Exjobbspresentation: Julia Stevrell, MPBIO

Examinator: ​Yvonne Nygård
Handledare: Alexandra Madsen

Opponent: Anton Johansson

Sammanfattning:

The CRISPR-Cas9 system for gene editing holds immense potential in a vast range of bioscience applications, such as for the therapeutic treatment of somatic genetic disorders by directly correcting the disease-causing mutations in vivo or ex vivo. However, the potential for CRISPR-Cas gene therapies is largely limited by their ability to perform a desired edit exclusively at the intended (on-target) gene locus. Unintended mutations at off-target sites in the genome, so-called off-target effects (OTE), and their downstream phenotypic implications are key safety concerns currently impeding the translation of CRISPR-Cas9 into the clinics. To date, the best strategy for addressing the issue of off-target activity is to carefully design and evaluate the specificity of the guide RNAs and Cas9 variants. However, previous research has shown that this is insufficient for circumventing lowly frequent off-target effects. Although rigorous research has been performed to better understand the mechanisms behind CRISPR-Cas9 OTE, many potential underlying factors remain unexplored. One such factor is the cell type, which, to the best of our knowledge, has not been explored as a potential variable in CRISPR-Cas9 off-target activity.
This thesis project aimed to investigate whether the cell type influences the off-target activity of CRISPR-SpCas9 in human cells. As a first step, three different cell lineages (ventricular cardiomyocytes, hepatocytes, and midbrain dopaminergic neurons) were generated and partly validated from human induced pluripotent stem cells (hiPSCs), through directed differentiation. hiPSC line generation was based on the ObLiGaRe- and Xential techniques, generating a two-part doxycycline-inducible CRISPR-SpCas9 construct. Gene editing was induced through the addition of doxycycline to mature cell types, and on/off-target editing was subsequently verified using targeted sequencing assays. Off-target editing profiling identified potential cell-specific off-target effects that were independent of the CRISPR nuclease specificity. Although further validation assays are needed, these results have implications for continued research into the safety of CRISPR-Cas9, and nevertheless its clinical applications for combating disease.