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Note that this study covers 'on-target' effects. As often covered in the news, CRISPR-mediated gene editing can be used to edit a DNA sequence to another, desi

Note that this study covers 'on-target' effects. As often covered in the news, CRISPR-mediated gene editing can be used to edit a DNA sequence to another, desired sequence. In these applications, it's very important that no other mutations besides the desired mutation are introduced. Such additional mutations would be 'off-target' mutations. In a second kind of experimental approach, researchers can use CRISPR technology to make arbitrary mutations at a desired site in the genome. This approach cares less about the resulting damage; the goal is simply to destroy the existing gene. The referenced article[1] seems to characterize the types of mutations that arise when people use CRISPR to ablate a gene (i.e., the second approach). It would be interesting to see if the range of DNA damage is restricted when CRISPR is instead used to change the sequence of a targeted DNA segment into another, desired sequence. [1] https://www.nature.com/articles/Nbt.4192 https://www.nature.com/articles/Nbt.4192

nonbel 8y ago
The first thing to realize is that the crispr/cas9 strategy only produces damaged DNA (double stranded breaks, DSBs). It is not supposed to repair the DNA or do anything else. You have five main responses to this: 1) Necrosis/Lysis (cell dies due to malfunction) 2) Apotosis (cell kills itself) 3) Senescence (cell doesn't die but stops dividing) 4) Non-homologous end joining (NHEJ, the free ends of the damaged DNA are just pasted back together by the cells repair machinery) 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used by the cells repair machinery to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) The second thing to realize is that cells containing the targeted DNA sequence will be more likely to have damaged DNA than those without. So once a cell is "mutated" (either because it just existed beforehand, or because it survived the crispr/cas9 treatment), it will have a survival advantage. The third thing to realize is that often these cells they use can divide multiple times per day, I've seen as high as 12 times a day for activated T-cells. That means in two days a single mutant cell could possibly produce 2^24 = 16,777,216 offspring cells containing the mutation. Anyway, I think the efficiency of this procedure may have been far overstated. There may be only a very few cells in each experiment that actually get edited, but a few days later when they check, those (already mutated) cells dominate the population due to their resistance to the crispr/cas9 treatment. Eg, look at extended data fig 1h from this recent paper: https://www.nature.com/articles/s41586-018-0326-5/figures/5 https://www.nature.com/articles/s41586-018-0326-5/figures/5 The more cells that die, the higher percent of "edited" cells later.
- ejstronge 8y ago
  > 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) I wanted to elide the details, but this is what I was referring to when I discussed CRISPR applications that try to change a nucleotide sequence. This of course leaves discussions of base-editing aside[1] 1. http://www.sciencemag.org/news/2017/10/novel-crispr-derived-base-editors-surgically-alter-dna-or-rna-offering-new-ways-fix http://www.sciencemag.org/news/2017/10/novel-crispr-derived-...
  - nonbel 8y ago
    Yes, you mentioned the two possibilities that everyone likes to focus on, but not the others which (I suspect) are much more common. EDIT: I really mean that I think in some of these experiments, where they only try to cause NHEJ, they just already had mutants in the population and selected for them. Its possible not a single cell was successfully edited but you still have so many cells with indels at the targeted sequence afterward.
    - azeotropic 8y ago
      Indel mutation rates aren't high enough, and the number of cells screened for mutations is too low for this explanation to suffice.
      - nonbel 8y ago
        Do you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.
        azeotropic 8y ago
        You must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.

nonbel 8y ago

The first thing to realize is that the crispr/cas9 strategy only produces damaged DNA (double stranded breaks, DSBs). It is not supposed to repair the DNA or do anything else. You have five main responses to this: 1) Necrosis/Lysis (cell dies due to malfunction) 2) Apotosis (cell kills itself) 3) Senescence (cell doesn't die but stops dividing) 4) Non-homologous end joining (NHEJ, the free ends of the damaged DNA are just pasted back together by the cells repair machinery) 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used by the cells repair machinery to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) The second thing to realize is that cells containing the targeted DNA sequence will be more likely to have damaged DNA than those without. So once a cell is "mutated" (either because it just existed beforehand, or because it survived the crispr/cas9 treatment), it will have a survival advantage. The third thing to realize is that often these cells they use can divide multiple times per day, I've seen as high as 12 times a day for activated T-cells. That means in two days a single mutant cell could possibly produce 2^24 = 16,777,216 offspring cells containing the mutation. Anyway, I think the efficiency of this procedure may have been far overstated. There may be only a very few cells in each experiment that actually get edited, but a few days later when they check, those (already mutated) cells dominate the population due to their resistance to the crispr/cas9 treatment. Eg, look at extended data fig 1h from this recent paper: https://www.nature.com/articles/s41586-018-0326-5/figures/5 https://www.nature.com/articles/s41586-018-0326-5/figures/5 The more cells that die, the higher percent of "edited" cells later.

ejstronge 8y ago
> 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) I wanted to elide the details, but this is what I was referring to when I discussed CRISPR applications that try to change a nucleotide sequence. This of course leaves discussions of base-editing aside[1] 1. http://www.sciencemag.org/news/2017/10/novel-crispr-derived-base-editors-surgically-alter-dna-or-rna-offering-new-ways-fix http://www.sciencemag.org/news/2017/10/novel-crispr-derived-...
- nonbel 8y ago
  Yes, you mentioned the two possibilities that everyone likes to focus on, but not the others which (I suspect) are much more common. EDIT: I really mean that I think in some of these experiments, where they only try to cause NHEJ, they just already had mutants in the population and selected for them. Its possible not a single cell was successfully edited but you still have so many cells with indels at the targeted sequence afterward.
  - azeotropic 8y ago
    Indel mutation rates aren't high enough, and the number of cells screened for mutations is too low for this explanation to suffice.
    - nonbel 8y ago
      Do you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.
      - azeotropic 8y ago
        You must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.