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FISSEQ: Fluorescent In Situ Sequencing

A highly multiplexed technology for precisely locating multiple RNA molecules simultaneously within cells and tissues

Working copies of active genes — called messenger RNAs or mRNAs —translate the genetic information present in DNA into proteins within the cells’ multiple compartments. They are often positioned strategically within cells in ways that contribute critically to how cells and tissues grow, develop and function, and their mislocation can lead to disease development. To analyze many or all mRNAs present in cell or tissue samples simultaneously, scientists usually physically disrupt the cells, isolate the mRNAs and then use next generation sequencing methods to determine the exact sequence of “letters”, or bases identifying each mRNA. Genome sequencing provides a list of genes that are present, however, the valuable spatial information regarding where each mRNA is located within cells and tissues is lost.

George Church, Ph.D., a Core Faculty member at the Wyss Institute and Professor of Genetics at Harvard Medical School, explains how fluorescent in situ sequencing could lead to new diagnostics that spot the earliest signs of disease, and how it could help reveal how neurons in the brain connect and function. Credit: Wyss Institute at Harvard University.

A method developed at the Wyss Institute allows scientists to both, pinpoint the location of thousands of mRNAs (and other types of RNAs) at once in intact cells and determine the sequence of bases that identify each one of them. The method is carried out with enzymes that copy each mRNA into a matching strand of DNA ‘on-site’ and multiplying that DNA strand many times to create tiny balls of replica DNA, still fixed to the original spot. Then, with the help of four different fluorescent dyes—one for each of the DNA’s four bases—a sequence of flashing colors reveals each replica DNA’s exact sequence under a super-resolution microscope. To better separate the color sequences from often densely packed replica DNA balls, the Wyss researchers have developed a technique called partition sequencing that assigns each ball of replica DNA an address code based on its base sequence allowing to only analyze a fraction of the replica DNA balls at a time.

By looking comprehensively at gene expression within cells, we can now spot numerous important differences in complex tissues like the brain that are invisible today. This will help us understand like never before how tissues develop and function in health and disease.

George Church


FISSEQ could be widely applied in many disease diagnostic and therapeutic areas. For example in cancer research, it might lead to earlier diagnosis, help track how gene mutations impact local cancer invasion and metastasis, better define responses to modern targeted therapies, and uncover new drug targets. In addition, the method could also help biologists understand how the spatial organization of distinct mRNAs relates to stem cell differentiation and tissue morphogenesis during embryonic development. FISSEQ is also used as a key technology in an Intelligence Advanced Research Projects Activity (IARPA)-funded effort to map neurons in the brain.

The FISSEQ technology has been licensed to ReadCoor Inc., a start up company spun out of the Wyss Institute, which will commercialize it as a new generation sequencing platform, allowing researchers to perform high throughput RNA sequencing and obtain the cellular locations of multiple RNAs simultaneously in intact cell and tissue samples of their choice.


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