Performing In Vivo CRISPR Screens Using the FITS Approach

By Guest Blogger

Guest Blogger May 02, 2024

FITS

In vitro CRISPR screens in immune cells have identified many putative regulators of immunity. However, in vitro screens cannot faithfully model the complex spatial, cellular, metabolic, and cytokine microenvironment that exists in vivo. Over the past five years, in vivo CRISPR screens have been used to interrogate T cells, an important immune cell type, in more complex and physiological microenvironments. These screens have primarily been performed in activated T cells, precluding analyses of T cell differentiation beginning with naïve T cells.

The Framework for In vivo T cell Screens (FITS) approach (Milling and Markson et al., 2024), developed and deposited at Addgene by Arlene Sharpe’s lab, enables in vivo screens in naïve T cells. More broadly, this system can be used to perform in vivo screens in other cell types. A key feature of this system is a lentiviral gRNA expression vector (pRDA_526) that (1) enables efficient knockout (KO) of genes in immune cells; (2) is not immunogenic, a necessity for in vivo studies; (3) is compatible with single gene KO and pooled screening approaches; (4) encodes a Thy1.1 selectable marker to facilitate the isolation of modified cells; and (5) is compatible with the introduction of unique molecular identifiers, which can improve the statistical power of in vivo screens.

Three panels. The first shows a mouse with lymph nodes and a tumor being injected with barcoded T cells, which are then enumerated and used to design an in vivo screen targeting an appropriate number of genes based on the set number of mice. The second panel shows in vivo screen hit calling using the FAUST algorithm in targeted (subgenome) screens. The third panel shows in vivo screen clonal dynamics, with Level 1 being gRNA and Level 2 being unique molecular identifier to track distinct cell clones, leading to unique molecular identifiers added to cells with Gene A or B knocked out, enabling tracking of gene KO clonal dynamics in lymph nodes and tumor cells.

Figure 1: Using FITS to perform in vivo CRISPR screens to assess T cell responses and clonal dynamics in lymph nodes (LN) and tumor cells. Figure used with permission from Milling and Markson et al., 2024.

Proof of concept

The FITS approach was used to optimize conditions for a screen that assessed genes that regulate antigen-specific CD8+ T cell responses to a murine tumor model in vivo. In this screen, we used the pRDA_526 vector to knock out 30 genes putatively involved in CD8+ T cell responses to cancer. The screen recovered known regulators, such as Ptpn2, Pdcd1, and Zc3h12a, demonstrating the robustness of the system and utility of the pRDA_526 vector. Moreover, the screen identified putative novel regulators of CD8+ T cell responses in tumors, demonstrating that this system can also reveal novel pathways.

The FITS deposit also includes pLX_305_Ovalbumin, a plasmid that enables introduction of the model antigen, Ovalbumin, into cell lines to study antigen-specific responses.

Find FITS plasmids here!

0136_Martin_Lafleur_001 (1)-min Marty LaFleur is a Postdoctoral Fellow in Arlene Sharpe’s laboratory at Harvard Medical School and is interested in improving CD8+ T cell responses to cancer.

References and resources

Milling, L. E., Markson, S. C., Tjokrosurjo, Q., Derosia, N. M., Streeter, I. S. L., Hickok, G. H., Lemmen, A. M., Nguyen, T. H., Prathima, P., Fithian, W., Schwartz, M. A., Hacohen, N., Doench, J. G., LaFleur, M. W., & Sharpe, A. H. (2024). Framework for in vivo T cell screens. J Exp Med, 221(4), e20230699. https://doi.org/10.1084/jem.20230699.