O-075 Deficiency of DNA double-strand break repair in human preimplantation embryos revealed by CRISPR-Cas9

Abstract Study question Is double-strand DNA break repair functional in human preimplantation embryos and what implications might a deficiency have for genome editing performed at early developmental stages? Summary answer CRISPR-Cas9 succeeds generating breaks with high efficiency, but these often remain unresolved, indicating that are deficient repair. What is known already Genome (GE) technologies, such as CRISPR-Cas9, raise the possibility of avoiding transmission inherited disorders by converting mutant genes back to wild-type (normal). The therapeutic application stage controversial due uncertainty over safety concerns about alteration germline. However, from technical perspective it remains highly attractive, since only where delivery every cell individual can be guaranteed. Currently, information on aspects efficacy lacking. Therefore, evaluation cellular response needed. design, size, duration 84 were generated research an IRB approved study. For this purpose, donor oocytes fertilised sperm using ICSI. 51 resulting served controls, while other 33 double strand (DSBs) created controlled fashion, directed specific genomic sites technology. Successfully underwent culture time-lapse incubator key events carefully timed. Participants/materials, setting, methods components targeted several non-coding genome. An artificial fragment was provided, which cells could potentially use homology (HDR). Embryos grown 60 hours, then disaggregated. Blastomeres whole amplification, followed next generation sequencing detect segmental aneuploidy related failure Additionally, amplified sequenced confirm whether had occurred distinguish mechanism employed. Main results role chance study successfully validated utilised three CRISPR ‘guides’ chromosomes 2, 3 12. Alterations detected 24/25 (96% targeting efficiency). Repair DSBs considered successful when embryo contained least one blastomere changes sequence site consistent nonhomologous end joining (NHEJ) or HDR. In total, 53 double-stranded during Of these, 51% (27) repaired NHEJ, 9% (5) HDR 40% (21) remained persistent breakage eventually causing breakpoint side. Segmental abnormalities detrimental embryonic viability risk congenital offspring. This shows embryos, prior activation genome, damage deficiency. Timings morphokinetic milestones altered unresolved damage, confirming checkpoint control active. continued mitotic division, despite presence DSBs, mechanisms usually preserve genetic integrity lack stringency, ultimately failing ensure provide warning against embryos. Limitations, reasons caution While remarkably efficient (DSBs introduced 96% embryos), majority NHEJ repair, process allowing introduction additional mutations rather than correcting existing ones. Only <10% used necessary removal mutations. Wider findings These reveal less able types. suggests should give consideration protecting damage. clinical technologies create CRISPR-Cas9. Trial registration number NA