3 ms·

>"What is your proposed selection mechanism for pre-existing indels, given CRISPR treatment?" This was explained earlier I believe, so I am not sure where the

by nonbel 10y ago

>"What is your proposed selection mechanism for pre-existing indels, given CRISPR treatment?" This was explained earlier I believe, so I am not sure where the confusion lies. 1) You start with a mixed population of cells. From the literature it looks like about 99-99.9% will lack indels at the target site, the rest have them. 2) The Cas9 will cut the DNA of cells lacking indels at that locus (ie the wt cells containing a sequence complementary to the guide RNA), thus killing and/or growth arresting those cells. 3) Meanwhile the cells with indels will continue living and proliferating since they lack the target sequence. These are "immune" to the CRISPR-induced damage. Thus the proportion of WT cells will decrease, while the "mutants" will increase. It may help to play with the code of the simple model I shared earlier.

AlexCoventry 10y ago
> The Cas9 will cut the DNA of cells lacking indels at that locus So you're stipulating the consensus understanding of Cas9's initial action, but arguing that this results in cell death rather than nonhomologous end joining repair?
- nonbel 10y ago
  In general, yes (except you left out "growth arrest"). I don't see how their experiments can tell the difference between the two mechanisms, or some combination of them. Also, since mine is simpler, it probably dominates.
  - AlexCoventry 10y ago
    Why are you skeptical about NHEJ repair of the DNA breaks caused by cas9?
    - nonbel 10y ago
      Because an alternative explanation exists that fits the data just as well or better. The way science works is via Bayes Rule: Pr(T[0]|O) = Pr(O|T[0])Pr(T[0])/sum(Pr(O|T[i:n])Pr(T[i:n])) This tells us the evidence for a given theory given the observations depends on the ratio of two things: 1) How well the observations fit a theory (you can substitute hypothesis/model/etc) and how plausible a theory would be without the observations in question. 2) The above for all other theories In other words, the default for the scientist is to be skeptical of any explanation until the others have been rendered implausible (ie ruled out). There is a bit more to it (eg the Pr(O|T[i]) terms depend on the precision of the predictions, which remain vague in the case of NHEJ despite generous funding), but that is pretty much it.
      - AlexCoventry 10y ago
        It appears that your prior is uninformed by the extensive research into NHEJ, and the other successful genetic engineering methods which ate based on it.
    - nonbel 10y ago
      >"It appears that your prior is uninformed by the extensive research into NHEJ, and the other successful genetic engineering methods which ate based on it." I don't see how it is even possible for you to gather that from what I have said? All you could possibly have is a rough estimate of the ratio between the priors for NHEJ vs selection. Selection is a far more common and well studied process... There also exist simple quantitative models of that process, which allow precise predictions, something lacking in the case of NHEJ models afaik, which must remain vague. So the likelihoods will also be narrower in the selection case. Anyway, it was productive to discuss the specific paper and model, but is now getting philosophical and pointless. I only mentioned the scientific thought process because you asked why I would be skeptical.
      - AlexCoventry 10y ago
        I'm afraid you've fallen into the same kind of quantitative cargo-cult reasoning which led people astray in their applications of NHST. Quantitative reasoning can give you a lot of leverage in systematizing and inferring from a body of knowledge, but if your reasoning doesn't start from that knowledge it will lead you nowhere. In this case, the necessary knowledge is the structure and mechanism of DNA repair. When Dirichlet computed the probability that the Sun wouldn't rise tomorrow, given a flat two-event Dirichlet prior and the observation that it had risen every day for the last 6,000 years, he added that of course, for people who understood the workings of the solar system, the probability is far, far, lower. Your arguments here are like that. They just don't take into account the relevant facts of molecular biology.
        nonbel 10y ago
        1) I do indeed deny you need to know the exact mechanism of toxicity to model it. In fact, having any kind of quantitative model is better than the qualitative A increases/decreases B stuff that is usual. The reason being that it is too vague and many explanations can account for such an observation. 2) But please, let us skip the philosophy and talk directly about this topic, because that argument is not even necessary. It sounds to me like you do not believe that double strand breaks can lead to cell death and growth arrest? You find this implausible? I am really surprised that this is an objection.

by nonbel 10y ago

AlexCoventry 10y ago

> The Cas9 will cut the DNA of cells lacking indels at that locus So you're stipulating the consensus understanding of Cas9's initial action, but arguing that this results in cell death rather than nonhomologous end joining repair?

nonbel 10y ago
In general, yes (except you left out "growth arrest"). I don't see how their experiments can tell the difference between the two mechanisms, or some combination of them. Also, since mine is simpler, it probably dominates.
- AlexCoventry 10y ago
  Why are you skeptical about NHEJ repair of the DNA breaks caused by cas9?
  - nonbel 10y ago
    Because an alternative explanation exists that fits the data just as well or better. The way science works is via Bayes Rule: Pr(T[0]|O) = Pr(O|T[0])Pr(T[0])/sum(Pr(O|T[i:n])Pr(T[i:n])) This tells us the evidence for a given theory given the observations depends on the ratio of two things: 1) How well the observations fit a theory (you can substitute hypothesis/model/etc) and how plausible a theory would be without the observations in question. 2) The above for all other theories In other words, the default for the scientist is to be skeptical of any explanation until the others have been rendered implausible (ie ruled out). There is a bit more to it (eg the Pr(O|T[i]) terms depend on the precision of the predictions, which remain vague in the case of NHEJ despite generous funding), but that is pretty much it.
    - AlexCoventry 10y ago
      It appears that your prior is uninformed by the extensive research into NHEJ, and the other successful genetic engineering methods which ate based on it.
  - nonbel 10y ago
    >"It appears that your prior is uninformed by the extensive research into NHEJ, and the other successful genetic engineering methods which ate based on it." I don't see how it is even possible for you to gather that from what I have said? All you could possibly have is a rough estimate of the ratio between the priors for NHEJ vs selection. Selection is a far more common and well studied process... There also exist simple quantitative models of that process, which allow precise predictions, something lacking in the case of NHEJ models afaik, which must remain vague. So the likelihoods will also be narrower in the selection case. Anyway, it was productive to discuss the specific paper and model, but is now getting philosophical and pointless. I only mentioned the scientific thought process because you asked why I would be skeptical.
    - AlexCoventry 10y ago
      I'm afraid you've fallen into the same kind of quantitative cargo-cult reasoning which led people astray in their applications of NHST. Quantitative reasoning can give you a lot of leverage in systematizing and inferring from a body of knowledge, but if your reasoning doesn't start from that knowledge it will lead you nowhere. In this case, the necessary knowledge is the structure and mechanism of DNA repair. When Dirichlet computed the probability that the Sun wouldn't rise tomorrow, given a flat two-event Dirichlet prior and the observation that it had risen every day for the last 6,000 years, he added that of course, for people who understood the workings of the solar system, the probability is far, far, lower. Your arguments here are like that. They just don't take into account the relevant facts of molecular biology.
      - nonbel 10y ago
        1) I do indeed deny you need to know the exact mechanism of toxicity to model it. In fact, having any kind of quantitative model is better than the qualitative A increases/decreases B stuff that is usual. The reason being that it is too vague and many explanations can account for such an observation. 2) But please, let us skip the philosophy and talk directly about this topic, because that argument is not even necessary. It sounds to me like you do not believe that double strand breaks can lead to cell death and growth arrest? You find this implausible? I am really surprised that this is an objection.