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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

Virally cleansing the pig genome

Transplants from pigs could be a solution to a shortage of human organs for transplantation. Unfortunately, porcine endogenous retroviruses (PERVs) are rife in pigs and can be transmitted to humans, risking disease. L. Yang et al. integrated CRISPR-Cas into the pig cell genome, where continuous induction of the Cas9 editing enzyme resulted in the mutation of every single PERV reverse transcriptase gene. This prevented replication of all copies of PERV, viral infection, and transmission to human cells.

Science, this issue p. 1101

Abstract

The shortage of organs for transplantation is a major barrier to the treatment of organ failure. Although porcine organs are considered promising, their use has been checked by concerns about the transmission of porcine endogenous retroviruses (PERVs) to humans. Here we describe the eradication of all PERVs in a porcine kidney epithelial cell line (PK15). We first determined the PK15 PERV copy number to be 62. Using CRISPR-Cas9, we disrupted all copies of the PERV pol gene and demonstrated a >1000-fold reduction in PERV transmission to human cells, using our engineered cells. Our study shows that CRISPR-Cas9 multiplexability can be as high as 62 and demonstrates the possibility that PERVs can be inactivated for clinical application of porcine-to-human xenotransplantation.

Pig genomes contain from a few to several dozen copies of PERV elements (1). Unlike other zoonotic pathogens, PERVs cannot be eliminated by biosecure breeding (2). Prior strategies for reducing the risk of PERV transmission to humans have included small interfering RNAs (RNAi), vaccines (35), and PERV elimination using zinc finger nucleases (6) or TAL effector nucleases (7), but these have had limited success. Here we report the successful use of the CRISPR-Cas9 RNA-guided nuclease system (810) to inactivate all copies of the PERV pol gene and effect a 1000-fold reduction of PERV infectivity of human cells

To design Cas9 guide RNAs (gRNAs) that specifically target PERVs, we analyzed the sequences of publicly available PERVs and other endogenous retroviruses in pigs (methods). Using droplet digital polymerase chain reaction (PCR), we identified a distinct clade of PERV elements (Fig. 1A) and determined that there were 62 copies of PERVs in PK15 cells (a porcine kidney epithelial cell line) (Fig. 1B). We then designed two Cas9 gRNAs that targeted the highly conserved catalytic center (11) of the pol gene on PERVs (Fig. 1C and fig. S1). The pol gene product functions as a reverse transcriptase (RT) and is thus essential for viral replication and infection. We determined that these gRNAs targeted all PERVs but no other endogenous retrovirus or other sequences in the pig genome (methods).

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.

Initial experiments showed inefficient PERV editing when Cas9 and the gRNAs were transiently transfected (fig. S2). Thus, we used a PiggyBac transposon (12) system to deliver a doxycycline-inducible Cas9 and the two gRNAs into the genome of PK15 cells (figs. S2 and S3). Continuous induction of Cas9 led to increased targeting frequency of the PERVs (fig. S5), with a maximum targeting frequency of 37% (~23 PERV copies per genome) observed on day 17 (fig. S5). Neither higher concentrations of doxycycline nor prolonged incubation increased targeting efficiency (figs. S4 and S5), possibly because of the toxicity of nonspecific DNA damage by CRISPR-Cas9. Similar trends were observed when Cas9 was delivered using lentiviral constructs (fig. S6). We then genotyped the cell lines that exhibited maximal PERV targeting efficiencies. We observed 455 different insertion and deletion (indel) events centered at the two gRNA target sites (Fig. 2B). Indel sizes ranged from 1 to 148 base pairs (bp); 80% of indels were small deletions (<9 bp). We validated the initial deep sequencing results with Sanger Sequencing (fig. S7).

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.

