On 2016 Jun 21, Evelina Tutucci commented:

We have also recently discussed Nelles et al. Nelles DA, 2016. Since we are interested in developing new techniques for studying gene expression and mRNA localization at the single molecule level, a potential tag-less system to detect mRNAs in fixed and live cells would be a further advance. As pointed out by the Duke RNA Biology journal club we think that Nelles et al. represents an attempt to apply the Cas9 System to detect endogenous mRNA molecules. Unfortunately, no evidence is presented to demonstrate that this system is ready to be used to study gene expression at the single molecule level, as the MS2-MCP system allows. The RNA letter by Garcia and Parker Garcia JF, 2015 showed that in S. cerevisiae the binding of the MS2 coat protein to the MS2-loops diminished tagged mRNA degradation by the cytoplasmic exonuclease Xrn1. However, these observations were not extended to higher eukaryotes. Previous work from our lab described the generation of the beta-actin-MS2 mouse, whereby all the endogenous beta-actin mRNAs were tagged with 24 MS2 loops in the 3’UTR (Lionnet T, 2011, Park HY, 2014). This mouse is viable and no phenotypic defects are observed. In addition, control experiments were performed to show that the co-expression of the MS2 coat protein in the beta-actin-MS2 mouse allowed correct mRNA degradation and expression (Supplementary figure 1b, Lionnet T. et al 2011). Furthermore, multi-color FISH (Supplementary figure 6, Lionnet T. et al 2011) showed substantial co-localization between the ORF FISH probes and MS2 FISH probes, demonstrating the validity of this model. We think that the observations by Garcia and Parker are restricted to yeast because of the short half-life of their mRNAs, wherein the degradation of the MS2 becomes rate-limiting. Based on our extensive use of the MS2-MCP system, we think that higher eukaryotes may have more time to degrade the high affinity complexes formed between MS2-MCP, providing validation for this system to study multiple aspects of gene expression. In conclusion, we think that the MS2-MCP system remains to date the best method to follow mRNAs at the single molecule level in living cells. For the use of the MS2-MCP system in S. cerevisiae we have taken the necessary steps to improve it for the study of rapidly degrading mRNAs and are preparing this work for publication.<br> Evelina Tutucci and Maria Vera, Singerlab

On 2016 May 17, Duke RNA Biology Journal Club commented:

These comments were generated from a journal club discussion:

We were excited to read and discuss this paper as many of us have questions pertaining to mRNA localization. This technique theoretically allows for imaging of mRNAs without genetic manipulation meaning mRNAs at native expression levels can be tracked in live cells. However, as with many cutting-edge papers, more work is needed before this will become commonplace in the lab.

Most current methods to track mRNAs in live cells involve aptamer based methods which require genetic manipulation of mRNA PMCID: PMC2902723. Additionally, the most commonly used aptamer system, the MS2-MCP system, has become controversial in light of recent findings that the MS2 coat protein stabilizes the aptamer bearing constructs Garcia JF, 2015. In this paper, Fig 1F and 1G replicated these findings and also reassured us that the RCas9 technique would not have the same downfall. While this is certainly a good thing, we were unconvinced this technique was better than FISH (Fig 2), other than having the potential for live cell imaging.

Unfortunately, we found the live cell imaging, which was limited to Fig 3B, to be disappointing. First, we observed that unless an mRNA is strictly localized, as in stress granules, live imaging shows a diffuse mass within the cytosol. Second, imaging was performed with ACTB mRNA which is highly abundant. We don’t think live cell imaging would work as well for low abundance mRNAs due to high background signal. Finally, while specialized imaging software can detect the pile-ups of mRNA in localized foci, we are concerned that tracking individual mRNA may prove a hurdle. Cell models for mRNA localization are large cells such as fibroblasts and neurons, we would be interested to see the ability of this system within these cell types.

One major flaw with this system is the lack of ability to monitor nuclear localized RNA such as lncRNA or splicing machinery. Since since the RCas9 and sgRNA have to first be produced in the nucleus, the majority of the signal in all the figures came from the nucleus. There is a split-GFP-PUM-HD system that has been used to successfully track mRNA in mitochondria Ozawa T, 2007. Perhaps a similar concept could be used with the Cas9 system. This would prove an advantage to the Pumilio system since only the sgRNA needs to be modified instead of the entire protein.

Overall, this is a great start towards a new, tagless, method of mRNA tracking. We look forward to future developments and improvements of this exciting technique.

