Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

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Science  27 Nov 2015:
Vol. 350, Issue 6264, pp. 1101-1104
DOI: 10.1126/science.aad1191
  • Fig. 1 CRISPR-Cas9 gRNAs were designed to specifically target the pol gene in 62 copies of PERVs in PK15 cells.

    (A) Phylogenetic tree representing endogenous retroviruses present in the pig genome. PERVs are highlighted in blue. (B) Copy number determination of PERVs in PK15 cells via digital droplet PCR. The copy number of pol elements was estimated to be 62, using three independent reference genes: ACTB, GAPDH, and EB2. n = 3 independent reference genes, mean ± SEM. (C) We designed two CRISPR-Cas9 gRNAs to target the catalytic region of the PERV pol gene. The two gRNA targeting sequences are shown below a schematic of PERV gene structure. Their PAM (protospacer adjacent motif) sequences are highlighted in red.

  • Fig. 2 Clonal PK15 cells with inactivation of all copies of PERV pol genes after Cas9 treatment.

    (A) A bimodal distribution of pol targeting efficiencies was observed among the single-cell–derived PK15 clones after 17 days of Cas9 induction. 45 out of 50 exhibited <16% targeting efficiency; 5 out of 50 clones exhibited >93% targeting efficiency. (B) PK15 haplotypes at PERV pol loci after CRISPR-Cas9 treatment. Indel events in the PERV pol sequence are represented in red. Shades of purple indicate endogenous PERVs.

  • Fig. 3 PK15 cells with all PERV pol genes inactivated lose the infection capacity of human HEK 293 cell lines.

    (A) Detection of PERV pol, gag, and env DNA in the genomes of HEK 293–green fluorescent protein (GFP) cells after coculturing with PK15 cells for 5 and 7 days (293G5D and 393G7D, respectively). A pig GGTA1 primer set was used to detect pig cell contamination of the purified human cells. (B) Quantitative PCR (qPCR) analysis of the number of PERV elements in 1000 293G cells derived from a population cocultured with WT PK15 cells using specific primer sets (n = 3 qPCR experiment replicates, mean ± SEM). (C) qPCR quantification of the number of PERV elements in PK15 clones 15, 20, 29, and 38, with high levels of PERV pol modification and minimally modified clones 40 and 41 (n = 3 qPCR experiment replicates, mean ± SEM). (D) Results of PCR on PERV pol on genomic DNA from various numbers of HEK 293–GFP cells (0.1, 1, 10, and 100) isolated from populations previously cultured with highly modified PK15 clone 20 and minimally modified clone 40 (see figs. S18 to S21 for a full panel of PCR reactions).

Supplementary Materials

  • Genome-wide inactivation of porcine endogenous retroviruses (PERVs)

    Luhan Yang, Marc Güell, Dong Niu, Haydy George, Emal Lesha, Dennis Grishin, John Aach, Ellen Shrock, Weihong Xu, Jürgen Poci, Rebeca Cortazio, Robert A Wilkinson, Jay A. Fishman, George Church

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Methods
    • Figs. S1 to S27
    • References

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