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This web page was produced as an assignment for an undergraduate course at Davidson College.

Image Courtesy of Wikimedia Commons via the United States Department of Agriculture

Harvard Medical School’s George Church, Ph.D. led a team of scientists in an attempt to remedy the problem of transmissible retroviruses with respect to transplantation of pig organs to humans (xenotransplantation). Church’s team specifically honed in on the porcine endogenous retrovirus (PERV) pol gene. The pol gene’s inability to be eliminated by “biosecure breeding” makes it a reasonable target for inactivation through genomic manipulation. Previous studies have reduced the risk of PERV transmission to humans using such strategies as small interfering RNAs and vaccines with limited success.

Church’s team incorporated a different strategy, making use of the CRISPR-Cas9 RNA-guided nuclease system.Using this system, Church and his team inactivated all 62 copies of the PERV pol genes they identified. Additionally, they examined the copies of the PERV pol genes that they inactivated and found no evidences of genomic rearrangements or mutations. This indicated that the team’s strategy did not incur dangerous genomic “instability.” (Glaser et al., 2015)

Church’s team studied strategies attempting to reduce/eliminate PERV transmission from pig organs to humans. They found that the strategies from prior studies resulted in low success rates. After this discovery, the team achieved genome-wide success using the CRISPR-Cas9 system. (Yang et al., 2015)

The researchers analyzed publically available PERV and other endogenous retrovirus sequences and determined that there were 62 copies of PERVs in the PK15 cell line. They determined this using droplet digital PCR. Afterwards, they used Sanger Sequencing to check the results of their sequencing whilst looking for any insertions or deletions. They then designed “two Cas9 guide RNAs (gRNAs)” that specifically targeted the pol gene. They thought this would be effective because the product of the pol gene acts as reverse transcriptase (RT), making it vital in processes such as replication or viral infection. At first they discovered that transient transfection yielded low success rates, so they used a PiggyBac transposon system to insert the gRNAs into the genome of the PK15 cells. They used cytometry and karyotyping of the clones that followed in order to check for any genomic rearrangements. (Yang et al., 2015)

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 sequences are highlighted in red.(Adapted from Yang et al., 2015)

By taking advantage of knowledge about cell pathways, we can target certain problems with greater specificity. In this study, the manipulation of the CRISPR system resulted in the genome-wide inactivation of PERVs. Because the strategy Church’s team used also resulted in no significant instances of genomic rearrangement, xenotransplantation could potentially be the answer to the shortage of organs for transplants without any repercussions involving viral transmission.

The overall research did well in using multiple genomic technologies in analysis and sequence checking as well as testing for in vitro transmission of PERVs with respect to their disruption of PERV pol genes. Future studies in this area should include analysis of other sources of PERV transmission.