DNA modification

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  • #3
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They say that the accuracy is low and even modify unwanted sites, does that mean a longer guide RNA in cas9 makes for a better search algorithm? Anyway I thought this video is quite helpful in understanding.
 
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  • #4
Ygggdrasil
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They say that the accuracy is low and even modify unwanted sites, does that mean a longer guide RNA in cas9 makes for a better search algorithm?
Unfortunately, no. This is limited by the intrinsic fidelity of the Cas9 enzyme. There have been efforts, however, to engineer CRISPR reagents that cut DNA only when two Cas9 molecules bind next to each other on the DNA (http://www.nature.com/nbt/journal/v32/n6/abs/nbt.2908.html), which has helped decrease off target cutting.
 
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  • #6
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So I get how they are able to cut the DNA at the desired location, but how do they go about inserting a new strand of DNA at the gap? Do they also cut off a strand of DNA at the process?
Gene editing by CRISPR makes use of the cell's normal DNA repair mechanisms to insert the new strand of DNA at the site of DNA cleavage. The cell will recognize double-stranded breaks in DNA and, through a process called homology-directed repair, it can recognize another strand of DNA with similar sequence to the sequences surrounding the break (usually this is the second copy of the DNA on the other chromosome, but in the case of gene editing, it is a foreign DNA molecule injected into the cell by researchers). The DNA repair machinery can then copy the sequence from that other DNA strand onto the broken DNA strand in order to repair the double stranded break.

Here's a short video explaining the process. The explanation is not as clear as it could be (it uses a lot of specialized jargon), but it gives a nice visual. The side bar has links to other longer youtube videos that may do a better job at explaining the process.
 
  • #7
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I've read that part in your article and it does a good explanation. The natural repair process should be pretty precise, but can a mismatch still happens? http://www.nature.com/news/mini-enzyme-moves-gene-editing-closer-to-the-clinic-1.17234 This article shows that they are able to package into an AAV virus along with cas9 encoding gene and RNAs. That means the cell can start producing cas9 as a part of the viral protein in the translation process? They're using different ways of to deliver the mechanism. For their test around 40% of the mice liver cell contains modified genes. I am not sure why it is tissue specific since the entire body contains the same DNA anyway, tell me if I'm wrong.
 
  • #8
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Yes, the AAV approach is a way of getting DNA into the cells of interest, then expressing the sgRNA and Cas9 from the viral RNA. In this case, tissue specificity can potentially come from a few sources:

1) Tropism of the virus. The main way researchers target gene therapy to specific tissues is by selecting a virus that infects only that specific tissue type. In the paper that the new article you cite references (http://www.nature.com/nature/journal/v520/n7546/full/nature14299.html), the researchers use AAV serotype 8 which infects liver cells.

2) The promoter sequences used for sgRNA and/or Cas9 expression. Not all promoter sequences will work in every cell type, so it is possible to use a promoter sequence that restricts Cas9 expression to a specific cell type. So, even if the virus delivers its DNA to the wrong cell type, if that cell type lacks the required transcription factors to turn on the promoter, the cell won't make any Cas9 and no editing will occur. Of course, sometimes it is preferable to edit the DNA of many different cell types, in which case a more promiscuous promoter (such as some from viruses) can be used which will allow for expression of the Cas9 in a wider range of cell types.

It's worth noting in the paper that the authors did not test other cell types to determine how specific their treatment was for liver cells, so at least from this study, it's unclear the extent to which these measures helped confine editing only to liver cells. In the case of editing the PCSK9 gene, there are living individuals who have loss of function mutation in all of the cells in their body, so tissue specificity is probably not an issue with the particular gene edits the researchers were trying to introduce in this study. However, in the case of other gene edits, it will be very important to confirm the tissue specificity of the editing before the technology can be considered safe.
 
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  • #9
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So there isn't other way to transfer besides cell division. I suppose all you need is to update the cell once to have a permanent effect.
 
  • #10
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Yes, the permanence of gene edits is certainly a concern. Some edits produce cells with increased fitness relative to unedited cells, which allows relatively inefficient editing procedures to still produce large numbers of edited cells through the division of the edited cells. However, if the edited cells have the same or decreased fitness relative to unedited cells, it's likely that the therapeutic efficiency of the treatment will decrease over time and would not be permanent.

Here's a review article from Feng Zhang's group at MIT discussing some of the issues related to translating CRISPR technologies to therapeutic applications that discusses this and other issues: http://www.nature.com/nm/journal/v21/n2/full/nm.3793.html
(free version available here if you can't access the version on the Nature Medicine website).
 
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  • #11
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I think gene editing with embryonic stem cell would be more potent in this case. I've seen research that reverse cell back to its embryonic state, but there really isn't a way to reverse growth it seems. Growth with cell division is the best way to distribute modified genes as well as making structural changes. I am not sure about cancer cells though, it seems they have some sort of uncontrolled growth. Well, I'm more interested in the actual effect after CRISPR is applied, does the mice grows and extra arm or head?
 
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