Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Can Maternal Spindle Transfer and Pronuclear Transfer Be Prohibited under eu Legislation?

The question whether maternal spindle transfer (mst) and pronuclear transfer (pnt) can be prohibited under eu legislation was examined by the non-governmental organisation European Bioethics Research (ebr). It did so by submitting an official complaint to the eu Commission proposing that the uk Human Fertilisation and Embryology (Mitochondrial Donation) Regulations 2015 breached the prohibition on the modification of a person’s germ line genetic identity of the eu Clinical Trials Directive 2001/20/EC and the new Regulation eu 536/2014. A discussion then took place, during 2016, between ebr and the eu Commission whether mst and pnt principally involved a ‘medicinal product’ in which case the eu Clinical Trials Directive 2001/20/EC and Regulation eu 536/2014 would be applicable or whether the procedures just involved a medical procedure in which case the Tissue and Cells Directive 2004/23/EC was applicable which did not include any prohibition on the intentional modification of a person’s germline.

Mitochondrial replacement therapy and the “three parent baby”

The mitochondria contained in eukaryotic cells have their own DNA, and heritable mutations in mitochondrial DNA (mtDNA) can cause a variety of disorders in humans. A new therapy, mitochondrial replacement therapy (MRT), is currently being developed to address these mitochondrial disorders by eliminating the mutated mtDNA from the germline. The two main MRT techniques are pronuclear transfer, conducted in the zygote after fertilization, and spindle-chromosomal complex transfer, conducted in the oocyte before fertilization. In pronuclear transfer, the pronuclei from a zygote affected by a mtDNA mutation are transferred to an enucleated normal zygote. In spindle-chromosomal complex transfer, the genetic material from an oocyte affected by a mtDNA mutation is inserted into the cytoplasm of a donor oocyte that contains healthy mitochondria. A third method, polar body genome transfer, attempts to increase the efficiency of the above techniques by using polar bodies to supply the genetic material. While MRT is legally and ethically controversial, it has recently been implemented successfully in a clinical setting.