5 ms·

I have never seen a paper on CRISPR that can distinguish between selecting pre-existing mutants and actually modifying genes. I have read probably a dozen or so

by nonbel 11y ago

I have never seen a paper on CRISPR that can distinguish between selecting pre-existing mutants and actually modifying genes. I have read probably a dozen or so at this point, and it is amazing that they always fail to address this either in citations or actual data. At first I thought it was an honest mistake, but now it would not surprise me if some of the main players know that their experiments with CRISPR have been misinterpreted. They are then pushing the gene "modification" label anyway because it is sexier. After all, CRISPR has received an extremely unusual amount of media coverage over the last year or so, which raises red flags. I suspect a marketing effort is being directly funded. That is not a honest use of funds meant for research, especially that which is not meeting minimum scientific standards (ruling out other explanations for the results rather than just a null hypothesis).

daemonk 11y ago
But there are plenty of knock-in experiments where foreign DNA was put into the cut site. Are you saying a pre-existing mutant with the foreign DNA was used in those cases too?
- nonbel 11y ago
  Please link to one/some of the papers you are referring to. From what I have seen, they always detect edits in the controls or fail to report enough information to say either way, eg: "Although rare (∼1–2%), edits were detected with Cas9-only control treatment, including at the predicted CXCR4 cut site, potentially indicating trace amounts of experimental contamination of the Cas9 RNPs." http://www.pnas.org/content/112/33/10437.full http://www.pnas.org/content/112/33/10437.full Note the curiously missing rows in dataset S1.
  - cowsandmilk 11y ago
    What other controls do you expect to see? Dataset S1 seems complete to me. There is a similar background level of indels both at the CXCR4 site and off-target 1 and off-target 2 sites. The experiment increases indels at the CXCR4 site, but not at the off-target sites.
    - nonbel 11y ago
      The proportion of HDR (ie HindIII reads only) without the template. What percent of cells will randomly mutate to get HindIII recognition sites? Also, seeing what happens using template only would be good. We would expect low baseline levels of HDR to occur right? I it is plausible few out of 10^5 or 10^6 cells will require repair at that locus even without any Cas9.
speeder 11y ago
My sister was sent by Brazil's government to MIT so she can bring back CRISPR technology to Brazil public universities to speed a research here about using gene editing to control stem cell expression. I've seen plenty of people and papers where dna was edited in across entirely different organism realms, bacteria dna in animals, animal dna in plants, and so on... I honestly don't understand how the technique ins't about editing, the only use I saw for it is editing. EDIT: I asked my sister to give me links to some papers, I will post them after she replies.
- nonbel 11y ago
  You'll have to link to the exact evidence and methods you are referring to.
- nonbel 11y ago
  >"EDIT: I asked my sister to give me links to some papers, I will post them after she replies." Thanks, I appreciate it. This has been bugging me for awhile now.
daughart 11y ago
It's not clear to me what you are saying. Cas9/CRISPR unambiguously cuts the genome at a target site determined by the guide RNA sequence. In the presence of a DNA oligo with partial complementarity to the cut site, the DNA repair mechanism will sometimes incorporate the "payload" oligo, causing the genetic locus to be engineered from one sequence to another, predetermined sequence.
- nonbel 11y ago
  Start with 10^6 cells. Say 0.1% (1 in 1000) are already mutants at that site. Then add something that kills 100% the non-mutants and you will be left with 10^3 mutants without any gene editing. Say it kills 50% of the non-mutants and renders the rest quiescent due to DNA damage (not dividing), then you are left with 10^3 mutants and 5 x 10^5 non-mutants at time t0. After eg 7 divisions you will have 10^3 x 2^7 = 1.28 x 10^5 mutants, corresponding to 25% of the total. It depends on the initial number of cells, initial proportion of mutants, division rates, and toxicity. I have also noted that the initial number of cells is usually reported without any uncertainty, which makes me think those numbers may be rather unreliable.
  - daughart 11y ago
    No pre-existing cells have the mutation at the site you're trying to engineer. It just doesn't happen. Otherwise selection alone would be good enough. But mice cells don't have that much intrinsic variation. Plus a lot of time they're inserting whole genes or larger payloads. The statistical probability of that arising from chance is zero.
