Richie Kohman, PhD, senior research scientist and lead, Synthetic Biology, Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, talks to contributing editor Tanuja Koppal, PhD, about the recent advances in in situ RNA sequencing (RNA-seq) and its emerging applications.

Q: What can you tell us about in situ RNA-seq and how it can be applied?

A:In situ sequencing allows RNA-seq to be performed directly on biological samples and therefore retains the spatial context of the transcripts. It follows a similar conceptual workflow as conventional RNA-seq, where RNA gets converted to DNA amplicons. Sequencing then occurs on custom microscopy equipment with integrated fluidics. This process essentially converts the biological sample into a next-generation sequencing flow cell. In situ sequencing provides a spatial context that is lost by other methods. As cells exist in a dynamic and complex microenvironment, preservation of their spatial relationship can be crucial for understanding their fundamental biology or being able to effectively study and diagnose diseases. In situ sequencing can be applied to many different sample types ranging from whole zebrafish to mouse brain sections to human induced pluripotent stem cells. The technique is agnostic to sample type and hence, the applications can be very diverse. The hope is that once commercialized, the technology can be made available to more laboratories.

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Q: How did you get interested in this technology?

A: My introduction to the field came through the in situ work I was doing with a team of researchers in Dr. George Church’s lab at the Wyss Institute. They were developing a foundational technology in the field and some members of the group founded the company ReadCoor Inc. to commercialize the technology. Since the spinout, I have been running a team at Wyss to create new variations of the technology, as well as find ways to apply it in new directions. For instance, I was recently co-PI with Dr. Church on the Machine Intelligence from Cortical Networks (MICrONS) project funded by Intelligence Advanced Research Projects Activity, which aims to use in situ sequencing to map the neural connectivity within the mouse visual cortex. I became interested in the technology because of the multidisciplinary challenges it posed, as well as the number of applications it can be used for. In particular, I was drawn to its use in studying the brain, as there are still many fundamental questions that remain unanswered in neuroscience.

Q: What are some of the current technical challenges when doing in situ RNA-Seq?

A: With regular RNA-seq, there is not much information obtained on spatial resolution at the sub-cellular level. However, doing in situ RNA-seq by coupling microscopy with sequencing is a formidable engineering challenge and is probably not something that can be set up in a lab for a single experiment or project. There are issues associated with sample handling, sample movement, heat transfer, fluidics, and more that need to be addressed before the imaging and sequencing come into play. There is also the challenge surrounding data acquisition and analysis that needs to be tackled after iterations of sequencing data are generated. The company, ReadCoor Inc., that is commercializing this technology is currently developing instruments and reagents so that laboratories can perform in situ sequencing themselves.

Q: What are some of the upcoming trends and technologies in sequencing that we should be aware of?

Image 1: A representative image of a sequencing round performed on a primary fibroblast.Credit: “Highly Multiplexed Subcellular RNA Sequencing in Situ” - Je Hyuk Lee et al. Science 343, 1360 (2014); DOI: 10.1126/science.1250212A: The inclusion of the 3D context for gene expression introduces many additional, multi-disciplinary challenges relating to instrument engineering, microscopy, and data analysis. Fortunately, the field has solved many of these problems and industrial in situ sequencing is emerging. Soon, it will be increasingly common to analyze the identity of endogenous transcripts within their spatial, biological context.

Two additional trends in the field can be seen emerging. One is that in situ sequencing will be coupled with synthetic biology approaches for writing molecular signatures into biological samples. In this case, the targets of interest will not be natural molecules but rather engineered ones. For instance, in the Synthetic Biology Platform at the Wyss Institute, we engineered a mouse that records its development through evolving CRISPR guide RNAs. By performing in situ sequencing of this RNA, one can obtain 3D lineage maps that are very difficult to obtain from other methods.

Another trend will be the coupling of in situ RNA detection with the simultaneous analysis of other molecular modalities such as proteins and genomic DNA. Because of the scalability of sequencing chemistry, it will be possible to analyze large numbers of different molecular types provided they can be made compatible with the current sequencing pipelines. By analyzing additional target types, a more complete molecular profile of cells and tissues can be obtained.

Richie Kohman, PhD, is a senior research scientist and the lead of the Synthetic Biology Platform at the Wyss Institute for Biologically Inspired Engineering at Harvard University. He received his BS from Santa Clara University and PhD from the University of Illinois Urbana-Champaign, both in chemistry. Subsequently, he was postdoctoral fellow in the Department of Biomedical Engineering at Boston University, an affiliate of the MIT Media Lab, and group leader at Expansion Technologies, Inc. Currently, he oversees all research conducted by the Synthetic Biology Platform including advances in nucleic acid synthesis, in situ sequencing, gene editing, genome recoding, neurotechnology, gene therapy, stem cell therapy, anti-aging, and all aspects relating to the intersection of synthetic biology and synthetic chemistry.