Category Archives: FISH

New CRIPSR-based RNA imaging tool

About a year and a half ago I wrote here about new uses of CRISPR/Cas9 as an imaging tool. In particular, I was excited about the possibility to use enzyme-dead Cas9 (dCas9) as an RNA binding protein for live imaging of mRNA. Unfortunately, in my hands this did not work (the dCas9 has exited the nucleus with non-targeting guide RNA at the same rate as with the specific guide RNA).

Last week, a new CRISPR tool was published in Nature, from Feng Zhang’s lab. Briefly, Zhang’s team found in the past a new type of Cas protein which was named C2c2 and later Cas13. Unlike Cas9, which typically binds and cuts DNA, Cas13 specifically binds and cuts RNA. In this paper, they isolated a Cas13a variant from Leptotrichia Wadei (LwaCas13a),  and used it as a programmable tool to knock-down expression. As a tool for knowckdown it is pretty awesome – it is comparable to shRNA in terms of knock-down (~40-90%), but it has three big advantages:

1. Unlike shRNA, LwaCas13a can also target nuclear RNA.

2. It can be used easily for multiplexed knock-down of sereval RNAs.

3. Amazingly, it seems that it has NO OFF-TARGET effect. They used RNA-seq to look at the change in expression level of all RNAs in the cells. Whereas shRNA showed hundreds of off-target knocked-down RNAs, LwaCas13a was very specific and only the targeted mRNA was knocked-down.

They then mutated LwaCas13a to create a dead variant (dLwaCas13a) and asked if it can be used for pull-down assays and for live imaging.

Just like the RNA-dCas9, the pull down efficiency of beta-actin mRNA wasn’t great (2-3 fold over non-targeting guide RNA). However, for Luciferase mRNA, this ratio was better (8-11 fold enrichment). So, this has good potential. Too bad they did not compare it to puul down using MS2-labeling and see if the same proteins are pulled down with the mRNA.

And now I come to my problem with this paper – the imaging.

Just like the RNA-Cas9 paper, they imaged beta-actin mRNA with a single guide RNA and single GFP fused to dLwaCas13a. dLwaCas13a was nuclear localized, but exited the nucleus when specific guide RNA was used. However, they claim only a 3-3.7 fold enrichment of cytoplasmic/nuclear ratio compared to non-targeting guide RNA. They acknowledge that there is neclaer escape due to nuclear off-target binding or other reasons. They added a negative feedback system to improve SNR. Still, the best they got was 3.7-fold. So, if you look at a specific cell – how can you tell if you are looking on RNA-bound or not? you can’t.

They also compared their labeling to FISH staining. Their FISH looks much better than in the RNA-Cas9 paper, really single spots. But to say that it co-localizes? There is more-or less uniform cytoplasmic green staining. Of course it will “co-localize” with the cytoplasmic beta-actin mRNA FISH spots. At the very least they should have done a control with FISH against another highly expressed mRNA (e.g. GAPDH), or perform beta-actin FISH of cells expressing a guide RNA against a differnt mRNA, and not just compare to the non-targeting control.

Last point – stress granules again? I guess it’s just an easy assay to do, that also gets the RNA in a concentrated blob: good if you don’t have single molecule resolution. But nobody really knows what SGs are and only now starting to figure out how and why they form.

They claim that when they induce stress granules (SG) formation, the GFP-dLwaCas13a co-localizes with the SG marker. However, their images are not very good. You see something in Fig 4 (only 2 cells in the control!) but Extended Fig 10 is terrible. Low resolution, small images – can anyone tell from this figure if there is or isnt co-localization, and get some qualitative, if not quantitative sense?

They do provide quantitative data, but although it could be statistically significant, it does not look striking. Really marginal differnece compared to the non-targeting guide RNA (or non-stressed cells).

Is this a good tool for RNA imaging? I don’t think so. Not until you can get single-molecule resolution.

