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> Cas-9 damages the DNA of some cells less because the the mutation was already there. The cells with no cas-9 are noise because they were never treated. If th

> Cas-9 damages the DNA of some cells less because the the mutation was already there. The cells with no cas-9 are noise because they were never treated. If the mutations were already there, then control in figure 7 in Lonowski et al. would not contain 0% "on-target" mutations. The presence of Cas9 is clearly causing a genetic change that is not already present in the population, and those cells that are changed do not all die. It is clearly not the case that the cells with the desired mutations completely overlapped with the cells transfected.

nonbel 9y ago
>"Although exhibiting background fluorescence, population 1 expresses some nuclease, as evidenced by a low level of editing. [...] The ‘shoulder’ indicated by an asterisk is not an indel, as it is also present in the control sample; it is probably due to incomplete 3´ A nucleotide addition to the IDAA amplicon (see Table 3, Step 21)." So they do see mutations in the control cells but decided to call it some kind of artefact. BTW, I also see a less shoulder in the left (on-target) site of the same figure. The point is that these mutants pre-exist in low frequencies of 1/100 to 1/100,000 cells (depends on cell type, environment, etc), almost regardless of the target site. If you look at enough cells you are guaranteed to find one with an indel at any site. In those cases, just as cas-9 doesn't just keep re-editing the dna after the target sequence has been cut, the cells are relatively "immune" to it. So for figure 7 they started with 50k cells, the questions are: 1) How many of those cells already contained a mutation at the target site? 2) What was the growth curve of the cells after treatment? 3) What is the division rate of these cells under these conditions? 4) What is the death rate of these cells under these conditions? 5) How many cells were there in the end for final analysis (sequencing, whatever)?

nonbel 9y ago

>"Although exhibiting background fluorescence, population 1 expresses some nuclease, as evidenced by a low level of editing. [...] The ‘shoulder’ indicated by an asterisk is not an indel, as it is also present in the control sample; it is probably due to incomplete 3´ A nucleotide addition to the IDAA amplicon (see Table 3, Step 21)." So they do see mutations in the control cells but decided to call it some kind of artefact. BTW, I also see a less shoulder in the left (on-target) site of the same figure. The point is that these mutants pre-exist in low frequencies of 1/100 to 1/100,000 cells (depends on cell type, environment, etc), almost regardless of the target site. If you look at enough cells you are guaranteed to find one with an indel at any site. In those cases, just as cas-9 doesn't just keep re-editing the dna after the target sequence has been cut, the cells are relatively "immune" to it. So for figure 7 they started with 50k cells, the questions are: 1) How many of those cells already contained a mutation at the target site? 2) What was the growth curve of the cells after treatment? 3) What is the division rate of these cells under these conditions? 4) What is the death rate of these cells under these conditions? 5) How many cells were there in the end for final analysis (sequencing, whatever)?