Cells of early human embryos are often unable to repair DNA damage caused by CRISPR/Cas gene editing. Report: Claire Robinson

Scientists have discovered that the cells of early human embryos are often unable to repair damage to their DNA caused by the CRISPR/Cas gene editing process. The researchers say that this has important implications for the proposed use of gene editing to repair mutated genes, which underlie serious inherited diseases, as well as for in vitro fertilisation (IVF) in general.

Commenting on her team's findings, lead researcher Dr Nada Kubikova from the University of Oxford, UK, said: “Gene editing has the potential to correct defective genes, a process that usually involves first breaking and then repairing the DNA strand. Our new findings provide a warning that commonly used gene editing technologies may have unwanted and potentially dangerous consequences if they are applied to human embryos.
         
“Our results show that the use of CRISPR/Cas9 in early human embryos carries significant risks. We have found that the DNA of embryo cells can be targeted with high efficiency, but unfortunately this rarely leads to the sort of changes needed to correct a defective gene. More often, the strand of DNA is permanently broken, which could potentially lead to additional genetic abnormalities in the embryo.”

The new research was presented at a conference and is not yet peer reviewed and published, but it has already created a worried stir, with the Science Media Centre putting out comments from scientists. While some of the scientists comment that any attempt to use gene editing clinically is “premature” and “clinically problematic”, others suggest that base and prime editing, which are gene editing methods that don’t make double-strand breaks in the DNA, might solve the problems found in this research.

GMWatch sought the opinion of molecular geneticist Dr Michael Antoniou, who uses all types of genetic manipulation techniques, including gene editing, in clinical research. What he told us suggests that Dr Kubikova’s findings likely will kill attempts to carry out human germline editing in humans using CRISPR/Cas – and that attempts to get around the problem by using alternative genetic manipulation methods may also prove problematic. He said, “These findings present serious problems for human germline [heritable] genetic engineering.”

Dr Antoniou said the findings have relevance to some agricultural gene-edited GMOs – certainly to animals and possibly to plants, casting yet more doubt on the viability and safety of these types of GMO. Regarding animals, he said, “Some gene-edited animals are created not by cloning but by injecting the gene-editing tool straight into the fertilised embryo.”

This is how Alison Van Eenennaam made her gene-edited cattle that were genetically manipulated to give birth only to male offspring. The experiment failed, creating chaos in the cattle’s genomes.

Dr Antoniou said this was not surprising, as gene editing the embryo is “like unmanned drone warfare: You can target the missile with apparent success, but it still can cause massive collateral damage”.

As for the alleged promise of base editing and prime editing, he said these methods are unlikely to make the issues go away: “They will also create unintended mutations – just perhaps at a lower level than using CRISPR/Cas to make double-strand DNA breaks. They may reduce but not eliminate the risks to the humans that would result from the editing process. And that level of risk is unacceptable.

“Furthermore, base and prime editing can be efficient in non-reproductive (somatic) cells growing in tissue culture, but in living embryos it might be different. We don’t know yet.

“Base editing is often presented as more precise and less destructive than gene editing involving double-strand DNA breaks, because it only results in a break of one strand of DNA and aims to change just a single DNA base unit. However, some serious human diseases, like sickle cell disease, are caused by a mutation in a single base. The lesson that we should draw from this is that using base editing to gene edit livestock animals is risky and will require careful screening for off-target DNA damage.

“In humans and other animals, many genetic diseases are caused by larger mutations – not single base mutations. In those cases, base editing to try to eliminate the genetic disease will be useless.”

Regarding gene-edited plants, Dr Antoniou said that scientists don’t yet know if plants are more or less capable than humans of repairing DNA breaks cleanly: “However, it has long been known that the tissue culture process that is an obligatory part of plant gene editing creates hundreds or thousands of unintended mutations (DNA damage) throughout the genome of the targeted organism. The new research findings raise the question: Are plant cells in tissue culture less able to repair DNA damage than plant cells in their natural environment, so the DNA damages accumulate? If so, that would explain why tissue culture is so mutagenic – it would be, at least in part, an issue of impaired DNA repair.

