Investigation of the DNA damage in haematopoiesis using CRISPR/Cas9 technology

DNA replication is a highly regulated process that ensures the faithful duplication of the genome. However, endogenous or exogenous factors, such as limited DNA replication components and chemotherapeutic drugs, respectively, can cause defects in its regulation resulting in the stalling or slowing of the replication fork progression; a conditioned called replication stress (RS). Persistent RS can be detrimental to cellular survival and functions causing accumulation of DNA damage often leading to genomic instability (GI) and tumourigenesis. Haematopoiesis, a highly regulated process for blood cell generation is strongly affected by physiological ageing; molecular changes, epigenetic alterations, functional decline and abnormal cellular behaviour. These changes are often accompanied with increased DNA damage predisposing cells to leukaemic transformation. However, there is still limited data on how DNA damage and RS can affect normal haematopoiesis. This work was aimed to elucidate the effects of DNA damage in normal murine haematopoietic cells using the CRISPR/Cas9 technology to generate efficient gene knockout (KO) models. Following a simple and rapid pipeline, our CRISPR/Cas9-mediated KO models targeting a DNA replication factor, MCM6, and an anti-ageing factor also involved in DNA damage repair (DDR), SIRT1, were successfully validated for both on-target efficiency and off-target activity at the bulk population and/or single cell level. Our data showed that KO haematopoietic cells exhibited increased levels of DNA damage. This further affected haematopoietic cellular properties and functions in a way that KO cells could share some characteristics of the physiological ageing phenotype predisposing them in profound GI. Finally, another aspect of this work was to model a common chromosomal translocation found in AML patients, MLL-AF9 (MA9), using CRISPR/Cas9 technology. Several techniques and methods have been established over the years for modelling human AML either using murine or human primary cells. Each one of them have their own caveats resulting in poor recapitulation of the actual disease phenotype reported in humans. We therefore used our validated CRISPR/Cas9-system to induce double strand breaks on both chromosomes around the breakpoint cluster region to de novo generate MA9 translocation in murine haematopoietic cells. We successfully confirmed the chromosomal translocation at both DNA and mRNA levels and showed that generated MA9 translocation could confer phenotypic and molecular characteristics of the MA9+ AML cells. Altogether, this data supports that the endogenous expression of a chromosomal or genetic aberration could generate a better, more convenient model to study human diseases. Overall, this study contributes to a better understanding of the role of DNA damage in normal haematopoietic functions and reveals a potential link with ageing-associated characteristics. Finally, this study further supports that CRIPSR/Cas9 technology is a powerful tool with high efficiency, specificity and minimal off-target activity to be used for gene silencing and editing purposes