5 ms·

CRISPR is a great example. Please see my other post on that topic in this thread, and if you are interested I will go find the refs that led me to that position

by nonbel 10y ago

CRISPR is a great example. Please see my other post on that topic in this thread, and if you are interested I will go find the refs that led me to that position.

AlexCoventry 10y ago
I would certainly be interested to know the references, but I don't find your argument here convincing. Testing for insertion of a defensive gene by exposing the organism to a hostile environment is an extremely well-developed and reliable method in genetics. But I suppose there must be a million ways to screw it up so I can't rule your mutation scenario out based on what I currently know of the research. On the other hand, I find your skepticism and independent thinking refreshing.
- nonbel 10y ago
  Thanks, not sure if I will get to it today. A lot can be found in my previous posts here on the topic if you want to search.
- nonbel 10y ago
  Actually this thread should do: https://news.ycombinator.com/item?id=10967719 https://news.ycombinator.com/item?id=10967719 I see now I had an earlier discussion with tstactplsignore, back then they were seemingly incapable of understanding what I was saying (they kept thinking I denied the endonuclease activity).
  - AlexCoventry 10y ago
    http://www.nature.com/nchembio/journal/v10/n8/abs/nchembio.1550.html http://www.nature.com/nchembio/journal/v10/n8/abs/nchembio.1... claims that resequencing of a CRISPR-targeted gene showed dramatic increases in indel mutations, consistent with non-homologous end joining repair of the CRISPR cuts. (Supplementary table 5.) I assume the resequencing happened before the drug screen, otherwise the paper is badly written.
    - nonbel 10y ago
      Well, from your table S5 and table S12, as is usual we see that about 0.1 - 1% of the cells were mutants in the control group (ie they came pre-"modified"). Interestingly, from table S12 and my (selection for pre-existing mutants) model we can also explain a mysterious result they observed: "Silent co-mutations in the repair oligonucleotides were introduced into >99% of D54H mutant alleles and ~3% of the F482S allele (Fig. 2c). Carryover of these silent SNPs indicates that the alleles are the product of HR and not de novo mutagenesis. The lower rate of coappearance of silent SNPs in F482S is presumably due to the larger distance between the two SNPs in the oligonucleotide and is rather common to see with single-stranded oligonucleotide donors19." Rather than that ad hoc explanation, it is simply that the D54H cells had more silent only mutations to begin with (0.1% vs 0.0%). Regarding that 0.0%, an annoying thing is that they only report these percentages to one decimal place. I'm not exactly clear on the number of cells present before the CRISPR-Cas9 treatment, but it sounds like 10^8, and then they let them grow for 72 hr + 7-12 days (total of 10-15 days) after the treatment. They also don't tell us how many cells were left at the end... but anyway if we assume these cells divide once a day, and 0.1% are preexisting mutants we could calculate the possible number of mutants thus: Nt = 10^8 p = 0.001 d = 0:15 Nt*p*2^d After 12 days we can get ~400 million cells from those initial pre-existing mutants, and after 15 days over as 3 billion. Of course other factors would probably come into play that limit this growth, I'm just saying it would be no problem for that small subset of the population to become dominant during the experiment. That is even if the 99,900,000 "WT" cells were just growth arrested rather than died. So I find those results to favor the "selection for pre-existing mutants" explanation over the "gene modification" one.
      - AlexCoventry 10y ago
        The key observation from my perspective is that they seem to have done the resequencing before any resistance selection. Why would the indel frequencies rise only in the CRISPR-treated cells during drug-free growth?
        nonbel 10y ago
        >"The key observation from my perspective is that they seem to have done the resequencing before any resistance selection. Why would the indel frequencies rise only in the CRISPR-treated cells during drug-free growth?" Because in cells that contain the target sequence (the complement to the guide RNA), Cas9 is damaging the DNA, leading to cell death and growth arrest. The small percentage of cells that already contained indels (thus reducing affinity for the guide RNA) are "immune", so they preferentially survive and divide to take over the population.

by nonbel 10y ago

CRISPR is a great example. Please see my other post on that topic in this thread, and if you are interested I will go find the refs that led me to that position.

