6 ms·

Lol, what, just what... the biophysics of the Cas9 system are understood pretty well. We literally get as close as you can get to observing the accepted mechani

Lol, what, just what... the biophysics of the Cas9 system are understood pretty well. We literally get as close as you can get to observing the accepted mechanism of action. How would you explain the consistency of site specific gene integration? You can't be selecting from existing mutants and "just happen" to get your transgenic product inserted at the exact site you specified. Your comment is just absurd. You also don't seem to realize that CRISPR is used at pretty much every university in dozens of different systems by thousands of different scientists. What a bizarre comment. Edit: uh, if you actually need a "source" for this: http://m.pnas.org/content/109/39/E2579.full http://m.pnas.org/content/109/39/E2579.full

nonbel 11y ago
>"You can't be selecting from existing mutants and "just happen" to get your transgenic product inserted at the exact site you specified. Your comment is just absurd." This is very easy. If you take a very many cells, some small percent will be mutants at any given site (unless you claim zero background rates of mutation, which is absurd and also directly contradicted by the data in these same papers). If you give a treatment that raises/causes the affinity of DNA damaging substances for a certain site, this will selectively damage the DNA of the non-mutant cells. The proliferation of the non-mutants will be suppressed and many will die off. The remaining mutants will proliferate to fill the gap. See my other post for a (very simple) mathematical model of this phenomenon. This is not absurd at all. It is basic logic and algebra. I will check that paper and get back to you. I actually have not read any using bacterial cells yet, thanks. Also, as far as I know there are no mathematical models of the standard proposed CRISPR mechanism that have been published, if you know of one that would be great.
- tstactplsignore 11y ago
  That might be possible if CRISPR was just a site specific knock out system. It's not. Your theory does not explain how site specific gene integration of transgenic products is possible if CRISPR/Cas is not an efficient site specific nuclease. If Cas9 is not cutting the DNA at the specific site so the transgenic product can integrate there, we wouldn't be getting the results seen.
nonbel 11y ago
What experiment in that paper do you think addresses the issue of selection vs modification? Both require the cleavage of specific DNA sequences, that is all I see reported in Gasiunas et al 2012.
- tstactplsignore 11y ago
  Here, listen. The following two papers conclusively "disprove" your idea. Both use single embryo injection and show multiple successful site specific mutagenesis in groups of no more than 5 to 25 cells. http://www.sciencedirect.com/science/article/pii/S0092867413010167 http://www.sciencedirect.com/science/article/pii/S0092867413... http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686313/#SD1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686313/#SD1
  - nonbel 11y ago
    Yang et al (2013): "To assess whether a marker transgene could be inserted into an endogenous locus, we coinjected Cas9 mRNA, sgRNA, and a double-stranded donor vector that was designed to fuse a p2AmCherry reporter with the last codon of the Nanog gene (Figure 2A). A circular donor vector was used to minimize random integrations. To assess toxicity and to optimize the concentration of donor DNA, we microinjected different amounts of Nanog-2A-mCherry vector. Injection with a high concentration of donor DNA (500 ng/ml) yielded mCherry-positive embryos with high efficiency, with most blastocysts being retarded, whereas injection with a lower donor DNA concentration (10 ng/ml) yielded mostly healthy blastocysts, most of which were mCherry-negative. When 200 ng/ml donor DNA was used, 75% (936/1,262) of the injected zygotes developed to blastocysts, 9% (86/936) of which were mCherry-positive (Figure 2C; Table S1)." So efficiency is inversely proportional to toxicity, they treated many more than 5-25 cells (the selection would occur at the level of the embryo), and there was only 1-10% rate of mutation detection. Also, they used "Superovulated female B6D2F1 mice". This procedure leads to chromosomal abnormalities and probably genetic instability so we would expect elevated presence of mutations at any given site: http://jhered.oxfordjournals.org/content/77/1/39.full.pdf http://jhered.oxfordjournals.org/content/77/1/39.full.pdf I'll have to look closer at the HDR aspect though (primers used, etc). But what may be going on that usually they detect the insertion via PCR: there is one primer to a sequence unique to the cassette and another upstream or downstream that should only be in the cells. Then the segment spanning the junction is amplified which supposedly is conclusive evidence of insertion at the correct location. The problem is you can get single primer amplification and also the homology arms required for HDR are likely to contain similar sequences to the "cell-only" primer. Eg: http://link.springer.com/protocol/10.1385%2F0-89603-258-2%3A307 http://link.springer.com/protocol/10.1385%2F0-89603-258-2%3A... Hwang et al 2013: "On the next day, injected embryos were inspected under stereoscope and were classified as dead, deformed or normal phenotypes. Only embryos that developed normally were assayed for target site mutations" They don't seem to tell us how many embryos were injected. And that study does not appear to use any type of control group at all. AFAICT, that is exactly the type of study that is consistent with a selection effect.

