NEW YORK (GenomeWeb) – Researchers have used genome editing for the first time to study gene function in early human embryonic development.
UK and South Korean researchers led by the Francis Crick Institute's Kathy Niakan aimed to elucidate the molecular mechanisms underlying the first cell fate decisions in the human embryo, which are not well understood, they wrote in their paper, published today in Nature.
To determine whether CRISPR/Cas9 can be used to understand gene function in human preimplantation embryo development, the team chose to target POU5F1, a gene encoding the developmental regulator OCT4, as a proof of principle. Zygotic POU5F1 is thought to be first transcribed at the four- to eight-cell stage, coinciding with embryo genome activation, and OCT4 protein is not detectable until approximately the eight-cell stage, the researchers wrote. They predicted that OCT4 perturbation would cause a clear developmental phenotype, based on studies in mouse and human embryonic stem cells.
The team selected four single-guide RNAs to target POU5F1, and tested their on-target editing efficiency and off-target activity in mouse embryos and human embryonic stem cells. Once they selected the most efficient sgRNA-Cas9 complex, they moved on to target POU5F1 in human preimplantation embryos.
To test whether OCT4 is required in human embryos, the researchers performed CRISPR/Cas9 editing on thawed in vitro fertilized zygotes that were donated from infertility treatments. They microinjected 37 zygotes with the sgRNA2b–Cas9 ribonucleoprotein complex and 17 zygotes with Cas9 protein alone to control for their microinjection technique.
Of the zygotes that were microinjected with the sgRNA2b-Cas9 complex, 30 embryos retained both pronuclei during microinjection, with pronuclear fading observed approximately six hours later and cytokinesis on average five hours later, the researcher noted. Genome editing was estimated to start after approximately three hours in vitro and to persist for 12 hours to 24 hours, so CRISPR/Cas9-induced double-strand breaks are likely to be formed during late S phase or subsequently at G2 phase.
"Time-lapse microscopy of the embryos showed that the timings of cleavage divisions following pronuclear fading was similar between embryos microinjected with Cas9 protein or sgRNA2b–Cas9. By the eight-cell stage, cleavage arrest was observed in 62 percent (23 out of 37) of sgRNA2b-Cas9-microinjected embryos compared to 53 percent (9 out of 17) of Cas9-microinjected control embryos," the authors wrote.
About 47 percent (8 out of 17) of the Cas9-microinjected control embryos developed to the blastocyst stage, a rate equivalent to that of uninjected embryos. However, only 19 percent (7 out of 37) of the sgRNA2b-Cas9-microinjected embryos developed to the blastocyst stage.
"The blastocysts that formed following sgRNA2b-Cas9 protein microinjection were of variable quality. Although all blastocysts had a discernible blastocoel cavity, only some possessed a small compact [inner cell mass], and all retained a thick zona pellucida, in contrast to Cas9-microinjected controls," the authors wrote. "Embryos arising from zygotes microinjected with sgRNA2b-Cas9 also went through iterative cycles of expanding and initiating blastocyst formation and then collapsing, until some embryos ultimately degenerated. These findings suggest that targeting OCT4 in human embryos reduces both viability and quality of blastocysts."
Importantly, the researchers noted, CRISPR/Cas9-mediated genome editing didn't appear to increase genomic instability or developmental arrest before embryo genome activation, which suggested to them that genome editing could be used to study the function of other genes involved in embryonic development as well. Further, this proof-of-principle study lays out a framework for future studies on early human biology, and could lead to improvements in the therapeutic use of stem cells and in IVF treatments, they added.
"The acquisition of this knowledge will be essential to develop new treatments against developmental disorders and could also help understand adult diseases such as diabetes that may originate during the early stage of life," study co-author and Wellcome Trust Sanger Institute researcher Ludovic Vallier said in a statement. "Thus, this research will open new fields of opportunity for basic and translational applications."