A landmark study, published in Nature on August 2, 2017, has described the first timein the U.S. that CRISPR was used to repair a germline mutation in human embryos created through in vitro fertilization. The study was a collaboration between the Salk Institute, Oregon Health and Science University (OHSU), and Korea's Institute for Basic Science. The scientists behind the work, led by Shoukhrat Mitalipov, Ph.D., of the Center for Embryonic Cell and Gene Therapy at OHSU, concluded that the technique to replace a mutation in the gene MYBP3C could help prevent hypertrophic myocardiopathy (HCM), a disease that eventually leads to heart failure. This study is the first in the country to demonstrate that modifying single-gene defects using the gene-editing technology CRISPR has the potential to be safe and accurate enough to use in human embryos.
"This research significantly advances scientific understanding of the procedures that would be necessary to ensure the safety and efficacy of germline gene correction," said Daniel Dorsa, Ph.D., senior vice president for research at Oregon Health & Science University (OHSU), in a press statement. "The ethical considerations of moving this technology to clinical trials are complex and deserve significant public engagement before we can answer the broader question of whether it's in humanity's interest to alter human genes for future generations."
In response to the publication, a workgroup of the American Society of Human Genetics (ASHG) comprising 10 other organizations released a statement advising caution, not a ban, of human germline editing. The statement, approved by the ASHG Board of Directors in March in anticipation of such research, noted "there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing.” However, the working group did posit that germline editing that would result in a human pregnancy would be "inappropriate."
The researchers set out to determine if they could prevent the germline transmission of a genetic mutation causing HCM. In samples with afflicted sperm—which were confirmed via preimplantation genetic diagnosis (PGD) to carry the mutation that causes HCM—scientists used CRISPR to cleave out damaged sections of DNA. Then, they replaced the mutation with new genetic information, trying a few different methods to incorporate the corrected gene. Once edited, 42 out of 58 embryos lacked the HCM mutation.
In an unexpected twist, the investigators discovered that once the paternal gene was excised, the genetic material originating from the mother (i.e., the homologous wild-type maternal gene) was more easily substituted than the synthetic DNA the scientists attempted to introduce.
To reduce mosaicism, which is characterized by a population of cells that originate from one egg but are genetically distinct, researchers injected sperm cells and CRISPR components directly into oocytes early in their cell-cycle phase, only 18 hours post-fertilization. The study authors assumed this would be the best time for genome editing to occur, as the sperm at that time only has a single mutant copy. In addition, injecting genetic material early, before DNA replication occurred, meant that the CRISPR components stayed in the cytoplasm longer. As a result of prolonged cytoplasm residency, the CRISPR components degraded quickly, before further replication of mutant alleles could occur.
Mosaicism, noted the authors, could have major negative effects and could restrict the clinical applications of the gene-editing technique in embryos, a fact that the authors identified as a limitation. In addition, the uncertainty surrounding the ability to reproduce the study’s findings was also a limitation, the authors acknowledged.
Employing CRISPR in embryos, rather than in stem cells, yielded better results: The overall targeting efficiency in human embryos was found to be 72.2% (13/18), which was higher than the rate in induced pluripotent stem cells (iPSCs) exposed to the same construct (27.9%, or 17/61). The higher targeting efficiency “suggests that human embryos employ different DNA repair mechanisms than do somatic or pluripotent cells, probably reflecting evolutionary requirements for stringent control over genome fidelity in the germline," the authors wrote in the paper.
Because off-target cutting is also a concern with CRISPR/Cas9, researchers evaluated all of the potential off-target sites via a whole-genome sequencing analysis. They determined that their technique did not produce “any detectable” off-target mutations in the blastomeres.
And, because Cas9 was used in purified protein form, and was not contained in a plasmid, off-site targeting was further reduced, investigators concluded.