nonbel 11y ago

>"You can't be selecting from existing mutants and "just happen" to get your transgenic product inserted at the exact site you specified. Your comment is just absurd." This is very easy. If you take a very many cells, some small percent will be mutants at any given site (unless you claim zero background rates of mutation, which is absurd and also directly contradicted by the data in these same papers). If you give a treatment that raises/causes the affinity of DNA damaging substances for a certain site, this will selectively damage the DNA of the non-mutant cells. The proliferation of the non-mutants will be suppressed and many will die off. The remaining mutants will proliferate to fill the gap. See my other post for a (very simple) mathematical model of this phenomenon. This is not absurd at all. It is basic logic and algebra. I will check that paper and get back to you. I actually have not read any using bacterial cells yet, thanks. Also, as far as I know there are no mathematical models of the standard proposed CRISPR mechanism that have been published, if you know of one that would be great.

tstactplsignore 11y ago
That might be possible if CRISPR was just a site specific knock out system. It's not. Your theory does not explain how site specific gene integration of transgenic products is possible if CRISPR/Cas is not an efficient site specific nuclease. If Cas9 is not cutting the DNA at the specific site so the transgenic product can integrate there, we wouldn't be getting the results seen.

nonbel 11y ago

What experiment in that paper do you think addresses the issue of selection vs modification? Both require the cleavage of specific DNA sequences, that is all I see reported in Gasiunas et al 2012.

tstactplsignore 11y ago
Here, listen. The following two papers conclusively "disprove" your idea. Both use single embryo injection and show multiple successful site specific mutagenesis in groups of no more than 5 to 25 cells. http://www.sciencedirect.com/science/article/pii/S0092867413010167 http://www.sciencedirect.com/science/article/pii/S0092867413... http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686313/#SD1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686313/#SD1
- nonbel 11y ago
  Yang et al (2013): "To assess whether a marker transgene could be inserted into an endogenous locus, we coinjected Cas9 mRNA, sgRNA, and a double-stranded donor vector that was designed to fuse a p2AmCherry reporter with the last codon of the Nanog gene (Figure 2A). A circular donor vector was used to minimize random integrations. To assess toxicity and to optimize the concentration of donor DNA, we microinjected different amounts of Nanog-2A-mCherry vector. Injection with a high concentration of donor DNA (500 ng/ml) yielded mCherry-positive embryos with high efficiency, with most blastocysts being retarded, whereas injection with a lower donor DNA concentration (10 ng/ml) yielded mostly healthy blastocysts, most of which were mCherry-negative. When 200 ng/ml donor DNA was used, 75% (936/1,262) of the injected zygotes developed to blastocysts, 9% (86/936) of which were mCherry-positive (Figure 2C; Table S1)." So efficiency is inversely proportional to toxicity, they treated many more than 5-25 cells (the selection would occur at the level of the embryo), and there was only 1-10% rate of mutation detection. Also, they used "Superovulated female B6D2F1 mice". This procedure leads to chromosomal abnormalities and probably genetic instability so we would expect elevated presence of mutations at any given site: http://jhered.oxfordjournals.org/content/77/1/39.full.pdf http://jhered.oxfordjournals.org/content/77/1/39.full.pdf I'll have to look closer at the HDR aspect though (primers used, etc). But what may be going on that usually they detect the insertion via PCR: there is one primer to a sequence unique to the cassette and another upstream or downstream that should only be in the cells. Then the segment spanning the junction is amplified which supposedly is conclusive evidence of insertion at the correct location. The problem is you can get single primer amplification and also the homology arms required for HDR are likely to contain similar sequences to the "cell-only" primer. Eg: http://link.springer.com/protocol/10.1385%2F0-89603-258-2%3A307 http://link.springer.com/protocol/10.1385%2F0-89603-258-2%3A... Hwang et al 2013: "On the next day, injected embryos were inspected under stereoscope and were classified as dead, deformed or normal phenotypes. Only embryos that developed normally were assayed for target site mutations" They don't seem to tell us how many embryos were injected. And that study does not appear to use any type of control group at all. AFAICT, that is exactly the type of study that is consistent with a selection effect.