LONDON – Scientists in the U.K. are laying claim to the creation of the first human gene knockout after using CRISPR/Cas 9 to delete the gene encoding for the transcription factor Oct4, in day-old embryos.

In doing so, the researchers said they have demonstrated that Oct4 is essential for embryos to develop from day one to reach the 200-cell blastocyst stage at day seven, and to have provided a methodology for future research to discover when and how specific genes are active in the earliest stages of human development.

Some of their findings are at odds with previous understanding of the function of Oct4 based on the study of mouse embryogenesis, justifying the use human embryos in the research, the scientists said.

"The research is the first time genome editing has been used to understand the role of a gene in early human embryo development," said Kathy Niakan, of the Francis Crick Institute in London, who led the research.

Oct4 (octamer-binding transcription factor 4) is the master regulator of the maintenance of pluripotency in embryonic stem cells and is used as a marker for undifferentiated cells. Given that, the work lays foundations to increase understanding of how embryonic stem cells arise, how to culture them to improve cell models, and to inform and improve in vitro fertilization, Niakan said.

The researchers describe in a paper in Nature this week how they microinjected 41 donated human embryos with a CRISPR/Cas9 construct designed to inactivate POU5F1, the gene encoding Oct4. The embryos were at the pronuclei stage, before the maternal and paternal DNA had merged.

A further 17 embryos, injected with Cas9 alone, acted as controls.

The subsequent development of the embryos was followed for seven days using live imaging. Development was then stopped and the embryos analyzed.

In embryos generated for IVF treatments, around 50 percent successfully develop to form a 200-cell blastocyst by day seven. At that stage, a blastocyst consists of three different cell types that are precursors of the placenta, the yolk sac and around 20 epiblast progenitor cells that give rise to the embryo.

In the experiment, 50 percent of the control embryos developed to the blastocyst stage, as expected from IVF data. Only 19 percent of the Oct4 knockout embryos reached the blastocyst stage. It transpired that in those that did form blastocysts, the editing was only partially successful and there was mosaicism, meaning some cells were expressing Oct4.

Subsequent analysis of the knockout embryos showed genes downstream of Oct4 that are associated with the maintenance of pluripotency, including Nanog and ETS1, were down-regulated.

Conversely, genes associated with differentiation, such as PAX6 and SOX17, were up-regulated after POU5F1 was knocked out, suggesting Oct4 normally restrains differentiation.

Previous studies in human embryonic stem cells and mice mean the finding that Oct4 is central to the development of the human blastocyst hardly comes as a surprise. Indeed, it was in expectation of demonstrating it plays such an important role that the researchers elected to attempt to knock out POU5F1 in the proof-of-principle experiment.

However, the effect of preventing expression of Oct4 in human and mice embryos is different in several respects. In mice, turning Oct4 off turns Nanog on, whilst in humans inactivating Oct4 blocks production of Nanog, for example.

In addition, in mouse embryos, Oct4 is not found in placental precursor cells, whereas it is expressed in human placental precursor cells.

"The effect of inactivating Oct4 in the two species is different. In other words, in order to get to the full picture, you need to study human embryos," Niakan said. "We now know you have to have Oct4 to make a blastocyst and it is critical for embryo progenitor cells and also for progenitor cells of the placenta."

More to learn

Having confirmed Oct4 is critical, the next stage of the research will be to fully understand what downstream genes it is regulating.

The finding could in the future reduce IVF failure rates, Niakan suggested. "Of those 50 percent of embryos that make a blastocyst, 50 percent fail to implant and we don't know why. So could we find a biomarker to predict if a blastocyst will implant?"

It also may be possible to alter the IVF embryo culture medium to provide factors that are essential for blastocyst formation and subsequent implantation.

Another avenue would be to investigate Oct4 expression in the control embryos that failed to form blastocysts.

One of the researchers, Kay Elder, from the Bourn Hall IVF clinic in Cambridge, U.K., where the donated embryos were fertilized said, "IVF is really very inefficient. There is so much more to learn about how to culture embryos and especially because most of what we know is based on mouse studies."

Niakan also would like to more precisely control the timing of the editing, by injecting CRISPR/cas9 into an egg at the same time as the sperm. However, that would require changes to the license she was granted to conduct germline gene editing by the U.K.'s Human Fertility and Embryology Authority.

It was by simultaneously injecting donor eggs with sperm from a man carrying the MYBPC3 hypertrophic cardiomyopathy mutation and a CRISPR/Cas9 construct designed to correct it that Shoukhrat Mitalipov, of Oregon Health and Science University, succeeded in generating embryos with two correct copies of the MYBPC3 gene, without causing any off-target effects or mosaicism. The MYBPC3 mutation is the commonest cause of heart failure and sudden death in apparently healthy young people. (See BioWorld, Aug 3, 2017.)

Niakan's paper describes a year of work in optimizing CRISPR/Cas9 to edit out POU5F1, testing various constructs in human embryonic stem cells and in mice. The researchers also tested different microinjection techniques. "We wanted to minimize the number of embryos used," Niakan said. "We tweaked every component."

The researchers also devoted significant effort to testing for off-target effects of CRISPR/Cas, concluding their construct was highly specific for POU5F1.

The result is that there is now a toolbox for other researchers to carry out related gene knockout experiments. "Now we have demonstrated an efficient way of doing this, we hope that other scientists will use it to find out the roles of other genes," Niakan said.