1. The gene editing tool CRISPR-Cas9 was used to correct a mutant paternal MYBPC3 allele in human preimplantation embryos.
2. No off-target effects were detected.
Evidence Rating Level: 1 (Excellent)
Study Rundown: A dominant mutation in the gene MYBPC3 causes hypertrophic cardiomyopathy (HCM), the most common cause of sudden death in otherwise healthy young athletes. While most current therapies focus on relieving symptoms of HCM, researchers in this study aimed to prevent transmission of the causative gene mutation by correcting it in preimplantation embryos.
Healthy donor eggs were injected with sperm that were heterozygous for the MYBPC3 mutation. After fertilization, recombinant Cas9 protein and single guide RNA that targeted MYBPC3 were microinjected into the zygotes. A majority of treated embryos survived and lost the mutation in this gene, without other genes being impaired. CRISPR-Cas9 targeting of MYBPC3 was found to be highly specific in the treated embryos.
This study was the first to use CRISPR-Cas9 to correct a harmful mutation without causing significant off-target effects. Although this genome editing technique is still far from clinical use and requires full discussion from a bioethics perspective, this research suggests the potential clinical efficacy of this therapy for in vitro fertilization and the correction of fatal mutations.
In-Depth [in vitro study]: Human zygotes were produced by fertilizing 70 oocytes without MYBPC3 mutations with sperm from an HCM patient with a heterozygous mutation in MYBPC3. Eighteen days after fertilization, recombinant Cas9 protein, short guide RNA, and single-stranded oligodeoxynucleotides were microinjected into the cytoplasm of the zygotes. A majority of zygotes survived this procedure, with a survival rate of 97.1%. Three days after injection of the Cas9 protein, 54 injected embryos were sequenced and 66.7% were found to be homozygous for the wild-type (WT) allele of MYBPC3. Almost half of the blastomeres from mosaic embryos were also found to be homozygous for the WT allele of this gene, demonstrating that the heterozygous mutation was repaired through homology-directed repair. These analyses demonstrated the efficient targeting by CRISPR-Cas9 in human embryos.
To improve the efficacy of gene correction, CRISPR-Cas9 was mixed with sperm and injected into 75 oocytes in metaphase II. This method resulted in an increase in WT embryos, with 72.4% successfully removing the mutation. Additionally, a majority of these oocytes developed into the eight-cell stage and then blastocysts, demonstrating no significant effect on embryonic development due to this therapy.
Finally, off-target effects were assessed through whole genome sequencing, digested genome sequencing, and whole exome sequencing. No insertions or deletions were detected in the WT blastomeres at 23 off-target loci, demonstrating the high targeting efficacy and potential safety of this treatment.
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