Uncovering Novel Regulators of Dendritic Cell Reprogramming Through Crispr/Cas9 Screening

Cellular identity generally remains stable and is regulated by specific transcription factors. However, perturbation of gene expression can alter cellular identity. Direct cell reprogramming induces a desired cell fate without transitioning through a pluripotent stage, making it a powerful approach for therapeutic development and mechanistic studies of cell fate conversion. Using PU.1, IRF8 and BATF3, mouse and human fibroblasts can be converted into type one conventional dendritic cell-like cells. However, the regulatory mechanisms of chromatin regulators or RNA modifiers in iDC1 reprogramming are still poorly understood. CRISPR/Cas9 screenings are a powerful approach to identify regulatory networks in biological processes. Therefore, we used CRISPR/Cas9 screening to target epigenetic regulators and characterize barriers and facilitators of DC reprogramming. Furthermore, 18 barriers and 13 facilitators were top candidates for individual validation. Therefore, multiplexed guide RNA vectors were cloned to induce gene knockouts, confirmed by sequencing and validated for most selected barriers. The expression levels of CD45 and HLA-DR were determined by flow cytometry on day 9. Out of the 13 candidate facilitators, 5 slightly increased the non-reprogrammed population. Moreover, 7 of the 18 candidate barriers increased the CD45 and HLA-DR positive reprogrammed population. Furthermore, those hits were stained for both reprogramming markers at various time points to study the kinetics and efficiency of DC reprogramming. CD40, CD226, XCR1, crucial for cDC1 cells, were stained for the assessment of further characteristics. For the selected facilitators, no change of the non-reprogrammed population could be characterized over time. Moreover, the identified candidate barriers increased the reprogrammed population over time, with SND1 being the most significant and HIRA showing interesting mechanisms. For the facilitators and barriers, CD40 and CD226 remain unaltered, and XCR1 could not be determined throughout the time course as XCR1 gene expression had not been identified and the alternative staining approach did not work. In the past, the histone chaperone HIRA has already been identified as a barrier in neuronal reprogramming. The oncogene SND1 could act as a barrier by having an important role in the influence of the NF-κB pathway and miR-221, which has been previously identified as targets by the characterized barrier c-Jun. Overall, our investigation into the effects of selected barriers and facilitators has revealed some interesting insights into the regulatory mechanisms in DC reprogramming. To gain deeper understanding of the mechanisms mediated through HIRA and SND1 silencing, further experiments, like ATAC-Seq analysis or RNA- sequencing, need to be conducted.