The Rewriting Revolution

Earlier this week, I discussed the ethical and safety implications of the potential for germline editing using CRISPR/Cas9 technology, referencing  the ‘rumors’ that international scientists had already begun experimenting with human embryos. On Wednesday, the rumors became fact, as Nature News reported the world’s first example of gene editing in human zygotes. The study, led by Junjiu Huang at Sun Yat-sen University, was published in the online journal Protein and Cell.

The scientists targeted the human beta globin gene (HBB) which, when mutated causes the blood disorder b-thalassemia. The investigators used non-viable embryos; eggs that are fertilized by two sperm and therefore do not develop. They are typically discarded by fertility clinics, providing a potential ethical alternative model system for normal human embryos.  While CRISPR/Cas9 was shown to be successful in cleaving the endogenous HBB gene, the paper details numerous obstacles to using this technique for clinical applications in human embryos.

First, the efficiency of the technology was found to be much lower than what would be necessary for use in normal embryos. The researchers injected 86 embryos with Cas9, guide RNA to target HBB, and single-stranded DNA (ssDNA) that, if successful, would be inserted into the HBB locus. Seventy-one of these embryos survived. Out of these, 28 were cleaved by Cas9, but only 4 or 14.3% were edited with the ssDNA. Instead, the double-stranded breaks induced by Cas9 were preferentially repaired by direct ligation of the broken ends, which would not correct the mutations and could also induce additional mutations into the target gene. For ethical reasons, the efficiency and survival would need to be close to 100% to be used in viable human embryos.

Second, numerous off-target effects were detected, meaning the guide RNA induced double-stranded breaks in other genes besides HBB. These unintended mutations were seen at much higher rate than what had been observed in stem cells or mouse embryos, which is alarming. Off-target editing can damage healthy DNA to cause numerous side-effects and diseases if propagated in human embryos.

The results of this study further underscore the need to halt the use of CRISPR/Cas9 in human embryos until the technique can be optimized in human cells or animal models to improve the efficiency of gene-editing and reduce off-target mutations. However, rumors continue to suggest that at least four other groups are editing human embryos.