Gene Editing Used to Repair Mutations in Embryos

The CRISPR/Cas9 gene editing tool was used to correct a mutation in the DNA of a human embryo, and the problem of mosaicism was avoided by carrying out the gene editing step while the embryo was still a single-cell fertilized egg.

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs.

Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand.

Investigators at Oregon Health & Science University (Portland, USA) sought to investigate human gamete and embryo DNA repair mechanisms activated in response to CRISPR/Cas9-induced double-strand breaks (DSBs). Their intent was to demonstrate the proof-of-principle that heterozygous gene mutations could be corrected in human gametes or early embryos.

In the August 2, 2017, online edition of the journal Nature they described the correction of the heterozygous MYBPC3 mutation - the cause of hypertrophic cardiomyopathy (HCM), a common genetic heart disease that can cause sudden cardiac death and heart failure - in human preimplantation embryos. This repair depended on precise CRISPR/Cas9-based targeting accuracy and high homology-directed repair efficiency that was obtained by activating an endogenous, germline-specific DNA repair response. Induced DSBs at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template.

By modulating the cell cycle stage at which the DSB was induced, the investigators were able to avoid mosaicism in cleaving embryos and achieved a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The solution to the mosaicism problem was to minimize their occurrence by the co-injection of sperm and CRISPR/Cas9 components into metaphase II oocytes.

"Every generation on would carry this repair because we have removed the disease-causing gene variant from that family's lineage," said senior author Dr. Shoukhrat Mitalipov, director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University. "By using this technique, it is possible to reduce the burden of this heritable disease on the family and eventually the human population."

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