CRISPR could eventually allow us to treat HIV, cancer, autism

Researchers have managed to devise a new method using gene-editing tool CRISPR-Cas9 that can track movement of RNA in living cells thereby enabling them to study a wide range of disease-related RNA processes.

The study, published in Cell, paves way for even greater promise of finding a cure for deadly diseases like HIV and cancer. The work, according to researchers at University of California, San Diego, is just the tip of the iceberg and holds immense potential as it opens a whole new door for treating diseases by correcting disease-causing RNA behaviors – something that wasn’t possible until now considering that CRISPR-Cas9 only targeted DNA.

Researchers acknowledge that there are methods that enable them to recognize and track RNA, but they lack specificity and are quite labor-intensive procedures. Further there are methods such as the molecular beacons, but they 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.

That’s where CRISPR-Cas9 comes into play. CRISPR-Cas9 is known for its precision and 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 of the key modifications that paved way for use of CRISPR-Cas9 in RNA tracking was inspired by previous work from the lab of study co-author Jennifer Doudna, of the University of California, Berkeley. Researchers at UC San Diego 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.