T7EN1 restriction enzymes. WT:wild type; g1:gRNA1; g3:gRNA3.

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  Figure 2. Mutations of APOE and LDLR genesis by CRISPR/Cas9 Detailed mutations of the modified alleles detected in PEF colonies. gRNA binding sites of APOE and LDLR gene are underlined; “Δ”: deletion; “+”: insertion; WT: wild-type allele.

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  Figure 3. ApoE/LDLR double-mutant cell colonies for SCNT The chromatogram for a partial genomic sequence of ApoE/LDLR double mutant PEF colonies. Sequencing was used to verify insertions or deletions produced by the non-homologous end joining repair pathway after the double strand break induced by the CRISPR/Cas9. gRNA binding sites are underlined; “Δ”: deletion; “+”: insertion; WT: wild-type allele of ApoE and LDLR gene, respectively.

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  Figure 4. ApoE/LDLR knockout pigs and serum biochemical status (A) Photographs and genotypes of the six ApoE-/-/LDLR-/- founder pigs. (B) Serum TC, TG, LDL-C and HDL-C in male founders and WT males. The data comprise fasting serum measurements at two-months and twelve-months of age in founders and age-matched WT pigs. (C) Serum APOB and ApoE levels at two-months and twelve-months of age. The data comprise fasting serum measurements at two-months and twelve-months of age in founders and age-matched WT pigs. * P<0.05, ** P<0.01.