On 2016 May 17, Duke RNA Biology Journal Club commented:

These comments were generated from a journal club discussion:

We were excited to read and discuss this paper as many of us have questions pertaining to mRNA localization. This technique theoretically allows for imaging of mRNAs without genetic manipulation meaning mRNAs at native expression levels can be tracked in live cells. However, as with many cutting-edge papers, more work is needed before this will become commonplace in the lab.

Most current methods to track mRNAs in live cells involve aptamer based methods which require genetic manipulation of mRNA PMCID: PMC2902723. Additionally, the most commonly used aptamer system, the MS2-MCP system, has become controversial in light of recent findings that the MS2 coat protein stabilizes the aptamer bearing constructs Garcia JF, 2015. In this paper, Fig 1F and 1G replicated these findings and also reassured us that the RCas9 technique would not have the same downfall. While this is certainly a good thing, we were unconvinced this technique was better than FISH (Fig 2), other than having the potential for live cell imaging.

Unfortunately, we found the live cell imaging, which was limited to Fig 3B, to be disappointing. First, we observed that unless an mRNA is strictly localized, as in stress granules, live imaging shows a diffuse mass within the cytosol. Second, imaging was performed with ACTB mRNA which is highly abundant. We don’t think live cell imaging would work as well for low abundance mRNAs due to high background signal. Finally, while specialized imaging software can detect the pile-ups of mRNA in localized foci, we are concerned that tracking individual mRNA may prove a hurdle. Cell models for mRNA localization are large cells such as fibroblasts and neurons, we would be interested to see the ability of this system within these cell types.

One major flaw with this system is the lack of ability to monitor nuclear localized RNA such as lncRNA or splicing machinery. Since since the RCas9 and sgRNA have to first be produced in the nucleus, the majority of the signal in all the figures came from the nucleus. There is a split-GFP-PUM-HD system that has been used to successfully track mRNA in mitochondria Ozawa T, 2007. Perhaps a similar concept could be used with the Cas9 system. This would prove an advantage to the Pumilio system since only the sgRNA needs to be modified instead of the entire protein.

Overall, this is a great start towards a new, tagless, method of mRNA tracking. We look forward to future developments and improvements of this exciting technique.

On 2016 Jun 21, Evelina Tutucci commented:

We have also recently discussed Nelles et al. Nelles DA, 2016. Since we are interested in developing new techniques for studying gene expression and mRNA localization at the single molecule level, a potential tag-less system to detect mRNAs in fixed and live cells would be a further advance. As pointed out by the Duke RNA Biology journal club we think that Nelles et al. represents an attempt to apply the Cas9 System to detect endogenous mRNA molecules. Unfortunately, no evidence is presented to demonstrate that this system is ready to be used to study gene expression at the single molecule level, as the MS2-MCP system allows. The RNA letter by Garcia and Parker Garcia JF, 2015 showed that in S. cerevisiae the binding of the MS2 coat protein to the MS2-loops diminished tagged mRNA degradation by the cytoplasmic exonuclease Xrn1. However, these observations were not extended to higher eukaryotes. Previous work from our lab described the generation of the beta-actin-MS2 mouse, whereby all the endogenous beta-actin mRNAs were tagged with 24 MS2 loops in the 3’UTR (Lionnet T, 2011, Park HY, 2014). This mouse is viable and no phenotypic defects are observed. In addition, control experiments were performed to show that the co-expression of the MS2 coat protein in the beta-actin-MS2 mouse allowed correct mRNA degradation and expression (Supplementary figure 1b, Lionnet T. et al 2011). Furthermore, multi-color FISH (Supplementary figure 6, Lionnet T. et al 2011) showed substantial co-localization between the ORF FISH probes and MS2 FISH probes, demonstrating the validity of this model. We think that the observations by Garcia and Parker are restricted to yeast because of the short half-life of their mRNAs, wherein the degradation of the MS2 becomes rate-limiting. Based on our extensive use of the MS2-MCP system, we think that higher eukaryotes may have more time to degrade the high affinity complexes formed between MS2-MCP, providing validation for this system to study multiple aspects of gene expression. In conclusion, we think that the MS2-MCP system remains to date the best method to follow mRNAs at the single molecule level in living cells. For the use of the MS2-MCP system in S. cerevisiae we have taken the necessary steps to improve it for the study of rapidly degrading mRNAs and are preparing this work for publication.<br> Evelina Tutucci and Maria Vera, Singerlab