The gene-editing tool CRISPR-Cas9 can now be used as a flexible and accessible means to target and track the movement of RNA in living cells. This new method, presented March 17, 2016 in Cell, could eventually be used to study a wide range of disease-related RNA processes and to manipulate gene transcription for disease modeling.
"We are just beginning to see the implications of genome engineering using the CRISPR technology, but many diseases, including cancer and autism, are linked to problems with another fundamental biological molecule: RNA," says senior study author Gene Yeo of the University of California, San Diego. "Future developments of this work could enable researchers to measure other features of RNA processing or support therapeutic approaches to correct disease-causing RNA behaviors."
Other attempts to engineer proteins to recognize certain RNA sequences have lacked specificity and required labor-intensive procedures. Meanwhile, short nucleic acids called molecular beacons are limited to imaging applications and are difficult to deliver into cells. Protein-binding molecules called aptamers enable RNA tracking in living cells but are limited in the number of RNA sequences that they can recognize.
To overcome the limitations of these approaches, Yeo and his team turned to CRISPR-Cas9, known for precisely editing virtually any genomic site of interest in a variety of organisms. The specificity of the technique relies on a single-guide RNA (sgRNA), which forms a complex with the Cas9 enzyme to generate mutations at target DNA sequences. In the new study, the researchers modified the CRISPR-Cas9 system in several ways to optimize it for RNA tracking.
One key modification was inspired by previous work from the lab of study co-author Jennifer Doudna, of the University of California, Berkeley. Based on her recent findings, they designed a separate, short nucleic acid called a PAMmer to allow Cas9 to efficiently recognize RNA rather than DNA without damaging the target molecule. They also used a catalytically inactive form of the Cas9 enzyme to avoid cleaving the transcriptome and tagged Cas9 with a fluorescent protein to monitor its movement under the microscope. Finally, they used an optimized sgRNA to improve the efficiency of RNA targeting. With these modifications, the technique enabled RNA tracking in live cells without altering RNA abundance or the amount of translated protein.
The researchers demonstrated that their approach could be used to track RNA movement in response to cellular stress in human cells. They were able to visualize specific RNA molecules accumulating in stress granules, which are dense aggregations of proteins and RNA that form in the cytosol in response to cellular stress and have been linked to neurodegenerative disorders such as amyotrophic lateral sclerosis.
Currently, Yeo and his team are exploring the ability of RNA-targeted Cas9 to alter and measure other features of RNA processing beyond RNA localization. Future development of this approach could shed new light on dysfunctional RNA processes implicated in cancer and neurodegenerative disorders such as spinal muscular atrophy, as well as neurodevelopmental disorders such as fragile X syndrome -- the most common inherited form of mental retardation.
"One potential application of this technique is to track RNA transport in diseased neurons over time in order to identify the molecular features of these diseases and support the development of therapies," says first study author David Nelles of the University of California, San Diego. "Just as CRISPR-Cas9 is making genetic engineering accessible to any scientist with access to basic equipment, RNA-targeted Cas9 may support countless other efforts for studying the role of RNA processing in disease or for identifying drugs that reverse defects in RNA processing."
This work was supported by grants from the National Institutes of Health and the California Institute of Regenerative Medicine. Additional support received by the National Science Foundation, the Howard Hughes Medical Institute, and the Alfred P. Sloan Foundation.
Cell, Nelles et al.: "Programmable RNA tracking in Live Cells with CRISPR/Cas9" http://dx.doi.org/10.1016/j.cell.2016.02.054
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