MAY 10, 2016 4:45 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
 
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