The role of new genetic technologies for the analysis of early human embryos: clinical application and research

Mutations of the beta-globin gene (HBB) are the most common cause of inherited disease in humans, causing ß-thalassaemia and sickle cell anaemia. Traditional preimplantation genetic testing (PGT) protocols for the detection of HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. Chapter 1 focuses on the development, validation and clinical implementation of a novel and universally applicable PGT method for the diagnosis of HBB gene mutations, utilising next generation sequencing (NGS). Employing this protocol HBB mutation status and associated single nucleotide polymorphism (SNP) haplotypes were successfully determined in all 21 embryos of three couples undergoing PGT for ß-thalassaemia. Analysis of 141 heterozygous sites showed no instances of allele dropout in the clinical samples and the test displayed 100% concordance compared with the data obtained using an established method (karyomapping). Taken together, the results suggest that the new method may deliver superior accuracy than typically achieved with traditional PGT methods. Furthermore, the test is streamlined and economical, which should improve patient access to PGT, reducing costs and waiting times. This will be especially important in less affluent parts of the world where diseases affecting hemoglobin synthesis are of high prevalence. An alternative to PGT for reducing the burden of inherited disorders is the correction of disease-causing mutations in human embryos using genome editing methods. However, this possibility is subject to numerous ethical and technical concerns, especially as intervention in gametes or preimplantation embryos would inevitably involve modification of the germline and a high likelihood of transmission of edited genes to future generations. CRISPR-Cas9 is currently the leading technology for introducing specific and heritable modifications into the genome. Chapter 2 evaluated the CRISPR-Cas9 system from the perspective of technical feasibility. It involved development of a methodological framework and computational pipelines for evaluation of on-target mutagenesis as well as potential off-target consequences of the editing in OCT4 (POU5F1) CRISPR-Cas9-targeted human zygotes and controls. A high efficiency of editing was observed with a wide variety of indel mutations, characteristic of non-homologous end-joining (NHEJ). No evidence of off-target activity was recorded. There is a possibility of unintended editing outcomes following the use of CRISPR-Cas9 that may have pathologic consequences, potentially exacerbated by insufficient DNA repair in early human embryos prior to embryonic genome activation (EGA). Chapter 3 investigated the repair outcomes of Cas9-induced double-strand breaks (DSBs) introduced in the POU5F1 locus. Strikingly, the results showed that 37.5% of the targeted zygotes present with breaks that remain unrepaired or participate in complex genomic rearrangements, resulting in segmental aneuploidy with breakpoints within the targeted 6p21.3 region. Gains and losses of large regions, stretching from 6p21.3 to the end of the short arm of chromosome 6, as well evidence indicating a complexity of DNA sequence mutations at on-target sites after CRISPR-Cas9 editing, provide a cautionary note to those considering the technology for clinical use and underscore the importance of basic research into DNA repair pathways and genomic stability in human embryos. Such research is not only relevant in the context of genome editing, but also has importance for assisted reproductive treatments and stem cell research. Furthermore, collaborative work of which this thesis is a part emphasizes that CRISPR-Cas9 remains a powerful molecular biology tool for the study of gene function and the biology of early human development.