AlexCoventry 10y ago

I would certainly be interested to know the references, but I don't find your argument here convincing. Testing for insertion of a defensive gene by exposing the organism to a hostile environment is an extremely well-developed and reliable method in genetics. But I suppose there must be a million ways to screw it up so I can't rule your mutation scenario out based on what I currently know of the research. On the other hand, I find your skepticism and independent thinking refreshing.

nonbel 10y ago
Thanks, not sure if I will get to it today. A lot can be found in my previous posts here on the topic if you want to search.
nonbel 10y ago
Actually this thread should do: https://news.ycombinator.com/item?id=10967719 https://news.ycombinator.com/item?id=10967719 I see now I had an earlier discussion with tstactplsignore, back then they were seemingly incapable of understanding what I was saying (they kept thinking I denied the endonuclease activity).
- AlexCoventry 10y ago
  http://www.nature.com/nchembio/journal/v10/n8/abs/nchembio.1550.html http://www.nature.com/nchembio/journal/v10/n8/abs/nchembio.1... claims that resequencing of a CRISPR-targeted gene showed dramatic increases in indel mutations, consistent with non-homologous end joining repair of the CRISPR cuts. (Supplementary table 5.) I assume the resequencing happened before the drug screen, otherwise the paper is badly written.
  - nonbel 10y ago
    Well, from your table S5 and table S12, as is usual we see that about 0.1 - 1% of the cells were mutants in the control group (ie they came pre-"modified"). Interestingly, from table S12 and my (selection for pre-existing mutants) model we can also explain a mysterious result they observed: "Silent co-mutations in the repair oligonucleotides were introduced into >99% of D54H mutant alleles and ~3% of the F482S allele (Fig. 2c). Carryover of these silent SNPs indicates that the alleles are the product of HR and not de novo mutagenesis. The lower rate of coappearance of silent SNPs in F482S is presumably due to the larger distance between the two SNPs in the oligonucleotide and is rather common to see with single-stranded oligonucleotide donors19." Rather than that ad hoc explanation, it is simply that the D54H cells had more silent only mutations to begin with (0.1% vs 0.0%). Regarding that 0.0%, an annoying thing is that they only report these percentages to one decimal place. I'm not exactly clear on the number of cells present before the CRISPR-Cas9 treatment, but it sounds like 10^8, and then they let them grow for 72 hr + 7-12 days (total of 10-15 days) after the treatment. They also don't tell us how many cells were left at the end... but anyway if we assume these cells divide once a day, and 0.1% are preexisting mutants we could calculate the possible number of mutants thus: Nt = 10^8 p = 0.001 d = 0:15 Nt*p*2^d After 12 days we can get ~400 million cells from those initial pre-existing mutants, and after 15 days over as 3 billion. Of course other factors would probably come into play that limit this growth, I'm just saying it would be no problem for that small subset of the population to become dominant during the experiment. That is even if the 99,900,000 "WT" cells were just growth arrested rather than died. So I find those results to favor the "selection for pre-existing mutants" explanation over the "gene modification" one.
    - AlexCoventry 10y ago
      The key observation from my perspective is that they seem to have done the resequencing before any resistance selection. Why would the indel frequencies rise only in the CRISPR-treated cells during drug-free growth?
      - nonbel 10y ago
        >"The key observation from my perspective is that they seem to have done the resequencing before any resistance selection. Why would the indel frequencies rise only in the CRISPR-treated cells during drug-free growth?" Because in cells that contain the target sequence (the complement to the guide RNA), Cas9 is damaging the DNA, leading to cell death and growth arrest. The small percentage of cells that already contained indels (thus reducing affinity for the guide RNA) are "immune", so they preferentially survive and divide to take over the population.