We next sorted single cells from PK15 cells with high PERV targeting efficiency using flow cytometry, and we genotyped the pol locus of the resulting clones via deep sequencing (13, 14). A repeatable bimodal (Fig. 2A and figs. S8 and S9) distribution was observed, with ~10% of the clones exhibiting high levels of PERV disruption (97 to 100%) and the remaining clones exhibiting low levels of editing (<10%). We then examined individual indel events in the genomes of these clones (Fig. 2B and figs. S10 and S11). For the highly edited clones (clone 20, 100%; clone 15, 100%; clone 29, 100%; clone 38, 97.37%), we observed only 16 to 20 unique indel patterns in each clone (Fig. 2B and fig. S11). In addition, there was a much higher degree of repetition of indels within each clone than across the clones (fig. S25), suggesting a mechanism of gene conversion in which previously mutated PERV copies were used as templates to repair wild-type PERVs cleaved by Cas9 (Fig. 2B and fig. S25). Mathematical modeling of DNA repair during PERV elimination (fig. S26) and analysis of expression data (figs. S22 to S24) supported this hypothesis and suggested that highly edited clones were derived from cells in which Cas9 and the gRNAs were highly expressed.

Next, we examined whether unexpected genomic rearrangements had occurred as a result of the multiplexed genome editing. Karyotyping of individual modified clones (figs. S12 to S14) indicated that there were no observable genomic rearrangements. We also examined 11 independent genomic loci with at most 2 bp mismatches to each of the intended gRNA targets and observed no nonspecific mutations (fig. S27). This suggests that our multiplexed Cas9-based genome engineering strategy did not cause catastrophic genomic instability.

Last, we examined whether our disruption of all copies of PERV pol in the pig genome could eliminate in vitro transmission of PERVs from pig to human cells. We could not detect RT activity in the cell culture supernatant of the highly modified PK15 clones (fig. S15), suggesting that modified cells only produced minimal amounts of PERV particles. We then cocultured wild-type (WT) and highly modified PK15 cells with human embryonic kidney (HEK) 293 cells to test directly for transmission of PERV DNA to human cells (15). After coculturing PK15 WT and HEK 293 cells for 5 days and 7 days (figs. S16 to S17), we detected PERV pol, gag, and env sequences in the HEK 293 cells (Fig. 3A). We estimated the frequency of PERV infection to be approximately 1000 PERVs per 1000 human cells (Fig. 3B). However, PK15 clones with >97% PERV pol targeting exhibited up to 1000-fold reduction of PERV infection, producing results that were similar to background levels (Fig. 3C). We validated these results with PCR amplification of serial dilutions of HEK 293 cells that had a history of contact with PK15 clones (Fig. 3D and figs. S18 to S21). We could consistently detect PERVs in single HEK 293 cells isolated from the population cocultured with minimally modified clone 40, but we could not distinctly detect PERVs in 100 human cells from the population cocultured with highly modified clone 20. Thus, we concluded that the PERV infectivity of the engineered PK15 cells had been reduced by up to 1000-fold.

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).

We successfully targeted the 62 copies of PERV pol in PK15 cells and demonstrated greatly reduced in vitro transmission of PERVs to human cells. Although in vivo PERV transmission to humans has not been demonstrated (16, 17), PERVs are still considered risky (18, 19), and our strategy could completely eliminate this liability. Because no porcine embryonic stem cells exist, this system will need to be recapitulated in primary porcine cells and cloned into animals by means of somatic cell nuclear transfer. Moreover, we achieved simultaneous Cas9 targeting of 62 loci in single pig cells without salient genomic rearrangement. To our knowledge, the maximum number of genomic sites previously reported to be simultaneously edited has been six (20). Our methods thus open the possibility of editing other repetitive regions of biological significance.

Supplementary Materials

REFERENCES AND NOTES

  1. ACKNOWLEDGMENTS: We thank Z. Herbert and M. Vangala at the Dana-Farber Genomics Core Facility for assistance with RNA analysis, Y. Shen and O. Castanon for volunteering in laboratory research, and S. Broder for scientific advice. M.G. is funded by a Human Frontier Science Program Long Term fellowship. This study was funded by NIH grant P50 HG005550. L.Y., G.C., and M.G. are inventors on a patent filed by Harvard University that covers the work in this manuscript. L.Y., G.C., and M.G. are founding members of eGenesisBio. Pig cells are available from G.C. under a materials transfer agreement with Harvard University.
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