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Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9

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Science  22 Sep 2017:
Vol. 357, Issue 6357, pp. 1303-1307
DOI: 10.1126/science.aan4187
  • Fig. 1 Pig-to-human and human-to-human PERV transmission.

    (A) PERV copy number in infected human cells increases over time when cocultured with PK15 cells. Human HEK293T-GFP cells were cocultured with equivalent numbers of porcine PK15 cells for 1 week. HEK293T-GFP cells that did not have any contact with PK15 cells were used as a negative control (negative). Error bars indicate SD. (B) Detection of PERV insertion sites in the human genome. Among the 22 PERV insertion sites detected by inverse PCR, 15 were mapped to the intragenic region. We tested a portion of the intragenic hits and validated 7 out of 12 by junction PCR (shown here). The 30–base pair human genomic sequences are shown in blue, whereas the PERV long terminal repeats are shown in red. (C) Detection of human-to-human PERV transmission. Individual clones of HEK293T cells were grown from the single cells isolated from the coculture of i-HEK293T-GFP cells with HEK293T cells through flow cytometry. The PCR gel image shows that three out of four randomly tested HEK293T clones were infected and contained PERV sequences (PERV pol, env, and gag) but no sequence of GFP or pig genomic DNA (tested by pig-specific GGTA). Sample order is as follows: (1) HEK293T clone 1; (2) HEK293T clone 2; (3) HEK293T clone 3; (4) HEK293T clone 4; (5) HEK293T-GFP control; (6) i-HEK293T-GFP; (7) PK15 WT; and (8) negative control. (D) Four different i-HEK293T-GFP clones have different infectious potential. Four infected parental i-HEK293T-GFP clones are cocultured with WT HEK293T cells. The PERV copy numbers of the four parental i-HEK293T-GFP clones are 15, 28, 27, and 28, respectively. The percentages of the infected WT HEK293T clones from the coculture of i-HEK293T-GFP and WT HEK293T cells varied from 20 to 97%. Primers used are listed in table S1.

  • Fig. 2 PERVs insertion site mapping and genome-wide inactivation.

    (A) Chromosome mapping of PERV locations in the FFF3 cell line. Chromosomal scaffolds are shown in gray. Red arrows represent PERVs in the forward or positive chain of chromosome; blue arrows denote PERVs in the reverse or negative chain. The y axis represents chromosomal coordinates. Two additional copies were mapped to repetitive regions, and two could not be mapped to the current pig genome assembly and are not shown (11% gaps, Sus scrofa build 10.2) (11). (B) Failure to obtain 100% PERV-inactivated FFF3 clones using CRISPR-Cas9. After targeting the PERVs in FFF3, single cells were sorted and immediately genotyped. We observed a bimodal distribution of PERV targeting frequencies among single cells (top), similar to that seen for the PK15 clones (5). 100% PERV-inactivated FFF3 cells were present among the single cells that we directly genotyped. However, this pattern changed after expansion of the single cells (bottom). Among the single-cell clones, we only obtained those with lower efficiency (≤39%; the average targeting efficiency in the population was 37%), not the ones with 100% PERV inactivation (bottom). NHEJ, nonhomologous end joining. (C) Treatment with PFTα and bFGF sustained the growth of highly modified FFF3 clones. The combined use of a p53 inhibitor, PFTα, and a growth factor, bFGF, rescued the highly modified cells. A population of FFF3 was treated with PFTα and bFGF during the gene editing experiment (materials and methods), after which single cells were sorted for direct genotyping and colony growth, followed by genotyping. Both the single cells and expanded clones showed similar distribution in PERV targeting efficiency, and highly modified clones survived under this condition. (D) Genotype of 100% PERV-inactivated clones. Several 100% PERV-inactivated clones were achieved from the PFTα- and bFGF-treated FFF3 population. The figure shows haplotypes of one of the 100% PERV-inactivated clones at PERV pol loci after CRISPR-Cas9 treatment. The y axis indicates PERV copy number; the x axis indicates the relative locations of the indels within the PERV loci. Aligned indel events in the PERV pol sequence are shown in red. Shades of purple indicate different haplotypes of PERVs.

  • Fig. 3 PERV-inactivated pigs.

    (A) Image of the first-born PERV-inactivated pig (Laika), 2 days after birth. (B) PERV inactivation at genomic DNA level. We genotyped PERV-inactivated pigs at different ages (up to 100 days) by deep sequencing of the PERV pol loci. All examined pigs showed ~100% PERV inactivation efficiency, which demonstrates that there is no detectable PERV reinfection from surrogate sows to cloned pigs. Blue, 0- to 4-day-old piglets; brown, 29- to 40-day-old piglets; purple, 91- to 100-day-old piglets. (C) PERV inactivation at the mRNA level. Total mRNA-generated cDNA was used to detect the PERV inactivation efficiency of the PERV-knockout pig transcripts. All pigs exhibited ~100% PERV eradication efficiency at the mRNA level. Brown, 1-day-old piglets; blue, 5- to 7-day-old piglets; green, 15- to 19-day-old piglets.

Supplementary Materials

  • Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9

    Dong Niu, Hong-Jiang Wei, Lin Lin, Haydy George, Tao Wang, I-Hsiu Lee, Hong-Ye Zhao, Yong Wang, Yinan Kan, Ellen Shrock, Emal Lesha, Gang Wang, Yonglun Luo, Yubo Qing, Deling Jiao, Heng Zhao, Xiaoyang Zhou, Shouqi Wang, Hong Wei, Marc Güell, George M. Church, Luhan Yang

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

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    • Materials and Methods
    • Figs. S1 to S16
    • Table S1
    • References

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