6 ms·

Response to “Unexpected mutations after CRISPR-Cas9 editing in vivo”

121 points by lucapinello 9y ago

cing 9y ago
A few other responses: http://www.biorxiv.org/content/early/2017/06/21/153338 http://www.biorxiv.org/content/early/2017/06/21/153338 http://www.biorxiv.org/content/early/2017/06/30/157925 http://www.biorxiv.org/content/early/2017/06/30/157925 http://www.biorxiv.org/content/early/2017/07/05/159707 http://www.biorxiv.org/content/early/2017/07/05/159707
Scaevolus 9y ago
They did whole-genome sequencing for mice treated with CRISPR-Cas9 and compared it to a different mouse to see if any mutations were caused? Why didn't they just take samples from the mouse before and after?
- gwern 9y ago
  Hard to believe, isn't it? You can see why the field was furious.
- 88e282102ae2e5b 9y ago
  Some of my colleagues speculated that this was not the intended study. Originally, they might have tried to delete a gene, but the expected phenotype didn't show up, which would have put an end to that line of research. But instead of completely abandoning the project they figured they could sequence the mice that were still around to see what the off-target effects were, and they thought they found something surprising and important.
  - rhizome 9y ago
    Does that qualify as "p-hacking?"
    - firebones 9y ago
      Of all the stupidity and hype around blockchain tech, the idea of committing the hypothesis of your research to an immutable record beforehand so as to avoid p-hacking seems to be one of the best ideas I've heard, provided you could avoid the problem of people simply committing a tournament of alternative answers...
      - reitanqild 9y ago
        Except we don't need a blockchain for it: just publish it in a well known place where others can collect it.
        jacquesm 9y ago
        They are suggesting a blockchain to prevent mutation of the publication (pun intended) after the fact.
        majewsky 9y ago
        The "where others can collect it" part solves that: Suppose that university A announces their study method and university B makes a mirror. Then if A were to forge the announcement after the fact, B could tell. (You would need cryptographic signing, though, to see if the record mirrored by B has not been tampered with.)
        jacquesm 9y ago
        > (You would need cryptographic signing, though, to see if the record mirrored by B has not been tampered with.) Yes, hence the blockchain, which provides all that in a fairly convenient package. You could even piggyback on any existing public blockchain, for a few fractions of a penny.
        Dylan16807 9y ago
        If you're making a notary system, the part you can get from a blockchain is a very minor percent of your code, and comes with a lot of complicated baggage. It's not convenient at all.
leemailll 9y ago
All begins with this comment https://www.ncbi.nlm.nih.gov/m/pubmed/28557981/ https://www.ncbi.nlm.nih.gov/m/pubmed/28557981/
- JohnJamesRambo 9y ago
  I love that. Xiaolin Wu is really going in on them. More science needs to be like that.
  - j7ake 9y ago
    In most labs there are journal clubs that function as this, but a lot of the discussion stays within the institute.
- deleted 9y ago
  [deleted]
- FrozenVoid 9y ago
  Non-mobile link to the above: https://www.ncbi.nlm.nih.gov/pubmed/28557981/?ncbi_mmode=std https://www.ncbi.nlm.nih.gov/pubmed/28557981/?ncbi_mmode=std
jostmey 9y ago
Great example of why the peer review system is broke. I've seen first hand how the peer review system works in the United States. Foreign Postdocs studying in the United States are desperate for a Green Card. They peer review as many papers as they can as quickly as they can to prove something or other for the purposes of a securing a Green Card. Some of these foreign postdocs do a good job, many do not. Also, overworked professors don't always take the time to carefully read a paper and don't care. The result is that a lot papers make it through peer review that shouldn't. I'd rather have my paper peer reviewed live on the internet by allowing people to comment on it beneath a PDF link than to suffer through another drawn out peer review process. People who care have a text-box where they say what they want, and if no one cares then it indicates I need to do research on something else!
- leemailll 9y ago
  Seriously, I don't know any top quality journals is using large quantity of postdocs as reviewers. In academy if one is invited by a top journal as a reviewer, that is an honor, meaning one is acknowledged by peers. In my field I can't think of any quality journals' reviewers are majorly post docs. Also your pointing fingers at foreigners is quite disturbing. Why you think they caused the problem? Do you have any evidence? I don't think in any academic workplace you can say this without unfavorable consequence.
  - FiReaNG3L 9y ago
    What does happen very frequently however is a prestigious researcher being asked to review papers, and passing them to his/her postdocs.
  - zionic 9y ago
    He mentioned them because their unique situation creates perverse incentives. Your last line is one of the most frustrating parts of academia. Not being able to criticize misaligned incentives simply because it involves foreigners is crazy.
    - leemailll 9y ago
hayabusa 9y ago
They have a response paper though : "Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness." http://www.biorxiv.org/content/early/2017/06/23/154450 http://www.biorxiv.org/content/early/2017/06/23/154450
- nonbel 9y ago
  Thanks, I was looking for this info earlier (https://news.ycombinator.com/item?id=14448371 https://news.ycombinator.com/item?id=14448371): >"In our case, 56 zygotes were harvested from six pregnant females bred to six stud males and injected with CRISPRCas9." http://www.biorxiv.org/content/early/2017/06/23/154450 http://www.biorxiv.org/content/early/2017/06/23/154450 So they started with 56 zygotes, of which 11 survived injection of Crispr/etc. Of these 11, five and two mice showed some evidence of containing NHEJ/HDR cells, respectively. In the case of NHEJ, they do not tell us what percent of cells were "edited" per mouse (or I missed it when I read that paper earlier). However for HDR it was only about 1/3 and 1/5 for the two mice they found. So this suggests the "editing" is occurring not at the zygote stage, but somewhat later (based on the percentages, perhaps at the 4 or 8 cell stages). Assuming the edit/mouse rate is similar for NHEJ and HDR (somewhat dubious), it looks like 4x56 = 224 to 8x56 = 448 cells were treated with crispr/etc and ~ 7 got "edited". This gives ~1-3% "edits" per treated cell.

