Spatial CUT&TAG is a molecular biology technique for mapping where specific proteins attach to DNA within a tissue sample while preserving the cells’ original locations. It provides a spatial map of the epigenome, the collection of chemical modifications to DNA and its associated proteins that regulate gene activity. This spatial dimension helps explain how cells with the same genetic code can form distinct tissues and perform different functions.
The Core Mechanism of Spatial CUT&TAG
The process begins with a thinly sliced section of fresh frozen tissue placed on a specialized glass slide. This slide is coated with a grid of oligonucleotides, short DNA strands that will later play a part in location tracking. The first step is permeabilization, where the cell membranes are made porous, allowing antibodies and other molecules to enter the cells and nuclei while keeping the overall tissue structure intact.
Following permeabilization, a primary antibody is introduced to the tissue slice. This antibody is highly specific, designed to bind to only one type of protein, such as a particular modified histone or a transcription factor. The specificity of this antibody ensures that subsequent steps only occur at the genomic locations where the target protein is bound.
Next, a secondary antibody carrying an enzyme called pA-Tn5 transposase is added. This secondary antibody binds to the primary antibody, delivering the Tn5 enzyme to the target protein. The Tn5 transposase is capable of cutting DNA and inserting other DNA sequences in a process called tagmentation, concentrating its activity where the protein is located.
With the enzyme in position, it is activated to perform in-situ tagmentation. The Tn5 transposase simultaneously cuts the DNA adjacent to the target protein and attaches sequencing adapters to the ends of the cut fragments. This reaction occurs within the cells of the tissue slice, preserving the spatial context of the DNA before the tagged fragments are released.
The released fragments are captured by the oligonucleotides on the slide’s surface. Each oligonucleotide contains a unique spatial barcode corresponding to its x-y coordinate on the grid. When a DNA fragment is captured, it is ligated to this barcode, linking the DNA sequence to a physical location within the tissue and creating a spatially indexed library.
Decoding the Spatial Epigenomic Map
Once the barcoded DNA fragments are collected from the slide, they are prepared for next-generation sequencing. This process reads the sequence of each DNA fragment along with its attached spatial barcode. The result is a dataset containing millions of short DNA sequences, each tagged with information about its original position within the tissue.
In the bioinformatic analysis, each sequencing read is separated into its two components: the genomic DNA sequence and the spatial barcode. The genomic portion is aligned to a reference genome to identify its origin on the chromosomes. This reveals which genes or regulatory regions were near the protein of interest.
Simultaneously, the spatial barcode from each read is decoded to its known coordinate on the tissue slide. Linking the genomic location of the DNA fragment with this spatial barcode maps each protein-DNA interaction back to its point of origin within the tissue.
The final output is a visual map of epigenetic modifications across the tissue, often displayed as a heatmap over a microscope image. The colors represent the density of the target protein, with warmer colors indicating high enrichment. This allows scientists to identify “spatially variable features,” which are epigenetic marks that are more prevalent in distinct anatomical regions.
Applications in Biological Research
In neuroscience, Spatial CUT&TAG helps explore the epigenetic underpinnings of brain function and disease. Scientists can map histone modifications in specific areas like the hippocampus versus the cortex, gaining insights into how epigenetic regulation contributes to learning and memory. The technique can also identify how these patterns are altered in neurological disorders, revealing the cellular basis of disease.
In cancer biology, the technique provides insights into the tumor microenvironment, which includes cancer, immune, and structural cells. Researchers can use Spatial CUT&TAG to distinguish the epigenetic profiles of malignant cells from surrounding healthy tissue. This can reveal how cancer cells reprogram their gene expression to promote growth and evade the immune system, offering potential targets for new therapies.
The method is also applied in developmental biology to study how epigenetic patterns guide embryo formation. As an organism develops, cells differentiate to form tissues and organs, a process driven by changes in gene regulation. Spatial CUT&TAG can track histone modifications across an embryo, showing how different regions are epigenetically programmed to become distinct parts of the body.
Comparison with Related Genomic Techniques
Spatial CUT&TAG offers advantages over methods like bulk CUT&TAG and ChIP-seq. These bulk techniques are effective at identifying protein-DNA interactions but require the tissue to be dissociated or ground up, averaging the signals from millions of cells. This process destroys the native spatial organization of the tissue, which the spatial method preserves to show how epigenetic marks are distributed.
The technique also differs from single-cell CUT&TAG, which provides high-resolution epigenetic information for individual cells but loses their original locations during isolation. Spatial CUT&TAG complements this by retaining positional information. This is useful for understanding how a cell’s function is influenced by its neighbors and local environment.
Spatial Transcriptomics is another related technique that measures gene expression by capturing messenger RNA (mRNA) on a spatially barcoded slide, revealing which genes are active. In contrast, Spatial CUT&TAG measures the upstream epigenetic modifications that regulate gene activation. The two techniques are highly complementary, as one maps regulatory potential while the other maps the resulting gene activity, providing a more complete picture of tissue function.