CRISPR/Cas9 targeting of single genes in human blood cell progenitors to study Natural Killer cell development

Abstract

Natural killer (NK) cells provide a valuable resource for use in cellular immunotherapy, due to their ability to destroy cancerous cells without prior sensitisation or damage to healthy host cells. Here I show that I can generate biallelic knockout (KO) of genes known to be involved in mouse NK cell development in cord blood CD34+ hematopoietic progenitor cells (HPCs) using a lentiviral CRISPR/Cas9 system. Biallelic KO of E4BP4, a transcription factor associated with NK cell commitment in mouse, resulted in the absence of human NK cell differentiation in vitro, indicating the importance of E4BP4 expression during early human NK cell development. Deletion of a single EOMES allele, a transcription factor associated with NK cell maturation, also resulted in a failure of NK cell differentiation, suggesting EOMES may impact early human NK cell development. Gene targeting of the inhibitory NKG2A receptor showed successful generation of a phenotypic NKG2A KO NK cell clone, allowing for investigation into the effect of NKG2A deficiency on human NK cell function. This methodology allows for the investigation of genes thought to be important in human NK cell development. Its further application will give insights into how such genes contribute to human NK cell production and function. By using cord blood CD34+ progenitors to produce NK cells, we can generate large quantities of human NK cells. This provides the possibility of generating large amounts of “engineered” human NK cells with enhanced effector function(s) for NK cell immunotherapy.