 

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MS2 mRNA imaging in yeast: more evidence for artefacts

Previously, on the story of MS2 in yeast: Last year, Roy Parker published a short article, in which he claimed that using the MS2 system in yeast causes the accumulation of 3′ RNA fragments, probably due to inhibition of mRNA degradation by the 5′ to 3′ exoribonuclease Xrn1. He argued that these findings put in question all the work on mRNA localization in yeast using the MS2 system. About a year later, we wrote a response to that article. We argued that, yes, such fragments exist, but 1. most of it stems from over-expression of the labeled mRNA. Parker agreed with that. 2. That these fragments accumulate in P-bodies, and are distinguishable from single mRNAs and we can discard cells which show these structures. 3. We argued that this might not be the case for every mRNA and should be tested on a case by case basis.  4. We and Parker agreed that the best way to determine if such fragments exist is by performing single-molecule FISH (smFISH) with double labeling – a set of probes for the length of the mRNA and a set of probes for the MS2 stem-loops. Now, a new paper from Karsten Weis’ lab shows more evidence, by doing smFISH, for the existence of these fragments.

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Does bound MS2 coat protein inhibit mRNA decay?

Roy Parker recently sent a  “Letter to the Editor“, published in RNA journal, in which he suggested that the MS2 system might not be best suited for live imaging of mRNA in budding yeast. According to Parker, the MS2 system inhibits the function of Xrn1, the major cytoplasmic  5′ to 3′ RNA exonuclease in budding yeast, causing us to image mostly the remaining 3’UTR fragments. Thus, he claims, it is possible that interpertation of mRNA localization data using this system in yeast can be faulty. We wrote a response to his letter which just opened the debate even further.

But lets start with his Letter:

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Imaging with CRISPR/Cas9

The hottest buzz-word in biology today is CRISPR: an adaptive immune system in bacteria and archea. At its basis is a nuclease, named Cas9, which is targeted to DNA by a short single-guide RNA (sgRNA). This turned out to be a very useful system for genome engineering in any organism due to its specificity (provided by the sgRNA) and its simplicity (all you need is to express the Cas9 and sgRNA in the cell). However, this system can also be used for other purposes. One such use is modulation of gene expression, for example by targeting a nuclease dead Cas9 (dCas9) fused to a transcription activator or repressor to promoter regions. Another such use is for imaging.

Here, I’ll described how Cas9 can be used to visualize specific DNA loci or specific RNA transcripts in fixed and live cells.

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ASCB15 – part 2

I ended Part 1 after the morning session on pushing the boundaries of imaging.

After the amazing talks on imaging, I browsed the halls, visited some exhibitors, sampled a couple of exhibitor tech-talks. I later went to a mycrosymposium (#2: signaling in health & disease). This was mainly to see how this ePoster thing works, but also I promised Qunxiang Ong – with whom I discussed optogenetics the day before – to be at his presentation. He used a light-induced dimerization of signaling proteins to study the effect on neurite growth. The nice thing in his system was that the cells were plated in wells which were partly dark – so light-induction cannot take place in these regions. This allowed for analysis of neurite growth in lit vs “light-protected” regions of the same cell.

After this session, I attended my first “discussion table”. Continue reading

ASCB15 part 1

The ASCB meeting brings scientists from all levels to talk about cell biology, which is actually almost anything “biology”. But there’s also a full program dedicated to other matters, like science careers, science publishing, science communications and science policy. This is also a great venue for companies to show their products, and for organizations/institutions to recruit new members. If I remember the numbers correctly, there were over 550 oral presentations and over 2,700 posters. I overheard someone saying there were ~6000 people attending the meeting. I typically go to RNA meetings that are mostly in the lower 100’s of participants. So, to me, that’s a large meeting.

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In the right place at the right time: visualizing and understanding mRNA localization

The title of this post is also the title of a review paper that I co-authored  with Adina Buxbaum, a recently graduated PhD student from Rob Singer’s lab. The review was published last week in Nature Reviews Molecular Cell biology.

In this paper we review some of the old and new methods to visualize mRNA. These include mostly FISH and MS2-like systems, which I’ve discussed extensively in this blog. There is also a short section (“box”) on quantitative analysis tools for mRNA localization imaging.

We then discuss the current knowledge on the mechanisms of mRNA localization and how it relates to the biology in two very distinct model systems – unicellular organisms (budding yeast) and the extremely polarized neuronal cell.  We also discuss examples in other organisms from bacteria through fly to frog and mammals.

I’m biased, of course, but I think this turned out to be a balanced, comprehensive, yet not too detailed review paper that will benefit both beginners which are unfamiliar with the RNA localization field, as well as experts which are used to a single method or a single model organism.
ResearchBlogging.orgBuxbaum, A., Haimovich, G., & Singer, R. (2014). In the right place at the right time: visualizing and understanding mRNA localization Nature Reviews Molecular Cell Biology DOI: 10.1038/nrm3918