    - nonbel 11y ago
      >"No pre-existing cells have the mutation at the site you're trying to engineer. It just doesn't happen." Not in any paper on CRISPR I have read, in fact just the opposite: there are always low levels of mutants found in the controls (eg Schumann et al 2015 linked below). Please link to the papers that have lead you to make this claim.
jimrandomh 11y ago
The papers I've looked at sequence and measure the on-target mutation rate, and don't have any steps in them that would select for mutants (because that would ruin the measurement). Where do you propose the selection for mutants would be happening? Unless I'm misunderstanding something about how the experiments are done, your theory would require many groups to be independently committing scientific fraud, which is very implausible.
- nonbel 11y ago
  No fraud is necessary, just sloppy interpretation of data. Staying with Schuman et al (2015) linked in this thread, they start with 2.5 x 10^5 cells and end up with 5 x 10^4 to 2 x 10^5 three to four days later. Why are there fewer cells even without accounting for any division? Because the treatment is toxic. This is reported in many papers. I don't know what the proliferation rate is like for the cells in the conditions of that study, but apparently up to 7 divisions in 4 days is considered plausible for T-cells: http://www.ncbi.nlm.nih.gov/pubmed/17367338 http://www.ncbi.nlm.nih.gov/pubmed/17367338
  - jimrandomh 11y ago
    If you're right and CRISPR doesn't actually work, then none of the papers published so far are replicable and you'll be vindicated in mere months. If that doesn't happen, then there's a flaw in the style of reasoning and research that led you to conclude the existing papers were flawed. I recommend jotting down a few notes about how you came to this conclusion, and making a calendar reminder to check how it turned out next year.
    - nonbel 11y ago
      >"If you're right and CRISPR doesn't actually work, then none of the papers published so far are replicable and you'll be vindicated in mere months." Not at all. I'm not sure you understand what I am saying. It appears to me the data can be interpreted in multiple ways. Two different "theories" can explain the same results. This is a much more insidious problem than mere non-replication (I haven't seen any direct replications regarding CRISPR either though). People can continue on the wrong path for a long time by interpreting good, reliable data incorrectly. https://en.wikipedia.org/wiki/Experimentum_crucis https://en.wikipedia.org/wiki/Experimentum_crucis
tstactplsignore 11y ago
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.
Obi_Juan_Kenobi 11y ago
This is a baseless critique. No one has to address alternative hypotheses that don't make any sense. Are you unaware that Sanger Sequencing exists, and the actual lesions can be read? Or that heterologous genes are being introduced with CRISPR methods? Neither of these common results can be explained by the spontaneous insertion of hundreds of nucleotides that happen to precisely match the sequence of the construct being inserted. Again, this is utter nonsense and demonstrates a complete absence of basic understanding in molecular biology.
- nonbel 11y ago
  >"Are you unaware that Sanger Sequencing exists, and the actual lesions can be read? Or that heterologous genes are being introduced with CRISPR methods? Neither of these common results can be explained by the spontaneous insertion of hundreds of nucleotides that happen to precisely match the sequence of the construct being inserted." The first is just as consistent with the selection mechanism, because low levels of baseline mutants ARE reported (see the Schumann et al and Hendel et al papers I linked to in this thread for examples). The second would indeed be difficult to explain with a selection mechanism, unfortunately I have not seen that actually published. Instead, the primers used can just as well be amplifying the template and/or the sequence of the inserted cassette is not shown (eg Figure 2D and S1 here: http://www.sciencemag.org/content/348/6233/442 http://www.sciencemag.org/content/348/6233/442 ) If you have a reference to a specific paper I would appreciate it.
  - daemonk 11y ago
    What do you mean by the primers can just well be amplifying the template? How does that explain knock-ins without an actual insertion? And there are plenty of knock-in CRISPR papers out there. Just literally search for "CRISPR knock-in".
    - nonbel 11y ago
      Ok, I did that search and here is the first paper I found: http://www.nature.com/articles/srep14253 http://www.nature.com/articles/srep14253 EDIT: Let me look again at this paper later.

