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>"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
by 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.
- AlexCoventry 10y agoOh. Interesting theory.
- AlexCoventry 10y agoDoes that argument assume the Cas9 activity you belive the experiment doesn't demonstrate?
- nonbel 10y agoI assume the Cas9 endonuclease activity is the cause of the toxicity... The theory does not require that though, only that the treatment kills/damages the cells containing the target sequence.
- AlexCoventry 10y agoBut you don't give an alternative etiology?
- nonbel 10y agoSorry, I have no idea what you are looking for. What is confusing you about my explanation? It seems very clear to me.
- AlexCoventry 10y agoWithout an alternative mechanism of toxicity, it's an untestable alternative hypothesis.
- nonbel 10y agoI still don't understand. What is wrong with the mechanism of double stranded breaks leading to cell death and/or growth arrest? Also, it is 100% possible to create a testable model of something at the level of toxicity without knowing the details of the toxicity. I mean, here would be a simple one (written in R) where the wild type cells divide at 1/10th the rate of the mutants for some reason, so the mutants take over the population: # Basic parameters of the cell culture Ntotal = 10^8 # Initial total number of cells Pmut0 = 0.001 # Propotion pre-existing mutants Rdiv_wt = 0.1 # Divisions/day Rdiv_mut = 1.0 # Divisions/day # Calculate initial numbers of WT and mutant cells Nwt0 = Ntotal*(1-Pmut0) Nmut0 = Ntotal*Pmut0 # Convert between days and divisions t = 0:15 Dwt = Rdiv_wt*t Dmut = Rdiv_mut*t # Calculate number of cells at each timepoint Nt_wt = Nwt0*2^Dwt Nt_mut = Nmut0*2^Dmut # Calculate proportion of mutant cells in population at each timepoint Pt_mut = Nt_mut/(Nt_wt + Nt_mut) # Plot proportion of mutants vs time plot(t, Pt_mut, type = "b", panel.first = grid(), xlab = "Days Since Treatment", ylab = "Proportion of Mutant Cells") If the parameters are known accurately enough (initial number of cells, initial proportion of mutants, division rates, etc) this is a perfectly testable quantitative model.
- AlexCoventry 10y agoWhat is your proposed selection mechanism for pre-existing indels, given CRISPR treatment?
- 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 agoIn 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 agoWhy are you skeptical about NHEJ repair of the DNA breaks caused by cas9?
- nonbel 10y agoBecause 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.