Orthogonal CRISPR Library Screening Combined with Single-Cell Sequencing in Immunological Research

Orthogonal CRISPR Library Screening Combined with Single-Cell Sequencing in Immunological Research

Orthogonal experiments are a type of experimental design method that involves multiple factors and levels. They can effectively reduce the number of experiments, allowing researchers to obtain sufficient information from a small number of experiments. Combining currently popular target screening tool—CRISPR libraries and single-cell sequencing technology—can detect differences between cells and identify potential disease treatment targets.

Today, we will share an article published in Nature Genetics (IF=31) by the team of Charles A. Gersbach from Duke University: "Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens." This article develops a pooled epigenetic CRISPR screening method to systematically evaluate the effects of activation or inhibition of 120 transcriptional and epigenetic regulators on the state of human CD8+ T cells.

Adoptive T cell therapy (ACT) redirects T cells to cancer cells by expressing engineered receptors that recognize and bind tumor-associated antigens, thus holding great potential for cancer treatment. The state and function of T cells are largely regulated by specific transcription factors (TFs) and epigenetic modifiers, which process intrinsic and extrinsic signals into complex and finely regulated gene expression programs. In this article, the authors developed a method for interference (CRISPRi) or activation (CRISPRa) screening in primary human T cells and applied it to systematically analyze the effects of 120 genes on the state of human CD8+ T cells. These screenings and subsequent characterizations revealed that overexpression of BATF3 supports the specific characteristics of memory T cells, combats T cell exhaustion, and improves tumor control.

The following is an introduction to the screening process using CRISPR library in this article:

The authors designed a gRNA library targeting 120 transcription factors and epigenetic modifiers related to T cell states. CCR7 is a well-characterized T cell marker and is highly expressed in specific T cell subsets (Figure 1). To detect whether specific gene perturbations changed the T cell state, the authors used CCR7 expression as a screening readout.

Figure 1: CRISPR interference or activation gene screening identifies transcriptional and epigenetic regulators of human CD8+ T cell states

Multiple gRNAs targeting BATF and BATF3 were enriched in opposite directions in CRISPRi and CRISPRa screenings, and BATF and BATF3 were among the most significant hits at the gene level, highlighting the efficacy of coupling loss-of-function or gain-of-function perturbations. Combined with single-cell RNA sequencing (scRNA-seq) analysis of the transcriptional effects of each candidate gene identified in the authors' CRISPRi or CRISPRa screenings (Figures 2-3) the analysis showed that gRNAs targeting BATF3 had the strongest perturbation effect in CRISPRa, with BATF3-induced genes enriched in DNA and messenger RNA metabolic processes, ribosome biogenesis, and cell cycle pathways, suggesting that BATF3 improves T cell fitness.

Figure 2: Changes in gRNA abundance between CCR7 HIGH and CCR7 LOW populations in CRISPRi and CRISPRa 

Figure 3: Average fold change in CCR7 expression compared to control cells for each gRNA in CRISPRi and CRISPRa

BATF3 can promote the survival and memory formation of mouse CD8+ T cells. Considering that BATF3 ORF delivery results in higher BATF3 expression than endogenous BATF3 activation, the authors used ectopic BATF3 expression for all subsequent experiments. In an in situ human HER2+ breast cancer model, CAR T cells co-expressing BATF3 significantly reduced tumor activity compared to control CAR T cells (Figure 4). Additionally, to further identify transcription factors that can amplify the effect of BATF3 overexpression (OE), the authors constructed a gRNA library targeting all human TF genes while overexpressing BATF3/mCherry (Figure 5). The authors performed a pooled CRISPR-KO screening of all human TF genes with and without BATF3 OE, combining TF OE with TFome KO screening in an orthogonal manner to dissect cofactors and downstream factors. This screening system can serve as the basis for designing the next generation of cancer immunotherapies, potentially bringing better outcomes for cancer treatment in the future.

Figure 4: BATF3 OE enhances CAR T cell efficacy

Figure 5: CRISPR KO screening of BATF3 cofactors and cancer immunotherapy targets

I guess you have gained a better understanding of the combination of library screening and single-cell sequencing through the above case. But if you still want to discuss further the library screening method and your screening project with our scientists, feel free to reach out by clicking the "Contact Us" on the right>>>

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Reference:

McCutcheon S R, Swartz A M, Brown M C, et al. Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens[J]. Nature Genetics, 2023, 55(12): 2211-2223.