The RNA-sensing platform could help detect and selectively kill tumors or edit the genome in specific cells

The RNA-sensing platform could help detect and selectively kill tumors or edit the genome in specific cells

Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have developed a system that can recognize a specific RNA sequence in living cells and produce an interesting protein in response. Using the technology, the team demonstrated 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, called Reprogrammable ADAR Sensors, or RADARS, even allowed the team to target and kill a specific type of cell. The team said RADARS could one day help researchers detect and selectively kill tumor cells or edit the genome in specific 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 of the study. "This has really allowed us to learn about cell types and states. But we often haven't been able to specifically manipulate these cells. RADARS is a big step in that direction."

At the moment, the tools we have to effectively use cell markers are difficult to develop and engineer. We really wanted to find a programmable way to sense a cell state and respond to it.”

Omar Abudayyeh, a fellow at the McGovern Institute and co-corresponding author of the study

Jonathan Gootenberg, who is also a McGovern Institute fellow and co-corresponding author, says their team was keen to develop a tool to take advantage of all the data provided by single-cell RNA sequencing, which has revealed a wide range of cell types and cell states in the body.

“We wanted to ask how we can manipulate cellular identities in a way that is as simple 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 recognizes a specific RNA by taking advantage of RNA editing that occurs naturally in cells.

The system consists of an RNA that contains two components: a guide region that binds to the target RNA sequence that scientists want to capture in cells, and a payload region that encodes the protein of interest, e.g. B. kill a fluorescence signal or a cell enzyme. When the guide RNA binds to the target RNA, this creates a short double-stranded RNA sequence that contains 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 RNA-acting adenosine deaminases (ADARs).

In RADARS, the AC mismatch appears within a “stop signal” in the guide RNA that prevents production of the desired payload protein. The ADARs edit and deactivate the stop signal, allowing translation of this protein. The order of these molecular events is key to RADARS' function as a sensor; The protein of interest is produced only after the guide RNA binds to the target RNA and the ADARs deactivate 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 usefulness as sensors.

Most systems successfully recognized 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 cases are potentially useful; Harnessing the cell's native editing proteins would minimize the likelihood of off-target editing in therapeutic applications, but supplementing them could help produce more potent effects when RADARS are used as a research tool in the laboratory.

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 constructed more complex RADARS, in which cells produced one protein when they sensed two RNA sequences and another when they sensed either one or the other RNA sequence. The team adds that similar RADARS could help scientists detect more than one cell type at a time, as well as complex cellular states that cannot be defined by a single RNA transcript.

Ultimately, the researchers hope to develop a set of design rules to make it easier for others to develop RADARS for their own experiments. They suggest that other scientists could use RADAR to manipulate the state of immune cells, 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,” Chen said. "We can't even predict what the best applications will be. That really comes from the combination of people with interesting biology and the tools you develop."

Source:

Broad Institute of MIT and Harvard

Reference:

Jiang, K., et al. (2022) Programmable eukaryotic protein synthesis with RNA sensors using ADAR. Natural biotechnology. doi.org/10.1038/s41587-022-01534-5.

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