CRISPR-Cas technology has been used in a broad range of applications, including multiplexing. Up to five genes have been targeted in the same cell1, but whether this number could be extended by much was not clear. In a new demonstration of the capabilities of the CRISPR-Cas system, George Church, Luhan Yang and colleagues recently reported simultaneously editing 62 related genes in pig cells2.
Engineering the genome of pigs is not a new idea. A strong incentive for altering the genetic makeup of these animals is to make them more suitable as donors for xenotransplantation. Pig organs are roughly the same size as those of humans and share similar vascular architecture. Tens of thousands of individuals could receive life-saving transplants every year if the remaining hurdles in xenotransplantation were overcome. One such challenge is the potential transmission of porcine endogenous retroviruses (PERVs) to humans.
Aiming to inactivate PERVs, Church, Yang and colleagues began by determining how many PERV elements are present in the genome of pig cells, and identified 62. Then, they designed two Cas9 guide RNAs that would target the pol gene, an essential and conserved PERV gene, in all 62 PERVs. They expressed the guide RNAs and Cas9 in PK15 cells, a pig kidney epithelial cell line, and sequenced clones to determine the extent of editing that had occurred. They found a small number of clones that had up to 100% disruption of the pol gene, along with evidence suggesting that this high efficiency was due to a mechanism in which previously edited pol genes were used as templates to disrupt wild-type pol genes. Elucidating the intricacies of this mechanism may uncover interesting biology and further potential applications of the approach.
“This illustrates the power of the CRISPR technology,” says Randall Prather, of the University of Missouri. “A few years ago we wouldn't have thought of doing this because inactivating 62 genes, or even a family of 10 genes, would have been quite complicated with traditional homologous recombination.”
Editing 62 genes, even if closely related, is certainly a feat, and the engineered cells showed a 1,000-fold reduction in PERV transmission to human cells. That said, whether PERV transmission is likely to be a problem in xenotransplantation is not clear. Kevin Wells, also from the University of Missouri, says that PERV transmission was on the list of things to examine, but probably not at the top. “We don't know what the actual risk is,” he says. Although there is evidence that PERV transmission can occur in vitro, “no one has observed transmission of PERVs in experiments where a nonhuman primate received a pig organ,” says Wells. In addition, alternative approaches could be used, such as employing pig lines that do not produce human-tropic, replication-competent PERVs3.
Organ rejection continues to be the major hurdle in xenotransplantation. Although acute organ rejection has been addressed, immune rejection is still a considerable problem. On that front, Wells points out that CRISPR-Cas9 genome editing could be used to target pig genes encoding antigenic carbohydrates or proteins. Inactivation of porcine circulating proteins that don't interact well with their human counterparts could also help efforts to make xenotransplantation a reality.
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Jarchum, I. Getting rid of PERVs. Nat Biotechnol 34, 46 (2016). https://doi.org/10.1038/nbt.3448