In an unexpected twist, the investigators discovered that once the paternal gene was excised, the genetic material originating from the mother (i.e., the homologous wild-type maternal gene) was more easily substituted than the synthetic DNA the scientists attempted to introduce.

To reduce mosaicism, which is characterized by a population of cells that originate from one egg but are genetically distinct, researchers injected sperm cells and CRISPR components directly into oocytes early in their cell-cycle phase, only 18 hours post-fertilization. The study authors assumed this would be the best time for genome editing to occur, as the sperm at that time only has a single mutant copy. In addition, injecting genetic material early, before DNA replication occurred, meant that the CRISPR components stayed in the cytoplasm longer. As a result of prolonged cytoplasm residency, the CRISPR components degraded quickly, before further replication of mutant alleles could occur.

Mosaicism, noted the authors, could have major negative effects and could restrict the clinical applications of the gene-editing technique in embryos, a fact that the authors identified as a limitation. In addition, the uncertainty surrounding the ability to reproduce the studys findings was also a limitation, the authors acknowledged.

Employing CRISPR in embryos, rather than in stem cells, yielded better results: The overall targeting efficiency in human embryos was found to be 72.2% (13/18), which was higher than the rate in induced pluripotent stem cells (iPSCs) exposed to the same construct (27.9%, or 17/61). The higher targeting efficiency suggests that human embryos employ different DNA repair mechanisms than do somatic or pluripotent cells, probably reflecting evolutionary requirements for stringent control over genome fidelity in the germline," the authors wrote in the paper.

Because off-target cutting is also a concern withCRISPR/Cas9, researchers evaluated all of the potential off-target sites via a whole-genome sequencing analysis. They determined that their technique did not produce any detectable off-target mutations in the blastomeres.

And, because Cas9 was used in purified protein form, and was not contained in a plasmid, off-site targeting was further reduced, investigators concluded.

Excerpt from:
CRISPR Corrects Disease Mutation in Human Embryos - Genetic Engineering & Biotechnology News (blog)