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Lol, I just realized you were that guy who denies CRISPR-Cas9 genome editing really works. It may be against the rules, but I just have to tell you that you are

Lol, I just realized you were that guy who denies CRISPR-Cas9 genome editing really works. It may be against the rules, but I just have to tell you that you are an insufferable idiot who brings down the quality of discourse on this site with your bizarro pseudoscience nonsense.

nonbel 10y ago
This is a strawman. What I have seen in the data presented by the CRISPR-Cas9 researchers seems consistent with the idea that ~1% to 0.1% of cells in culture are mutants at any given site, the treatment selectively kills or growth arrests the non-mutants (at that site), and then the surviving (because they lacked the target sequence) pre-existing mutants divide and multiply to take over the population. I believe this may account for the vast majority of the "editing" they are measuring, and claims of increased efficiency over earlier methods (eg TALEN) are due to this mechanism. They have not done proper due diligence of ruling out other explanations and prematurely starting running with their favorite, most hype-able, hypothesis. Can you find one paper where they report the efficiency of inserting a cassette via HDR, or do they all only look at NHEJ when talking about that? I have only seen the latter. Also, this alternative explanation has real-life consequences. If correct, the very mechanism via which CRISPR-Cas9 works is toxic, meaning all the hundreds of millions of dollars (billions?) currently being spent trying to make it less toxic will be wasted.
- toufka 10y ago
  You do realize that the power of Cas9 is not it's ability to cut or even induce non-homologous-end-joining, but rather to co-localize with an exact sequence of DNA (that can be changed in a day for a few dollars). Cutting DNA is certainly a great self-defense mechanism used by the bacteria that causes strep throat to attack other bacteria. But in the ways that Cas9 will eventually be used, likely its entire endonuclease activity will be stripped, leaving only its homing ability. And for that you can swap in any other useful protein functions that now happen at a particular sequence location. And that is what makes Cas9 so exciting. That NHEJ works at all is just a bonus to get us to some early results quickly. fCas9 or dCas9, or a nickase (different endonuclease, and endonuclease-dead respectively) as well as a host of other variants actually hold great promise precisely and solely in their ability to locate a sequence.
  - nonbel 10y ago
    That may be. My explanation also requires relatively precise co-localization. Assuming the toxicity is only due to the endonuclease activity, those plans shouldn't be affected either way.

nonbel 10y ago

This is a strawman. What I have seen in the data presented by the CRISPR-Cas9 researchers seems consistent with the idea that ~1% to 0.1% of cells in culture are mutants at any given site, the treatment selectively kills or growth arrests the non-mutants (at that site), and then the surviving (because they lacked the target sequence) pre-existing mutants divide and multiply to take over the population. I believe this may account for the vast majority of the "editing" they are measuring, and claims of increased efficiency over earlier methods (eg TALEN) are due to this mechanism. They have not done proper due diligence of ruling out other explanations and prematurely starting running with their favorite, most hype-able, hypothesis. Can you find one paper where they report the efficiency of inserting a cassette via HDR, or do they all only look at NHEJ when talking about that? I have only seen the latter. Also, this alternative explanation has real-life consequences. If correct, the very mechanism via which CRISPR-Cas9 works is toxic, meaning all the hundreds of millions of dollars (billions?) currently being spent trying to make it less toxic will be wasted.

toufka 10y ago
You do realize that the power of Cas9 is not it's ability to cut or even induce non-homologous-end-joining, but rather to co-localize with an exact sequence of DNA (that can be changed in a day for a few dollars). Cutting DNA is certainly a great self-defense mechanism used by the bacteria that causes strep throat to attack other bacteria. But in the ways that Cas9 will eventually be used, likely its entire endonuclease activity will be stripped, leaving only its homing ability. And for that you can swap in any other useful protein functions that now happen at a particular sequence location. And that is what makes Cas9 so exciting. That NHEJ works at all is just a bonus to get us to some early results quickly. fCas9 or dCas9, or a nickase (different endonuclease, and endonuclease-dead respectively) as well as a host of other variants actually hold great promise precisely and solely in their ability to locate a sequence.
- nonbel 10y ago
  That may be. My explanation also requires relatively precise co-localization. Assuming the toxicity is only due to the endonuclease activity, those plans shouldn't be affected either way.