121 points by lucapinello 9y ago

cing 9y ago

A few other responses: http://www.biorxiv.org/content/early/2017/06/21/153338 http://www.biorxiv.org/content/early/2017/06/21/153338 http://www.biorxiv.org/content/early/2017/06/30/157925 http://www.biorxiv.org/content/early/2017/06/30/157925 http://www.biorxiv.org/content/early/2017/07/05/159707 http://www.biorxiv.org/content/early/2017/07/05/159707

Scaevolus 9y ago

They did whole-genome sequencing for mice treated with CRISPR-Cas9 and compared it to a different mouse to see if any mutations were caused? Why didn't they just take samples from the mouse before and after?

gwern 9y ago
Hard to believe, isn't it? You can see why the field was furious.
88e282102ae2e5b 9y ago
Some of my colleagues speculated that this was not the intended study. Originally, they might have tried to delete a gene, but the expected phenotype didn't show up, which would have put an end to that line of research. But instead of completely abandoning the project they figured they could sequence the mice that were still around to see what the off-target effects were, and they thought they found something surprising and important.
- rhizome 9y ago
  Does that qualify as "p-hacking?"
  - firebones 9y ago
    Of all the stupidity and hype around blockchain tech, the idea of committing the hypothesis of your research to an immutable record beforehand so as to avoid p-hacking seems to be one of the best ideas I've heard, provided you could avoid the problem of people simply committing a tournament of alternative answers...
    - reitanqild 9y ago
      Except we don't need a blockchain for it: just publish it in a well known place where others can collect it.
      - jacquesm 9y ago
        They are suggesting a blockchain to prevent mutation of the publication (pun intended) after the fact.
        majewsky 9y ago
        The "where others can collect it" part solves that: Suppose that university A announces their study method and university B makes a mirror. Then if A were to forge the announcement after the fact, B could tell. (You would need cryptographic signing, though, to see if the record mirrored by B has not been tampered with.)
        jacquesm 9y ago
        > (You would need cryptographic signing, though, to see if the record mirrored by B has not been tampered with.) Yes, hence the blockchain, which provides all that in a fairly convenient package. You could even piggyback on any existing public blockchain, for a few fractions of a penny.
        Dylan16807 9y ago
        If you're making a notary system, the part you can get from a blockchain is a very minor percent of your code, and comes with a lot of complicated baggage. It's not convenient at all.

leemailll 9y ago

All begins with this comment https://www.ncbi.nlm.nih.gov/m/pubmed/28557981/ https://www.ncbi.nlm.nih.gov/m/pubmed/28557981/

