Defining Regulators of Human Hematopoietic Stem Cells using CRISPR/Cas9 Gene Targeting

Projekt: Avhandling


Hematopoietic stem cells (HSCs) in the adult human bone marrow (BM) have the ability to self-renew and to give rise to all blood cells of the different lineages, thereby being responsible for the replenishment of blood cells throughout life. Due to these characteristics, HSCs have been extensively used in the clinic to treat hematological disorders and malignancies. However, a significant limitation in the transplantation setting is an insufficient number of stem cells. Therefore, work to understand HSC regulation aiming to find strategies to facilitate HSC expansion is of great importance, and could be applicable in clinical settings of bone marrow transplantation and gene therapy. In our work to understand positive and negative regulators of this important cell type, genome engineering systems, which have been recently highlighted as a possible highly specific and effective tools, can be used.

Over the last few years our laboratory has developed strategies to screen for modifiers of self-renewal/proliferation in human hematopoietic stem and progenitor cells (HSCPs) using complex viral vector libraries expressing shRNA. Previous work in the lab has demonstrated the feasibility of pooled shRNA screens in conjunction with next generation sequencing to identify genes and pathways that regulate primary human stem cell populations and that can be targeted to enhance ex vivo HSC expansion (Ali et al, Blood 2009; Baudet et al, Blood 2012)5,6. We have further identified genes with central roles in both normal and malignant HSPC function, such as JARID2, an important component of the polycomb repressive complex (Kinkel et al, Blood 2015)7. More recently our shRNA screens identified the cohesin genes as novel major players in regulation of human HSCs, which together with the recent discovery of recurrent cohesin mutations in myeloid malignancies, point toward a direct role of perturbed cohesin function as a true driver event in myeloid leukemogenesis (Galeev et al, Cell Reports 2016)8.

Here, we aim to further exploit this discovery tool using the CRISPR/Cas9 system as well as more stringent assays for human HSC function. Due to its simplicity and adaptability, CRISPR has rapidly become one of the most popular approaches for genome engineering. The ease of generating sgRNAs targeting basically every locus in the genome makes CRISPR/Cas9 an ideal genome editing system for large-scale forward genetic screens. Before the discovery of CRISPR/Cas9, RNA interference using shRNAs was commonly used for genetic screens. However, this method is prone to off-target effects and incomplete knock-down of the target can result in false negative results.
Gällande start-/slutdatum2018/05/01 → …