“In gene editing, the tissue culture process-induced mutations cause a far greater number of unintended mutations than the gene variations that occur in natural breeding. Claims that gene editing causes fewer mutations than natural reproduction ignore the tissue culture-induced mutations.

“Also, the gene editing-induced mutations are random, so no region of the genome is immune. Therefore, there is a high risk of hitting important gene functions.

“In contrast, the genetic variations from natural breeding are not random. Some areas of the genome are protected from variation and mutation. The variations happen in other areas and are directed towards adaptation, as has been shown in the model plant Arabidopsis.”

The problem with unrepaired genetic mutations in gene-edited GM crop plants is that important biochemical functions could be disrupted, resulting in plants that are toxic, allergenic, have altered nutritional value, or have unexpected effects on wildlife. This is why numerous scientists recommend that plants produced using gene editing should continue to be subjected to risk assessments for health and the environment.

The human embryo experiments in detail

Dr Kubikova and her colleagues fertilised donated eggs with donated sperm to create 84 embryos. In 33 of the embryos, they used CRISPR/Cas9 to create breaks in the two strands that make up the DNA molecule – known as DNA double-strand breaks. The remaining 51 embryos were kept as controls.

The researchers detected alterations at the targeted DNA sites in 24 out of 25 embryos, indicating that CRISPR is efficient at creating breaks at targeted sites of the DNA in the cells of human embryos. However, only nine percent of targeted sites were repaired using the clinically useful process of homology directed repair. Fifty-one percent of broken DNA strands underwent a different type of repair called non-homologous end joining, producing mutations (DNA damage) where the strands were reconnected. The remaining 40% of broken DNA strands failed to be repaired.

The unrepaired breaks In the DNA strands eventually led to large pieces of chromosome, which extend from the site of the break to the end of the chromosome, being lost or duplicated. Abnormalities of this type affect the viability of embryos and if affected embryos were transferred to the uterus and produced a baby, they would carry a risk of serious congenital abnormalities.

Dr Kubikova explained, “Our study shows that homology directed repair is infrequent in early human embryos and that, in the first few days of life, the cells of human embryos struggle to repair broken DNA strands. CRISPR/Cas9 was remarkably efficient in targeting the DNA site” (the part of CRISPR gene editing that gives rise to misleading claims of precision and predictability). However, Dr Kubikova continued, “The majority of cells repaired the DNA break induced by CRISPR using non-homologous end joining, a process that introduces additional mutations rather than correcting existing ones. This would be a challenge if there were attempts to use CRISPR/Cas9 to correct inherited disorders in human embryos, as it suggests that most times when it is attempted, it will not be successful.”

“Unwanted and unexpected surprises”

Professor Karen Sermon, Head of the Reproduction and Genetics Research Group, Vrije Universiteit Brussel in Belgium, who was not involved with the research, commented, “I think it’s likely that gene editing will become a useful tool at some point in the future for preventing babies from being born with serious genetic diseases in a restricted number of cases where preimplantation genetic testing would not apply. However, this research shows one of the ways that it can go wrong. It will be some time before we can be confident that we really understand how to use it successfully without any unwanted and unexpected surprises. It will require stringent regulation. In the meantime, careful research such as this brings us one step closer, and may also help with understanding how to improve fertility treatments.”

Human germline gene editing takes massive step backwards

Based on GMWatch’s inquiries among scientists, contrary to Prof Sermon’s suggestion, there may be no cases at all in which preimplantation genetic testing for serious genetic diseases would not apply but where gene editing could be successful. This makes human germline gene editing pretty much redundant.

And contrary to Prof Sermon’s conclusion, it looks as if the new research will trigger a massive step backwards from the implementation of human germline gene editing. Given the strongly eugenicist trends around human germline genetic engineering, that’s a good thing. There are safer and more ethical ways to prevent and treat inherited genetic diseases, including somatic gene therapy, in which the genetic changes made are confined to the patient and don’t get passed on to future generations.

Meanwhile, in the sphere of agricultural GMOs, the question arises of just how much more evidence is needed before pro-GMO lobbyists and politicians are forced to abandon the narrative of “precise, predictable, controllable" for gene editing techniques.