The Innovator Blog Series celebrates research conducted by 10x Genomics customers who have demonstrated scientific ingenuity by adapting Chromium Single Cell or Visium Spatial sequencing-based assays. These innovations are distinguished by their originality and potential impact on scientific discovery, including providing access to novel analytes, advancing multiomic analysis techniques, and demonstrating critical applications to human health and disease research. These techniques are customer developed, meaning they are not officially supported by 10x Genomics.
Giving CRISPR screens context
The human genome contains approximately 20,000 protein-coding genes (1), but the real treasure and impact of this knowledge will be found in characterizing the role of those genes—at increasing resolution with cellular and tissue specificity—in health and disease.
In its early development, CRISPR screening technology provided the ability to edit specific genes and assess the subsequent effects on a cell’s survival. With the introduction of single cell CRISPR screening, researchers were able to get a single cell view of the effects that hundreds of perturbations had on whole transcriptome gene expression and cell surface proteins. This view provides deeper functional insights into how a gene influences the expression of other genes and the overall cellular phenotype.
CRISPR screening, in this form, is a powerful solution to study cell-intrinsic genes, meaning genes whose effects are seen predominantly within the cell. However, the beauty (and challenge) of biology is that cells are only individual players in a larger tissue system, whose architecture and function is dictated by a complex network of cellular interactions and molecular events that occur outside of the cell. Studying the genes that control these interactions requires spatial context—a view of the morphology of the tissue—alongside a readout of the cellular and molecular composition of the tissue and the genetic perturbations in question. However, current CRISPR screening methods require tissue dissociation and therefore loss of spatial context.
In a recent Cell publication (2), Brian Brown, PhD, Director of the Icahn Genomics Institute, Icahn School of Medicine at Mount Sinai, with lead authors Maxime Dhainaut, PhD, and Samuel Rose, PhD, described this limitation to current CRISPR screening methods. Their research focuses on the tumor microenvironment (TME), and specifically, the genetic basis of tumor immune composition, which has not been well defined, despite being a critical determinant of immunotherapy response and cancer outcomes. Identifying dysregulated genes responsible for controlling tumor immune composition could be an important source of new therapeutic targets.
“...Many genetically distinct subclones exist in proximity to tumors and can have different TME compositions; however, how specific genes influence the TME, or how neighboring clones influence one another is not well defined… While some genes involved in orchestrating the TME have been identified, the potential roles of many genes in influencing the architecture and immune composition of different tumors are not established” (2).
New probes maintain tissue morphology of CRISPR edits
Seeking to fill this gap in our understanding of the genes that control immune infiltration and composition in the TME, Dr. Brown and his team used a spatial technology platform to perform functional genomics screens. Leveraging high-dimensional spatial immunohistochemical staining and Visium Spatial Gene Expression, they developed a modified protocol to visualize the cellular, molecular, and architectural effects in tumor lesions of knocking out genes associated with the tumor immune response, including cytokine signaling and immune cell interactions.
One key modification that enabled this approach, called Perturb-map, was the development of Pro-Codes (3). The Pro-Code is a cell-intrinsic combinatorial protein barcode system introduced via a lentiviral vector that can be used to identify the CRISPR guide RNA expressed within each individual cell. These Pro-Codes are composed of triplet combinations of peptides that are expressed together in a protein scaffold displayed on the cell surface. Each Pro-Code is expressed from a lentiviral vector that also expresses a specific guide RNA. Detection of a Pro-Code using antibody-based methods (immunofluorescence, CyTOF, or imaging mass cytometry) then enables inference of the associated CRISPR edit.
“We stain tissues all the time with antibodies and image them,” Dr. Brown shared in a recent GEN webinar (4). This would make translating the Pro-Code system to spatially resolved tissue analysis straightforward, given the accessibility of highly multiplexed immunohistochemical tissue staining methods. Using a mouse model of lung cancer as an example, Dr. Brown and his team cultured cancer cell lines with CRISPR knockouts (KOs) of key tumor immune response genes. They injected those cells into mice, then harvested the tumors for sectioning and multiplex imaging. This revealed a strong clonal structure of mouse lung tumor lesions, where it appeared specific cancer clones with a single CRISPR edit were seeding distinct tumor lesions.
This spatially resolved imaging readout identified a core set of genes for which loss of function seemed to influence tumor biology. In order to deeply characterize the results of those CRISPR edits in specific lesions, the team combined their Perturb-map method with Visium Spatial Gene Expression, sectioning tumor samples onto Visium slides to enable unbiased gene expression analysis of spatial regions corresponding to the lesions (3).
Tgfbr2 loss results in immune exclusion
Spatial gene expression analysis of CRISPR KO tumor lesions revealed new insights into the part the tumor microenviroment plays in helping cancer resist anti-tumor immune responses. Annotating each lesion’s gene signature with the corresponding gene perturbation revealed that tumors bearing Tgfbr2 or Ifngr2 KOs had a distinct gene signature compared to other tumors. Focusing on Tgfbr2, an immune receptor for TGF-b, a regulatory cytokine associated with immune suppression, the team observed that knocking out Tgfbr2 led to a highly fibromucinous tumor structure and marked exclusion of immune cells in tumor lesions (2).
Despite the loss of Tgfbr2 as an agent of immune suppression in cancer cells, spatial transcriptomics data revealed that fibroblast populations in the TME were compensating, bearing an enriched gene expression signature for TGF-b activation (2). TGF-b pathway activation in the TME may therefore represent a mechanistic explanation for the observed immune exclusion in these Tgfbr2 KO tumor lesions, and a new option for therapeutic intervention.
True to the spirit of the Innovator Series, Perturb-map combined with Visium Spatial Gene Expression represents the next evolution of CRISPR screening technology, providing access to CRISPR gRNA and the transcriptomic effects of perturbation in the tissue context. With these powerful insights into extracellular gene function and the effects of genetic perturbation on the cellular environment, this spatial CRISPR technique promises to reveal much more about the genes and pathways involved in tumor biology and many other areas of study.
Read the publication here →
And explore Visium Spatial Gene Expression →
- Nurk S, et al. The complete sequence of a human genome. Science 376: 44–53 (2022). doi: 10.1126/science.abj6987
- Dhainaut M, et al. Spatial CRISPR genomics identifies regulators of the tumor microenvironment. Cell 185: 1223–1239.e20 (2022). doi: 10.1016/j.cell.2022.02.015
- Wroblewska A, et al. Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens. Cell 175: 1141–1155.e16 (2018). doi: 10.1016/j.cell.2018.09.022
- Brown B. Spatial Functional Genomics: CRISPR Screens in the Spatial Era. GEN Webinar.