by nonbel 11y ago

daemonk 11y ago

But there are plenty of knock-in experiments where foreign DNA was put into the cut site. Are you saying a pre-existing mutant with the foreign DNA was used in those cases too?

nonbel 11y ago
Please link to one/some of the papers you are referring to. From what I have seen, they always detect edits in the controls or fail to report enough information to say either way, eg: "Although rare (∼1–2%), edits were detected with Cas9-only control treatment, including at the predicted CXCR4 cut site, potentially indicating trace amounts of experimental contamination of the Cas9 RNPs." http://www.pnas.org/content/112/33/10437.full http://www.pnas.org/content/112/33/10437.full Note the curiously missing rows in dataset S1.
- cowsandmilk 11y ago
  What other controls do you expect to see? Dataset S1 seems complete to me. There is a similar background level of indels both at the CXCR4 site and off-target 1 and off-target 2 sites. The experiment increases indels at the CXCR4 site, but not at the off-target sites.
  - nonbel 11y ago
    The proportion of HDR (ie HindIII reads only) without the template. What percent of cells will randomly mutate to get HindIII recognition sites? Also, seeing what happens using template only would be good. We would expect low baseline levels of HDR to occur right? I it is plausible few out of 10^5 or 10^6 cells will require repair at that locus even without any Cas9.

speeder 11y ago

My sister was sent by Brazil's government to MIT so she can bring back CRISPR technology to Brazil public universities to speed a research here about using gene editing to control stem cell expression. I've seen plenty of people and papers where dna was edited in across entirely different organism realms, bacteria dna in animals, animal dna in plants, and so on... I honestly don't understand how the technique ins't about editing, the only use I saw for it is editing. EDIT: I asked my sister to give me links to some papers, I will post them after she replies.

nonbel 11y ago
You'll have to link to the exact evidence and methods you are referring to.
nonbel 11y ago
>"EDIT: I asked my sister to give me links to some papers, I will post them after she replies." Thanks, I appreciate it. This has been bugging me for awhile now.

daughart 11y ago

It's not clear to me what you are saying. Cas9/CRISPR unambiguously cuts the genome at a target site determined by the guide RNA sequence. In the presence of a DNA oligo with partial complementarity to the cut site, the DNA repair mechanism will sometimes incorporate the "payload" oligo, causing the genetic locus to be engineered from one sequence to another, predetermined sequence.

nonbel 11y ago
Start with 10^6 cells. Say 0.1% (1 in 1000) are already mutants at that site. Then add something that kills 100% the non-mutants and you will be left with 10^3 mutants without any gene editing. Say it kills 50% of the non-mutants and renders the rest quiescent due to DNA damage (not dividing), then you are left with 10^3 mutants and 5 x 10^5 non-mutants at time t0. After eg 7 divisions you will have 10^3 x 2^7 = 1.28 x 10^5 mutants, corresponding to 25% of the total. It depends on the initial number of cells, initial proportion of mutants, division rates, and toxicity. I have also noted that the initial number of cells is usually reported without any uncertainty, which makes me think those numbers may be rather unreliable.
- daughart 11y ago
  No pre-existing cells have the mutation at the site you're trying to engineer. It just doesn't happen. Otherwise selection alone would be good enough. But mice cells don't have that much intrinsic variation. Plus a lot of time they're inserting whole genes or larger payloads. The statistical probability of that arising from chance is zero.
  - nonbel 11y ago
    >"No pre-existing cells have the mutation at the site you're trying to engineer. It just doesn't happen." Not in any paper on CRISPR I have read, in fact just the opposite: there are always low levels of mutants found in the controls (eg Schumann et al 2015 linked below). Please link to the papers that have lead you to make this claim.

