In situ sequencing (ISS) is a new method by which mRNA is sequenced directly in a section of fixed tissue or cell sample. The key to ISS’s power is the linkage between sequencing information and its location—in some cases, subcellular location. This differs from conventional sequencing, which analyzes samples after removing them from their endogenous environments and consequently losing location information.
Developed by Mats Nilsson’s group at the Science for Life Laboratory at Stockholm University, ISS quantifies several hundred mRNA transcripts simultaneously and spatially resolves them with single-cell resolution. The method uses four fluorescent dyes to indicate nucleic acid bases, padlock probes for RNAs of interest, and enzymes that catalyze the formation of circularized DNA at the locations of the padlock probes, in a mechanism called rolling circle amplification. The resulting fluorescent DNAs are read using imaging technologies. The ability to generate mRNA expression profiles with location information within tissues is a powerful tool. Here is a look at some new ISS developments and applications.
Better spatial detection
Nilsson’s research group has developed a new method called hybridization-based in situ sequencing (HybISS). Like previous ISS methods, HybISS uses rolling circle amplification of padlock probes. Its different probe designs allow for a new barcoding approach that uses sequencing-by-hybridization chemistry. According to Chika Yokota, manager of the In Situ Sequencing Facility Unit at the Science for Life Laboratory, HybISS gives improved sensitivity in spatial detection in addition to greater flexibility and multiplexing.
Nilsson’s group suggests that HybISS can be applied to create a comprehensive spatial reference map for cell atlas projects. “However, it still requires a lot of time to build a complete gene expression profile of the whole organ or tissue that is necessary for studying organ/tissue atlases,” says Yokota. “Further increases in throughput need to be addressed.”
It’s also possible that HybISS can help to fight the novel coronavirus. “HybISS can be used with probes selected to detect distinct immune cell subsets, as well as to look at different immune responses in different tissue samples from COVID-19 patients,” says Yokota. “This will enable us to generate spatially resolved immune-cell profiling of the disease.”
Commercialized ISS tools
The Swedish company Cartana has commercialized molecular tools developed by Nilsson’s group. Cartana’s tools for spatial analysis of transcripts are mainly aimed at cell type mapping, using a targeted approach directed at any nucleic acid sequence. “It’s fundamental biology and can also be applied to a specific question, such as if I have my potential drug target, in what type of cell is it expressed?” says Malte Kühnemund, Cartana’s executive vice president and head of R&D.
Cartana’s technology has been used in at least twelve tissue types in mouse and human in addition to different types of tumors, and they’re working to identify immune cells inside tissues. “If you can map and identify immune cells in the tumor, for example, then you can look at how well a tumor is infiltrated and assess how much immune response there is,” says Kühnemund. “That’s a hot application because then you can gear it toward tailoring cancer therapies.”
Cartana also created padlock probes for the novel coronavirus. “Using fixed tissue samples from COVID-19 patients, we’re trying to map the virus itself, as well as look at what types of cells it overlaps with,” Kühnemund says.
Data analysis for ISS is challenging. “You get a localized map of the expression of hundreds of genes in the form of an information spreadsheet with gene counts and coordinates,” says Kühnemund. Cartana is addressing this challenge by building software, and Kühnemund believes that the difficulties will be overcome as the new field matures and more people are using the technology and developing open-source tools.
In the future, Kühnemund hopes to develop Cartana’s technology to measure the efficiency of a cancer treatment and to develop better drugs. “Our technology can be used to monitor and quantify the efficacy of a treatment, and maybe ultimately, longer down the road, this could also have diagnostic potential,” he says.
A possible turnkey solution
In contrast to Cartana’s targeted ISS approach, an untargeted approach known as fluorescent in situ sequencing (FISSEQ) was developed in George Church’s lab at the Wyss Institute for Biologically Inspired Engineering at Harvard Medical School. Since then, Richie Kohman, lead senior scientist for synthetic biology at the Wyss Institute, has led the development of a variety of different versions of in situ single-cell sequencing, including the targeted analysis of RNA, genomic DNA, viral RNA, and barcoded antibodies against proteins. “We can even get super-resolution data by embedding our targets into a hydrogel and physically enlarging it,” he says. “These differences in the versions manifest themselves in the early steps of the protocol, whereas the sequencing iterations, imaging, and data analysis are similar for the various methods.”
Kohman believes that industry may soon help to address some of the challenges of ISS. “Our protocols for target capture and sample preparation are quite refined and will work well in anyone’s hands, but for a lab new to the technology, it is a large feat to overcome the hardware engineering and image analysis challenges of in situ sequencing,” he says. ReadCoor, aWyss Institute spinoff company, licensed FISSEQ and is currently developing it for commercialization into a turnkey solution, combining targets with 3D images of cell or tissue samples.
ISS and RNA binding proteins
The lead author on the original FISSEQ publication was Je Lee, now an assistant professor at Cold Spring Harbor Laboratory developing other ISS methods. The Lee group’s newest method is INSTA-seq (in situ transcriptome accessibility sequencing; manuscript in review), for which they invented a bidirectional paired-end sequencing chemistry that reads short molecular barcodes of individual cDNA molecules in situ (rather than a targeted detection of probes as in previous ISS methods).
After fluorescent imaging of cells or tissues, they use 12-base barcodes to precipitate the cDNA molecules of interest, leading to gene expression analysis with read lengths of over 300 bases in only one day of imaging. The method can also detect regulatory information in the form of footprints of RNA binding proteins, so it can map interactions between mRNA and regulatory RNA binding proteins in situ with sub-cellular resolution. Lee’s lab is collaborating with Gene Yeo, professor of cellular and molecular medicine at UCSD, to develop an automated workflow for the INSTA-seq method with a turnkey device.
Lee believes ISS has the potential to revolutionize functional genomics. “The moment you can precisely pinpoint a molecular mechanism with nucleotide resolution inside individual cells in an actual biological specimen in situ, it changes the workflow of how to answer the question of how cell–cell interactions influence gene regulation,” says Lee. “I think spatial functional genomics are the future of in situ sequencing.”