SAN DIEGO—With the rise of genomics, scientists have been motivated to sequence the human genome and develop ways to DNA; however, many diseases are linked to RNA. As RNA is the intermediary genetic material that carries the genetic code from the cell’s nucleus, a challenge has been to find an efficient method for targeting RNA in living cells. In a study published March 17 in Cell, researchers at the University of California, San Diego (UCSD), School of Medicine say they have achieved this by applying the popular DNA-editing technique CRISPR-Cas9 to RNA.
“This work is the first example, to our knowledge, of targeting RNA in living cells with CRISPR-Cas9,” said senior author Dr. Gene Yeo, associate professor of cellular and molecular medicine. “Our current work focuses on tracking the movement of RNA inside the cell, but future developments could enable researchers to measure other RNA features or advance therapeutic approaches to correct disease-causing RNA behaviors.”
David Nelles is a UCSD Jacobs School of Engineering graduate student in Yeo’s lab and first author of the study. Study co-authors also include Dr. Jennifer Doudna and Mitchell R. O’Connell of the University of California, Berkeley, and Mark Fang, Jia L. Xu and Sebastian J. Markmiller of UCSD.
RNA’s location in a cell—and how and when it gets there—can influence whether proteins are produced in the right location and at the appropriate time. For instance, proteins important to neuronal connections in the brain, known as synapses, are produced from RNAs located at these contacts. Defective RNA transport is linked to conditions ranging from autism to cancer, and researchers need ways to measure RNA movement in order to develop treatments for these conditions.
Efforts to edit and measure DNA got a boost a few years ago when researchers discovered they could take CRISPR-Cas9, a naturally occurring defense mechanism bacteria use to fend off other invading bacteria, and use it to edit genes in mammalian systems. Normally with CRISPR-Cas9, researchers design a “guide” RNA to match the sequence of a specific target gene. The RNA directs the Cas9 enzyme to the desired spot in the genome, where it cuts the DNA. As Nelles tells DDNews, “CRISPR-Cas9 is a type of adaptive bacterial immune system that allows cells to remember viral DNA sequences so that they can be recognized and destroyed to prevent infection.”
“This is a great example of an elegant solution in the natural world: bacteria remember past viral infections by incorporating viral DNA directly into their own genome, and then using this DNA to guide Cas9, a scissor-like protein, to cut viral DNA at a later time,” he continues. “Since CRISPR-Cas9 is specific enough to recognize specific DNA sequences, scientists can now cut and correct disease-causing DNA sequences in human cells.”
Until now, CRISPR-Cas9 could only be used to manipulate DNA. Yeo and colleagues, though, developed a flexible means of targeting RNA in live cells, also called RNA-targeted Cas9 (RCas9). In order to target RNA instead of DNA, the researchers altered several features of the CRISPR-Cas9 system. Building upon previous work by Doudna, they designed a short nucleic acid called the PAMmer that, along with the guide RNA, directs RCas9 to an RNA molecule.
Says Nelles: “The PAMmer is an important feature of the RCas9 system—it allows strong recognition of cellular RNA while avoiding the DNA that encodes it. Cas9 requires two things to bind a nucleic acid sequence: a short DNA sequence known as the PAM (protospacer adjacent motif) and an adjacent sequence that complements the single guide RNA. The PAMmer is a short DNA fragment (with some chemical modifications) that carries the PAM and hybridizes to the target RNA. By placing a PAM sequence on the RNA, the PAMmer then allows Cas9 and its single guide RNA to bind the RNA.”
To test the system, Yeo’s team targeted the RNA that encodes the proteins ACTB, TFRC and CCNA2. Then they watched as Cas9, fused with a fluorescent protein, revealed the movement of RNA into stress granules, a cluster of proteins and RNAs that form in a cell’s cytosol when the cell is under stress. This system allowed the team to track RNA over time, in live cells, without the need for artificial tags commonly used in other RNA-tracking techniques—an approach that can interfere with normal cellular processes.
“To measure RNA distribution in cells, researchers have to kill cells and treat them with fluorescent probes, or add extra sequences to RNAs which can be used to tag the RNA with fluorescent proteins,” says Nelles. “While powerful, these techniques are limited by the fact that either cells must be killed, or the RNAs need to be genetically modified. RNA is constantly being produced and trafficked around cells and genetic modification can be laborious, so a way to track RNA in living cells without genetic tagging would be ideal. By fusing a version of Cas9 that does not cut RNA to a fluorescent protein, we were able to track RNA movement in living cells using RCas9.”
RCas9 could allow researchers to measure and manipulate a wide range of RNA processing features like RNA transport, splicing, and turnover—neurogenerative diseases and certain types of autism are linked to dysfunctional RNA transport in neurons. According to Nelles, one potential application of this technique would be tracking RNA transport in diseased neurons over time, in order to identify the molecular features of diseases and support the development of therapies.