MAY 10, 2016 04:45 AM PDT

CRISPR/Cas9 Can Now Be Used to Track RNA In Vivo

WRITTEN BY: Carmen Leitch
Since the development of the gene-editing tool CRISPR/Cas9, researchers have found many ways to both improve and vary the technique for many applications. Now, the power of this revolutionary procedure is being used to conquer a hurdle often faced by investigators. In a new paper in Cell, investigators have reported that following the endogenous activity of RNA in live cells can finally be done using the CRISPR system, a technique previously only used on DNA. The authors of this study, including Jennifer Doudna at the University of California, Berkeley and Gene Yeo at the University of California, San Diego, have called this technology RNA-targeted Cas9 or RCas9.
Components for RNA-targeting Cas9 (RCas9) recognition of mRNA are: a nuclear localization signal-tagged nuclease-inactive Cas9 fused to a fluorescent protein, a modified sgRNA (expression is driven by the U6 promoter) and a PAMmer. The sgRNA and PAMmer are antisense to adjacent regions of the target mRNA whose encoding DNA doesn't have a PAM sequence. The RCas9/mRNA complex forms in the nucleus and is then exported to the cytoplasm.
CRISPR/Cas9 usually uses the Cas9 enzyme, which cuts DNA, and what’s called a guide RNA to direct it to the proper place in the genome where it’s supposed to make the cut. In this new report, they used a short nucleic acid called a PAMmer to hybridize an altered version of the enzyme to the targeted RNA molecules. The Cas9 they used has been inactivated so it will not make any cuts - dCas9 (deadCas9) and is also fused to a fluorescent molecule so that when it binds, the RNA can be visualized. The PAMmer is composed in such a way that will allow it to avoid degradation by cellular machinery, and it will not target DNA sequences. The dCas9 has a nuclear localization signal and so using the sequence provided by the guide RNA, the dCas9 associates with the target mRNA in the nucleus, then forms a complex and moves to the cytoplasm.
 
They had to verify that it could be used to visualize localization of RNA in cells. They first checked that system by targeting GAPDH with an mCherry-tagged dCas9 and indeed saw the mCherry signal exported from the nucleus. Next, they targeted the mRNA transcripts of ACTB, TFRC and CCNA2 genes. They then compared the localization patterns observed to the patterns they saw after visualizing those same genes using FISH (fluorescence in situ hybridization.) Indeed, they saw the targeted mRNAs in the same places where it was shown using FISH. They also took care to ensure that tagging the RNA does not interfere with the abundance of mRNA or the amount of translated protein.
CRISPR/Cas9 can bind RNA to allow endogenous RNA tracking in living cells; nuclear-localized RNA-targeting Cas9 (RCas9) gets exported to the cytoplasm in the presence of sgRNAs targeting mRNA
The authors of the study go on to suggest a host of future experiments, such as the investigation of splice site alteration, multiplexing the technique to target multiple RNAs at once. They even suggest that the Cas9 molecule could be altered with sensors to detect aberrant transcripts and potentially edit them. It’s known that some diseases have to do with problems not with DNA, but with RNA, thus further expanding the potential applications of this exciting new procedure into disease therapy.

       
 
Source: Cell
 
About the Author
  • Experienced research scientist and technical expert with authorships on 28 peer-reviewed publications, traveler to over 60 countries, published photographer and internationally-exhibited painter, volunteer trained in disaster-response, CPR and DV counseling.
You May Also Like
SEP 25, 2018
Cell & Molecular Biology
SEP 25, 2018
Creating Circuits to Detect and React to Conditions in Live Cells
Researchers at Caltech have taken an interesting approach to synthetic biology....
SEP 27, 2018
Genetics & Genomics
SEP 27, 2018
Learning What Causes Algae Blooms to Turn Toxic
According to the EPA, algal blooms threaten every state and in our changing climate, they may be more common....
SEP 29, 2018
Videos
SEP 29, 2018
CRISPR Technology may Have Serious Drawbacks
The CRISPR gene-editing system has been touted as a miraculous tool, but it has its problems....
OCT 14, 2018
Videos
OCT 14, 2018
An Ancient Genetic Element can Help Fight Cancer
Researchers have been trying a variety of ways to boost the immune system we all have so it's able to fight off cancer....
OCT 20, 2018
Videos
OCT 20, 2018
Understanding how the Immune System is Controlled
Scientists are trying to learn more about a gene, PTPN22, that is especially critical to the immune system....
OCT 22, 2018
Videos
OCT 22, 2018
Mad Cow Disease Identified in Scottish Cow
Stringent detection systems appear to be working, but cases still happen....
Loading Comments...