3 ms·

> 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 ea

> 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
        Thats not what I said. I said some significant percent of cells (I remember as high as 10% from one paper, but usually its closer to 0.1%...) seem to have indels at any given locus. Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates.
        azeotropic 8y ago
        How is the per-base indel rate not 10% if 10% of cells carry an indel at any given locus?
        nonbel 8y ago
        Reference: GAATTC Variants: Cell 1: TAATTC Cell 2: GTATTC Cell 3: GATTTC Cell 4: GAAGTC Cell 5: GAATGC Cell 6: GAATTT Cell 7: GAATTC Cell 8: GAATTC 7/8 cells contain the reference nucleotide at any given site. Ie, 12.5% of cells contain an indel wherever you look. For six cells, 1/6 nucleotides differ from the reference sequence (16.7% per base indel rate). For two cells, there is no difference (0 % per base indel rate). Even though 12.5% of cells contain an indel at any given site, no single cell contains 12.5% indels. There is no reason the two values should be the same.
        azeotropic 8y ago
        You clearly have no idea what you are talking about. 1) There are no indels in your example, only nucleotide substitutions. 2) There are six substitutions in 48 bases, for a per-base substitution rate of 12.5%. The fact that none of the individual cells contains 12.5% substitutions is totally irrelevant.

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
      Thats not what I said. I said some significant percent of cells (I remember as high as 10% from one paper, but usually its closer to 0.1%...) seem to have indels at any given locus. Either that or the methods used to measure this are too noisy to detect mutants that exist at those rates.
      - azeotropic 8y ago
        How is the per-base indel rate not 10% if 10% of cells carry an indel at any given locus?
        nonbel 8y ago
        Reference: GAATTC Variants: Cell 1: TAATTC Cell 2: GTATTC Cell 3: GATTTC Cell 4: GAAGTC Cell 5: GAATGC Cell 6: GAATTT Cell 7: GAATTC Cell 8: GAATTC 7/8 cells contain the reference nucleotide at any given site. Ie, 12.5% of cells contain an indel wherever you look. For six cells, 1/6 nucleotides differ from the reference sequence (16.7% per base indel rate). For two cells, there is no difference (0 % per base indel rate). Even though 12.5% of cells contain an indel at any given site, no single cell contains 12.5% indels. There is no reason the two values should be the same.
        azeotropic 8y ago
        You clearly have no idea what you are talking about. 1) There are no indels in your example, only nucleotide substitutions. 2) There are six substitutions in 48 bases, for a per-base substitution rate of 12.5%. The fact that none of the individual cells contains 12.5% substitutions is totally irrelevant.