CRISPR-based functional genomics in human dendritic cells

Abstract

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.

Emerging strategies for treating autoimmune disease with genetically modified dendritic cells

“…TLRs are prime examples, and these molecules have been demonstrated to be a co-stimulatory molecule for CTL response, which can be deleted using these approaches. Indeed, the benefits of CRISPR-mediated deletion of TLRs and its constraining effect on T cell responses have been validated in response to LPS stimulation, and clinical trials are currently investigating the potential advantages of this approach [ 99 ]. Another advantage of utilizing directed DNA gene editing technologies is that the directed gene deletion system is co-delivered with a DNA template to facilitate homologous recombination, they can be employed to insert genes of interest into specified genomic loci.…”

“…Unfortunately, this approach also renders the cells susceptible to NK cell-mediated killing, a consequence of “loss of self”. Furthermore, DCs derived from different donors exhibit distinct responses to the same stimulus, such as LPS, suggesting the combined influence of genetic factors and environmental exposure in determining DC functionality and size [ 99 ]. Therefore, it is crucial to comprehensively characterize the inflammatory, transcriptomic, epigenomic, and metabolic profiles of each disease and conduct in-depth phenotypic analysis to discern the disparities between monocytes and TolDCs generated from healthy individuals versus those with autoimmune diseases.…”

Innovative Strategies of Reprogramming Immune System Cells by Targeting CRISPR/Cas9-Based Genome-Editing Tools: A New Era of Cancer Management

“…In addition, the identification of candidate genes that control this specificity to pinpoint the determinants of inter-individual variations in immunity has been revealed. 200 …”

Advancements in CRISPR screens for the development of cancer immunotherapy strategies

“… Jost et al. (2021) 115 PTPN6 / SHP-1 PTPN6 PTPN6 knockout strongly increased TNF-α secretion and moderately decreased IL-10 secretion. human myeloid cell line U937 loss of function self-designed 10-sgRNA-per-gene CRISPR-Cas9 deletion library lentivirus transduction phagocytosis ability NHLRC2 NHLRC2 NHLRC2 negatively regulates RHOA, enabling RAC1-mediated cytoskeletal changes that are critical for phagocytosis.…”

“…CRISPR screen studies using lentivirus transduction have focused primarily on TNF-α secretion pathways in macrophages and moDCs, revealing the roles of METTL3-mediated m6A modification of Irakm mRNA and the KO of TLR2 and PTPN6. 113 , 115 DC studies, using retrovirus transduction, have emphasized antigen cross-presentation, uncovering the impact of Wdfy4 loss on cDC1s in mice. 114 However, challenges persist, such as the absence of sgRNA barcodes for pooled screens in DCs.…”