Nobody likes a PERV – they just make everyone uncomfortable. Porcine (pig) Endogenous RetroViruses (PERVs) are a creepy concern when it comes xenotransplantation (transplanting organs from a different species), since the perks of not having to wait for an organ transplant take a hit when PERVs get all up in your genome.
In order to take that pesky PERV problem out of your prospective organs, the lab of George Church and eGenesis Biosciences took on a porcine kidney epithelial cell line (PK15):
- They determined the PERV copy number was 62 using digital droplet PCR.
- Using CRISPR-Cas9, two sgRNAs were created to target the PERVs at their conserved pol gene, a reverse transcriptase critical for retroviral activity.
- Editing wasn’t easy, and a number of different delivery approaches were given a go.
- After sorting cells using flow cytometry, they noticed a bimodal distribution of editing success and focused in on the highly-edited cells.
- Lending insight into the mechanism:
- There was a lot of repetition in the specific indels found within a single clone.
- This suggested the repair pathways use successfully mutated PERV copies as templates to repair wild-type sequences that are cut by Cas9.
- A quick check for off-target effects revealed that the multiplexed approach “did not cause catastrophic genomic instability.”
They also went in vitro and examined whether the edited cell line would infect human (HEK 293) cells. Interestingly, while there had not been in vivo evidence for PERV transmission to human cells, the results showed human cells exposed to wild-type pig cells express PERV transcripts. As expected, PERVs in edited cells were reduced to background levels.
While transplantable organs await, all in all, the team demonstrated a knockout of 62 loci using 2 sgRNAs – a record-breaking effort that opens the door to multiplex editing of repetitive sequences.
Go pig out in Science, October 2015