| Zusammenfassung: | <p>CRISPR (clustered regularly interspaced short palindromic repeats) offers an unprecedented array of genetic tools for treating and modelling haematological disorders. However, there are concerns about its safety. CRISPR-based editing can induce upregulation of TP53 associated pathways, a finding particularly important for clinical applications. The impact of CRISPR-based editing on haematopoietic stem cells (HSCs) has not, however, been interrogated in detail.</p>
<p>In the work described in this thesis, I initially optimised the CRISPR approach in mouse haematopoietic stem and progenitor cells (HSPCs) by targeting the fluorescent reporter tdTomato, as this enabled lineage tracking of edited HSCs. I then sorted edited HSCs for RNA-Sequencing (RNA-Seq) 24 hours post-editing. This allowed me to assess the immediate transcriptional consequences of genome editing. Using in vivo transplantation, I serially tracked the contribution of edited cells to all blood lineages. I carried out RNA-Seq on edited HSCs 18 weeks post-transplant which allowed me to assess whether the short-term changes in transcriptional profiles were maintained long-term.</p>
<p>To demonstrate the applicability of the genome editing approach I had optimised to a disease model setting, I targeted the NF-1 gene which harbours loss of function mutations in 15% of JMML patients. While mouse models exist for this disorder, most lack a human fetal context. I first optimised targeting of the NF-1 locus in a GM-CSF dependent cell line (TF-1). I then carried out in vitro NF-1 targeting studies using human cord blood (CB) to establish a GM-CSF hypersensitivity phenotype. Finally, I set up in vivo transplants of NF-1 edited human CB to establish whether this could propagate the myeloid biased expansion characteristic of JMML.</p>
<p>In this way, I applied the CRISPR genome editing approach to study both normal and malignant haematopoiesis: first to establish the molecular and functional consequences of genome editing on normal haematopoietic output and second to model the consequence of NF-1 loss of function in a human fetal-like context.</p>
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