Tumor immunology CRISPR screening: present, past, and future

Abstract

Recent advances in immunotherapy have fundamentally changed the landscape of cancer treatment by leveraging the specificity and selectivity of the adaptive immune system to kill cancer cells. These successes have ushered in a new wave of research aimed at understanding immune recognition with the hope of developing newer immunotherapies. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technologies and advancement of multiomics modalities have greatly accelerated the discovery process. Here, we review the current literature surrounding CRISPR screens within the context of tumor immunology, provide essential components needed to conduct immune-specific CRISPR screens, and present avenues for future research.

Figure 1.

This schematic summarizes a simplified conceptual framework of immune cell interactions in cancer and the utility of CRISPR screen for identifying key regulators of tumor immunology. Macrophages and dendritic cells (DCs) recognize damage-associated molecular patterns (DAMPs) prior to the activation of the adaptive immune response via major histocompatibility complex (MHC) antigen presentation and cytokines expression. Neoantigens from tumor cells are presented by DCs to CD4 and CD8 T cells. CD8 T cells would get activated and mediated malignant cells killing. CD4 T helper cells release IFN-γ to mount innate immune response against cancer cells, like cytotoxicity mediated by natural killer cells (NK cells). B cells also get activated by tumor neoantigens and secret antibodies against the neoantigens, which would lead to antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells. As genetic regulations play crucial roles across all these immunological processes, CRISPR screens can in principle be applied in all these cell types for each specific process.

Figure 2.

This schematic summarizes the three main modes of CRISPR gene editing or gene expression perturbations used in mammalian cell biology including tumor immunology.

Figure 3.

This schematic outlines the general workflow of CRISPR screen in various settings. 1) CRISPRi, CRISPRa or CRISRPko components (guide RNA library included) can be introduced by viral vectors like AAV, lentivirus or retrovirus into different cell types. 2) Depending on the phenotypes of interest, either in vitro or vivo based assays can be adopted for guide RNA selection. 3) Next generation sequencing (NGS) is then performed to check guide RNA enrichment or deletion. 4) Top hits are then validated by cloning guide RNAs individually to target gene of interest, followed by similar in vitro or in vivo assays used for the screen initially. 5) The final goal of these screens is to identify targets that are able to increase tumor cell susceptibility or to improve immune cells mediated anti-tumor responses.