Exploring the Potential of CRISPRa and CRISPRi to Systematically Dissect Fate Options in Hematopoiesis

Abstract: Advances in our understanding of hematopoietic stem cells (HSCs) have enabled the establishment of differentiation pathways throughout the hematopoietic hierarchy. To further explore the molecular cues governing cell fate options, there is a need to manipulate gene function in a controllable manner. Gene manipulation systems based on CRISPR activation (CRISPRa) or interference (CRISPRi) could be used to achieve this requirement. Here, we have generated doxycycline-inducible CRISPRa and CRISPRi mouse models. To functionally validate these systems, we aimed to induce or repress the chosen genes C/EBPβ and Notch1, respectively. An initial requirement to this end was to obtain functional high titer lentiviruses that could be used to deliver guide RNAs (gRNAs) to cells from the mouse lines, and work was conducted to optimize the production of lentiviruses. Furthermore, we demonstrate the ability to induce the CRISPRa system. However, initial experiments show difficulties in C/EBPβ mediated transdifferentiation of B cells from the CRISPRa mouse model. Thus, further detailed validations are necessary to address the potential problems.