Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have developed a system that can detect a particular RNA sequence in live cells and produce a protein of interest in response. Using the technology, known as reprogrammable ADAR sensors (RADARS), the team showed how they could identify specific cell types, detect and measure changes in the expression of individual genes, track transcriptional states, and control the production of proteins encoded by synthetic mRNA.
The platform even allowed the team to target and kill a specific cell type, which points to the potential future use RADARS to help detect and selectively kill tumor cells, or carry out cell-specific genome editing.
“One of the revolutions in genomics has been the ability to sequence the transcriptomes of cells,” said Fei Chen, PhD, a core institute member at the Broad, Merkin Fellow, assistant professor at Harvard University, and co-corresponding author of the researchers’ study, which is published in Nature Biotechnology. “That has really allowed us to learn about cell types and states. But, often, we haven’t been able to manipulate those cells specifically. RADARS is a big step in that direction.”
Chen and colleagues described their RADARS technology in a paper titled “Programmable eukaryotic protein synthesis with RNA sensors by harnessing ADAR,” in which they concluded, “Overall, RADARS forms the basis of a reprogrammable biological sensor platform with many applications for biomedical research, diagnostics, and therapeutics.” The research team was led by co-first authors Jeremy Koob, PhD, Xi Chen, PhD, and Yifan Zhang, PhD, at Broad, and Rohan Krajeski, PhD, and Kaiyi Jiang, PhD, at MIT.
Programmable approaches to sense and respond to the presence of specific RNAs in biological systems will have broad applications in research, diagnostics, and therapeutics, the authors noted in their paper. “For basic research, programmable cell-state targeting of transgene expression allows for tracking and perturbing specific cell states to understand their function. Translationally, cell-state sensing and targeting can be critical for cell and gene therapies and minimally invasive diagnostics.” But, as Omar Abudayyeh, PhD, a McGovern Institute Fellow and co-corresponding author on the study, commented, “Right now, the tools that we have to leverage cell markers are hard to develop and engineer. We really wanted to make a programmable way of sensing and responding to a cell state.”
Jonathan Gootenberg, PhD, who is also a McGovern Institute Fellow and co-corresponding author, added that the team was looking to build a tool that could take advantage of all the data provided by single-cell RNA sequencing, which has revealed a vast array of cell types and cell states in the body. “We wanted to ask how we could manipulate cellular identities in a way that was as easy as editing the genome with CRISPR.” As the team further noted in their paper, “A technology that senses and responds to specific RNA markers via simple RNA base-pairing rules would provide a direct, programmable path to RNA control of transgene translation.”
The RADARS platform developed by the team generates a desired protein when it detects a specific RNA, by taking advantage of RNA editing that occurs naturally in cells. “RADARS harness simple base-pairing rules and RNA editing to conditionally translate an mRNA cargo in the presence of a target RNA species,” the investigators continued.
The system consists of an RNA containing two components: a guide region, which binds to the target RNA sequence that scientists want to sense in cells, and a payload region, which encodes the protein of interest, such as a fluorescent signal or a cell-killing enzyme. “We designed RADARS to consist of a ‘guide’ region containing one or more hybridization regions complementary to the desired target, an in-frame stop codon within the guide, and a downstream region containing the cargo protein sequence of the guide,” the scientists noted in their paper. When the guide RNA binds to the target RNA, this generates a short double-stranded RNA sequence containing a mismatch between two bases in the sequence —adenosine (A) and cytosine (C). This mismatch attracts a naturally occurring family of RNA-editing proteins called adenosine deaminases acting on RNA (ADARs).
In RADARS, the A-C mismatch appears within a “stop signal” in the guide RNA, which prevents the production of the desired payload protein. The ADARs edit and inactivate the stop signal, allowing for the translation of that protein. The order of these molecular events is key to the function of RADARS function as a sensor; the protein of interest is produced only after the guide RNA binds to the target RNA and the ADARs disable the stop signal.
The team tested RADARS in different cell types and with different target sequences and protein products. They found that RADARS distinguished between kidney, uterine, and liver cells, and could produce different fluorescent signals as well as a caspase, an enzyme that kills cells. RADARS also measured gene expression over a large dynamic range, demonstrating their utility as sensors. “We apply RADARS in multiple contexts, including tracking transcriptional states, RNA-sensing-induced cell death, cell-type identification, and control of synthetic mRNA translation,” they commented.
Most systems successfully detected target sequences using the cell’s native ADAR proteins, but the team found that supplementing the cells with additional ADAR proteins increased the strength of the signal. Abudayyeh says both of these cases are potentially useful; taking advantage of the cell’s native editing proteins would minimize the chance of off-target editing in therapeutic applications, but supplementing them could help produce stronger effects when RADARS are used as a research tool in the lab.
Abudayyeh, Chen, and Gootenberg suggest that because both the guide RNA and payload RNA are modifiable, others can easily redesign RADARS to target different cell types and produce different signals or payloads. They also engineered more complex RADARS, in which cells produced a protein if they sensed two RNA sequences and another if they sensed either one RNA or another.
The team noted that similar RADARS could help scientists detect more than one cell type at the same time, as well as complex cell states that can’t be defined by a single RNA transcript. Ultimately, the researchers hope to develop a set of design rules so that scientists can more easily develop RADARS for their own experiments. “ … we’re excited to see what the field does with it,” Gootenberg noted. The investigators commented that other scientists could use RADARS to manipulate immune cell states, track neuronal activity in response to stimuli, or deliver therapeutic mRNA to specific tissues. “The advantages of the RADARS platform, including high sensitivity, reprogrammability, minimal perturbation to cells, combinatorial logic on inputs, and modularity, make it ideal for a myriad of applications in fundamental and translational research,” the team wrote. “For RADARS cargoes, multiple different classes of protein outputs can be used, including fluorescent proteins, luminescent proteins, caspases, recombinases, and small peptides. Transcription factors could be used in models of cell differentiation, or triggering responsive transcription-factor cascades.”
“We think this is a really interesting paradigm for controlling gene expression,” said Chen. “We can’t even anticipate what the best applications will be. That really comes from the combination of people with interesting biology and the tools you develop.”
