Discipline: Biological Sciences
Lauren Reich - University of Delaware
Hematopoietic stem cells (HSC) are the precursor of all blood and immune cells. HSCs utilize a metabolic shift from glycolysis to oxidative phosphorylation in order to meet the energy demand for differentiation. The AK2 gene encodes adenylate kinase 2, an enzyme responsible for supplying substrate for ATP synthase in the intermembrane space of the mitochondria. A bi-allelic mutation of AK2 is defined as Reticular Dysgenesis (RD), which causes defects in the differentiation of myeloid and lymphoid lineages of blood cells. In order to elucidate the metabolic requirements of HSC differentiation, we engineer bi-allelic AK2 mutations in primary human HSCs using CRISPR/Cas9 genomic editing. Utilizing single-guide RNAs to target AK2 and control safe harbor gene AAVS1, we employ a dual GFP/BFP reporter system to visualize cell populations with bi-allelic knockouts. Single reporter (GFP) integrated populations were found to have as low AK2 protein expression as a GFP/BFP double knockout. Sequencing confirmed >90% INDEL frequency in GFP+ populations due to a link between homologous recombination and insertion and/or deletion frequency. Due to the rarity of GFP/BFP double knockout populations, we determined the use of a single reporter gene (GFP) is sufficient to achieve a model of RD. This model will allow for further insight into the metabolic requirements regulating hematopoietic stem cell fate and guide the development of novel treatment therapies for immune disorders. Future studies will involve cellular-wide analyses, such as metabolomic studies and RNA-seq to identify differences between control and experimental cellular populations. Future experiments will also investigate the prevalence of byproducts/intermediates of metabolism that are also epigenetic modifiers, such as S-adenosyl methionine, acetyl-coA, and alpha-ketoglutarate.
Funder Acknowledgement(s): The Department of Genetics at Stanford University
Faculty Advisor: Katja G. Weinacht, firstname.lastname@example.org
Role: I maintained various cell cultures, utilized CRISPR to modify cellular genomes, assisted in Fluorescence Activated Cell Sorting, stained cells for future microscopic assessment, determined overall cellular protein concentration amongst treatment groups as well as AK1 and AK2 protein expression levels via western blot.