Researchers at the McGovern Institute for Brain Research at MIT and the Broad Institute at MIT and Harvard have created a system that can identify a specific RNA sequence in living cells and produce an interesting protein in response.
The scientists demonstrated how the technique may be used to distinguish between different cell types, spot and quantify changes in gene expression, monitor transcriptional states, and manage the creation of proteins encoded by synthetic mRNA.
The team was even able to target and eradicate a particular cell type using the platform, referred to as Reprogrammable ADAR Sensors, or RADARS. According to the team, RADARS could one day aid in the detection and selective killing of tumor cells, as well as the editing of the genome in particular cells. The study appears today in Nature Biotechnology and was led by co-first authors Kaiyi Jiang (MIT), Jeremy Koob (Broad), Xi Chen (Broad), Rohan Krajeski (MIT), and Yifan Zhang (Broad).
“One of the revolutions in genomics has been the ability to sequence the transcriptomes of cells,” said Fei Chen, a core institute member at the Broad, Merkin Fellow, assistant professor at Harvard University, and co-corresponding author on the study. “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.”
“Right now, the tools that we have to leverage cell markers are hard to develop and engineer,” added Omar Abudayyeh, a McGovern Institute Fellow and co-corresponding author on the study. “We really wanted to make a programmable way of sensing and responding to a cell state.”
Jonathan Gootenberg, who is also a McGovern Institute Fellow and co-corresponding author, says that their team was eager to build a tool to 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 think this is a really interesting paradigm for controlling gene expression. 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.
Fei Chen
“We wanted to ask how we could manipulate cellular identities in a way that was as easy as editing the genome with CRISPR,” he said. “And we’re excited to see what the field does with it.”
Repurposing RNA editing
The RADARS platform generates a desired protein when it detects a specific RNA by taking advantage of RNA editing that occurs naturally in cells.
The system is made up of an RNA with two parts: a guide area that binds to the target RNA sequence that researchers wish to detect in cells and a payload region that encodes the desired protein, such as a fluorescent signal or a cell-killing enzyme.
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 RADARS’s 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 group experimented with RADARS using several cell types, target sequences, and protein products. They discovered that RADARS could discriminate between kidney, uterine, and liver cells and was capable of producing various fluorescence signals as well as the cell-killing enzyme caspase. RADARS also measured gene expression over a large dynamic range, demonstrating their utility as sensors.
The natural ADAR proteins of the cell were used by the majority of systems to detect target sequences, but the scientists discovered that adding extra ADAR proteins to the cells strengthened 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.
On the radar
Abudayyeh, Chen, and Gootenberg say 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 created more sophisticated RADARS, in which cells would generate one protein if they detected two distinct RNA sequences and another if they detected just one. According to the study, comparable RADARS may be used to identify many cell types simultaneously as well as complex cell states that aren’t captured by a single RNA transcript.
Ultimately, the researchers hope to develop a set of design rules so that others can more easily develop RADARS for their own experiments. They suggest other scientists could use RADARS to manipulate immune cell states, track neuronal activity in response to stimuli, or deliver therapeutic mRNA to specific tissues.
“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.”
