A new tool in the CRISPR toolbox doubles the number of single-base RNA edits on researchers’ wish lists. The tool—aptly named RNA Editing for Specific C-to-U Exchange (RESCUE)—can be programmed to convert targeted cytosine bases into uridine bases, complementing existing RNA base editors, which convert targeted adenine bases into inosine bases.
RESCUE, its creators declare, is a particularly welcome development because many of the transcript modifications within RESCUE’s purview correspond to protein modifications relevant to cell signaling and cancer-linked pathways. For example, RESCUE enables modulation of more posttranslational modifications, such as phosphorylation, glycosylation, and methylation, as well as expanded targeting of common catalytic residues, disease mutations, and protective alleles.
RESCUE was developed by a team of scientists led by Feng Zhang, PhD, an investigator at the McGovern Institute and a core member of the Broad Institute of MIT and Harvard. Zhang and colleagues made use of a deactivated Cas13 to guide RESCUE to targeted cytosine bases on RNA transcripts. Also, the scientists used a novel, evolved, programmable enzyme to convert unwanted cytosine into uridine—thereby directing a change in the RNA instructions.
“To treat the diversity of genetic changes that cause disease, we need an array of precise technologies to choose from,” noted Zhang. “By developing this new enzyme and combining it with the programmability and precision of CRISPR, we were able to fill a critical gap in the toolbox.”
Details about RESCUE appeared July 26 in the journal Science, in an article titled, “A cytosine deaminase for programmable single-base RNA editing.” The article emphasizes a key advantage of programmable RNA editing: reversability. Unlike changes made at the DNA level, which are permanent, changes at the RNA level are transient. Thus, RESCUE could be deployed in situations where a modification may be desirable temporarily.
The article also indicates that RESCUE complements adenine-to-inosine RNA base-editing platforms, such as the Zhang team’s REPAIR platform. (REPAIR stands for RNA Editing for Programmable A-to-I (G) Replacement.) According to Zhang and colleagues, RESCUE and REPAIR may work together, accomplishing multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.
“Although natural enzymes capable of catalyzing C-to-U conversion have been harnessed for DNA base editing, they operate only on single-stranded substrates, exhibit off-targets, and deaminate multiple bases within a window,” the article’s authors wrote. “Therefore, we took a synthetic approach to evolve the adenine deaminase domain of ADAR2 (ADAR2dd), which naturally acts on double-stranded RNA (dsRNA) substrates and preferentially deaminates a target adenine mispaired with a cytidine, into a cytidine deaminase.”
When this evolved cytidine deaminase was fused to catalytically inactive Cas13 in mammalian cells, the result was RESCUE.
To demonstrate RESCUE’s reversible action, Zhang and colleagues used RESCUE to target specific sites in the RNA encoding β-catenin in human cells. These sites are known to be phosphorylated on the protein product, leading to a temporary increase in β-catenin activation and cell growth. If such a change was made permanently, it could predispose cells to uncontrolled cell growth and cancer, but by using RESCUE, transient cell growth could potentially stimulate wound healing in response to acute injuries.
The researchers also targeted a pathogenic gene variant, APOE4. The APOE4 allele has consistently emerged as a genetic risk factor for the development of late-onset Alzheimer’s disease. Isoform APOE4 differs from APOE2, which is not a risk factor, by just two differences (both C in APOE4 vs. U in APOE2). Zhang and colleagues introduced the risk-associated APOE4 RNA into cells, and showed that RESCUE can convert its signature Cs to an APOE2 sequence, essentially converting a risk to a non-risk variant.
To facilitate additional work that will push RESCUE toward the clinic as well as enable researchers to use RESCUE as a tool to better understand disease-causing mutations, the Zhang lab plans to share the RESCUE system broadly, as they have with previously developed CRISPR tools. The technology will be freely available for academic research through the non-profit plasmid repository Addgene. Additional information can be found on the Zhang lab’s webpage.
