3029 – COMBINATORIAL GENE TARGETING IN PRIMARY HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Abstract

The CRISPR/Cas9 system offers enormous versatility for functional genomics. However, many applications, such as combinatorial perturbations for disease modelling and studies of gene interactions, have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, we addressed the challenge of multiplexed gene perturbations in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs) by co-delivering lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The use of fluorescent markers allows for tracing of single- or double-edited cells and we achieved robust double knockout of the cell surface markers CD45 and CD44 with an efficiency of around 70%. As a functional proof of concept, we demonstrated the use of this system to study gene dependencies for cell survival by simultaneously targeting the cohesin genes STAG1 and STAG2, as a model of synthetic lethality. Furthermore, we showed that this system can be used to study cooperative genetic events, and found a combinatorial effect with potential synergy for HSPC expansion when targeting the Aryl Hydrocarbon Receptor (AHR) in combination with members of the epigenetic modifier CoREST. Taken together, we established a paradigm for multiplexed CRISPR/Cas9 editing in primary human HSPCs, which enables studies of genetic dependencies and cooperation. This traceable multiplexed system has important implications for modelling polygenic diseases and for investigating gene interactions. The CRISPR/Cas9 system offers enormous versatility for functional genomics. However, many applications, such as combinatorial perturbations for disease modelling and studies of gene interactions, have proven to be challenging in primary human cells compared to cell lines or mouse cells. Here, we addressed the challenge of multiplexed gene perturbations in primary human cord blood-derived hematopoietic stem and progenitor cells (HSPCs) by co-delivering lentiviral sgRNA vectors expressing either Enhanced Green Fluorescent Protein (EGFP) or Kusabira Orange (KuO), together with Cas9 mRNA, to simultaneously edit two genetic loci. The use of fluorescent markers allows for tracing of single- or double-edited cells and we achieved robust double knockout of the cell surface markers CD45 and CD44 with an efficiency of around 70%. As a functional proof of concept, we demonstrated the use of this system to study gene dependencies for cell survival by simultaneously targeting the cohesin genes STAG1 and STAG2, as a model of synthetic lethality. Furthermore, we showed that this system can be used to study cooperative genetic events, and found a combinatorial effect with potential synergy for HSPC expansion when targeting the Aryl Hydrocarbon Receptor (AHR) in combination with members of the epigenetic modifier CoREST. Taken together, we established a paradigm for multiplexed CRISPR/Cas9 editing in primary human HSPCs, which enables studies of genetic dependencies and cooperation. This traceable multiplexed system has important implications for modelling polygenic diseases and for investigating gene interactions.