jimrandomh 11y ago

The papers I've looked at sequence and measure the on-target mutation rate, and don't have any steps in them that would select for mutants (because that would ruin the measurement). Where do you propose the selection for mutants would be happening? Unless I'm misunderstanding something about how the experiments are done, your theory would require many groups to be independently committing scientific fraud, which is very implausible.

nonbel 11y ago
No fraud is necessary, just sloppy interpretation of data. Staying with Schuman et al (2015) linked in this thread, they start with 2.5 x 10^5 cells and end up with 5 x 10^4 to 2 x 10^5 three to four days later. Why are there fewer cells even without accounting for any division? Because the treatment is toxic. This is reported in many papers. I don't know what the proliferation rate is like for the cells in the conditions of that study, but apparently up to 7 divisions in 4 days is considered plausible for T-cells: http://www.ncbi.nlm.nih.gov/pubmed/17367338 http://www.ncbi.nlm.nih.gov/pubmed/17367338
- jimrandomh 11y ago
  If you're right and CRISPR doesn't actually work, then none of the papers published so far are replicable and you'll be vindicated in mere months. If that doesn't happen, then there's a flaw in the style of reasoning and research that led you to conclude the existing papers were flawed. I recommend jotting down a few notes about how you came to this conclusion, and making a calendar reminder to check how it turned out next year.
  - nonbel 11y ago
    >"If you're right and CRISPR doesn't actually work, then none of the papers published so far are replicable and you'll be vindicated in mere months." Not at all. I'm not sure you understand what I am saying. It appears to me the data can be interpreted in multiple ways. Two different "theories" can explain the same results. This is a much more insidious problem than mere non-replication (I haven't seen any direct replications regarding CRISPR either though). People can continue on the wrong path for a long time by interpreting good, reliable data incorrectly. https://en.wikipedia.org/wiki/Experimentum_crucis https://en.wikipedia.org/wiki/Experimentum_crucis

tstactplsignore 11y ago

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.

Obi_Juan_Kenobi 11y ago

This is a baseless critique. No one has to address alternative hypotheses that don't make any sense. Are you unaware that Sanger Sequencing exists, and the actual lesions can be read? Or that heterologous genes are being introduced with CRISPR methods? Neither of these common results can be explained by the spontaneous insertion of hundreds of nucleotides that happen to precisely match the sequence of the construct being inserted. Again, this is utter nonsense and demonstrates a complete absence of basic understanding in molecular biology.

nonbel 11y ago
>"Are you unaware that Sanger Sequencing exists, and the actual lesions can be read? Or that heterologous genes are being introduced with CRISPR methods? Neither of these common results can be explained by the spontaneous insertion of hundreds of nucleotides that happen to precisely match the sequence of the construct being inserted." The first is just as consistent with the selection mechanism, because low levels of baseline mutants ARE reported (see the Schumann et al and Hendel et al papers I linked to in this thread for examples). The second would indeed be difficult to explain with a selection mechanism, unfortunately I have not seen that actually published. Instead, the primers used can just as well be amplifying the template and/or the sequence of the inserted cassette is not shown (eg Figure 2D and S1 here: http://www.sciencemag.org/content/348/6233/442 http://www.sciencemag.org/content/348/6233/442 ) If you have a reference to a specific paper I would appreciate it.
- daemonk 11y ago
  What do you mean by the primers can just well be amplifying the template? How does that explain knock-ins without an actual insertion? And there are plenty of knock-in CRISPR papers out there. Just literally search for "CRISPR knock-in".
  - nonbel 11y ago
    Ok, I did that search and here is the first paper I found: http://www.nature.com/articles/srep14253 http://www.nature.com/articles/srep14253 EDIT: Let me look again at this paper later.