JohnJamesRambo 9y ago
I love that. Xiaolin Wu is really going in on them. More science needs to be like that.
- j7ake 9y ago
  In most labs there are journal clubs that function as this, but a lot of the discussion stays within the institute.
deleted 9y ago
[deleted]
FrozenVoid 9y ago
Non-mobile link to the above: https://www.ncbi.nlm.nih.gov/pubmed/28557981/?ncbi_mmode=std https://www.ncbi.nlm.nih.gov/pubmed/28557981/?ncbi_mmode=std

jostmey 9y ago

Great example of why the peer review system is broke. I've seen first hand how the peer review system works in the United States. Foreign Postdocs studying in the United States are desperate for a Green Card. They peer review as many papers as they can as quickly as they can to prove something or other for the purposes of a securing a Green Card. Some of these foreign postdocs do a good job, many do not. Also, overworked professors don't always take the time to carefully read a paper and don't care. The result is that a lot papers make it through peer review that shouldn't. I'd rather have my paper peer reviewed live on the internet by allowing people to comment on it beneath a PDF link than to suffer through another drawn out peer review process. People who care have a text-box where they say what they want, and if no one cares then it indicates I need to do research on something else!

leemailll 9y ago
Seriously, I don't know any top quality journals is using large quantity of postdocs as reviewers. In academy if one is invited by a top journal as a reviewer, that is an honor, meaning one is acknowledged by peers. In my field I can't think of any quality journals' reviewers are majorly post docs. Also your pointing fingers at foreigners is quite disturbing. Why you think they caused the problem? Do you have any evidence? I don't think in any academic workplace you can say this without unfavorable consequence.
- FiReaNG3L 9y ago
  What does happen very frequently however is a prestigious researcher being asked to review papers, and passing them to his/her postdocs.
- zionic 9y ago
  He mentioned them because their unique situation creates perverse incentives. Your last line is one of the most frustrating parts of academia. Not being able to criticize misaligned incentives simply because it involves foreigners is crazy.
  - leemailll 9y ago

hayabusa 9y ago

They have a response paper though : "Here we provide additional confirmatory data and clarifying discussion, including sequencing data showing extensive heterozygous mutations throughout the genome in the CRISPR treated mice, which are all progeny of inbred mice purchased from a commercial vendor (JAX). The heterozygosity in these cases cannot be parentally inherited. The summary statements in our Correspondence reflect observations of a secondary outcome following successful achievement of the primary outcome using CRISPR to treat blindness in Pde6b/rd1 mice. As the scientific community considers the role of WGS in off-target analysis, future in vivo studies are needed where the design and primary outcome focuses on CRISPR off-targeting. We agree that a range of WGS controls are needed that include parents, different gRNAs, different versions of Cas9, and different in vivo protocols. We look forward to the publication of such studies. Combined, these results will be essential to fully understand off-targeting and can be used to create better algorithms for off-target prediction. Overall, we are optimistic that some form of CRISPR therapy will be successfully engineered to treat blindness." http://www.biorxiv.org/content/early/2017/06/23/154450 http://www.biorxiv.org/content/early/2017/06/23/154450

nonbel 9y ago
Thanks, I was looking for this info earlier (https://news.ycombinator.com/item?id=14448371 https://news.ycombinator.com/item?id=14448371): >"In our case, 56 zygotes were harvested from six pregnant females bred to six stud males and injected with CRISPRCas9." http://www.biorxiv.org/content/early/2017/06/23/154450 http://www.biorxiv.org/content/early/2017/06/23/154450 So they started with 56 zygotes, of which 11 survived injection of Crispr/etc. Of these 11, five and two mice showed some evidence of containing NHEJ/HDR cells, respectively. In the case of NHEJ, they do not tell us what percent of cells were "edited" per mouse (or I missed it when I read that paper earlier). However for HDR it was only about 1/3 and 1/5 for the two mice they found. So this suggests the "editing" is occurring not at the zygote stage, but somewhat later (based on the percentages, perhaps at the 4 or 8 cell stages). Assuming the edit/mouse rate is similar for NHEJ and HDR (somewhat dubious), it looks like 4x56 = 224 to 8x56 = 448 cells were treated with crispr/etc and ~ 7 got "edited". This gives ~1-3% "edits" per treated cell.