8 ms·

Potential DNA damage from CRISPR has been ‘seriously underestimated’ study finds

288 points by valeg 8y ago

aldoushuxley001 8y ago
And noone was shocked
ejstronge 8y ago
Note that this study covers 'on-target' effects. As often covered in the news, CRISPR-mediated gene editing can be used to edit a DNA sequence to another, desired sequence. In these applications, it's very important that no other mutations besides the desired mutation are introduced. Such additional mutations would be 'off-target' mutations. In a second kind of experimental approach, researchers can use CRISPR technology to make arbitrary mutations at a desired site in the genome. This approach cares less about the resulting damage; the goal is simply to destroy the existing gene. The referenced article[1] seems to characterize the types of mutations that arise when people use CRISPR to ablate a gene (i.e., the second approach). It would be interesting to see if the range of DNA damage is restricted when CRISPR is instead used to change the sequence of a targeted DNA segment into another, desired sequence. [1] https://www.nature.com/articles/Nbt.4192 https://www.nature.com/articles/Nbt.4192
- nonbel 8y ago
  The first thing to realize is that the crispr/cas9 strategy only produces damaged DNA (double stranded breaks, DSBs). It is not supposed to repair the DNA or do anything else. You have five main responses to this: 1) Necrosis/Lysis (cell dies due to malfunction) 2) Apotosis (cell kills itself) 3) Senescence (cell doesn't die but stops dividing) 4) Non-homologous end joining (NHEJ, the free ends of the damaged DNA are just pasted back together by the cells repair machinery) 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used by the cells repair machinery to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) The second thing to realize is that cells containing the targeted DNA sequence will be more likely to have damaged DNA than those without. So once a cell is "mutated" (either because it just existed beforehand, or because it survived the crispr/cas9 treatment), it will have a survival advantage. The third thing to realize is that often these cells they use can divide multiple times per day, I've seen as high as 12 times a day for activated T-cells. That means in two days a single mutant cell could possibly produce 2^24 = 16,777,216 offspring cells containing the mutation. Anyway, I think the efficiency of this procedure may have been far overstated. There may be only a very few cells in each experiment that actually get edited, but a few days later when they check, those (already mutated) cells dominate the population due to their resistance to the crispr/cas9 treatment. Eg, look at extended data fig 1h from this recent paper: https://www.nature.com/articles/s41586-018-0326-5/figures/5 https://www.nature.com/articles/s41586-018-0326-5/figures/5 The more cells that die, the higher percent of "edited" cells later.
  - ejstronge 8y ago
    > 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) I wanted to elide the details, but this is what I was referring to when I discussed CRISPR applications that try to change a nucleotide sequence. This of course leaves discussions of base-editing aside[1] 1. http://www.sciencemag.org/news/2017/10/novel-crispr-derived-base-editors-surgically-alter-dna-or-rna-offering-new-ways-fix http://www.sciencemag.org/news/2017/10/novel-crispr-derived-...
    - nonbel 8y ago
      Yes, you mentioned the two possibilities that everyone likes to focus on, but not the others which (I suspect) are much more common. EDIT: I really mean that I think in some of these experiments, where they only try to cause NHEJ, they just already had mutants in the population and selected for them. Its possible not a single cell was successfully edited but you still have so many cells with indels at the targeted sequence afterward.
      - azeotropic 8y ago
        Indel mutation rates aren't high enough, and the number of cells screened for mutations is too low for this explanation to suffice.
        nonbel 8y ago
        Do you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.
        azeotropic 8y ago
        You must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.
aaavl2821 8y ago
CRISPR is a revolutionary research tool that enables researchers to efficiently study the effect of changing genes, commonly by "knocking them out". CRISPR basically cuts a specific piece of DNA from the genome, so you can study how a cell / animal / disease model is different with and without the gene -- testing causality of gene and phenotype when CRISPR knocks out a gene, the cell tries to repair the DNA using its natural dna repair mechanisms. this can lead to unexpected insertions or deletions of DNA. researchers are widely aware of this risk and have studied it however, this paper exposes shortcomings of prior assessments of this risk. specifically, prior studies thought that the unexpected insertions or deltions of DNA were usually small, less than 20 nucleotides. there were observations of larger "indels" but these were thought to be rare. there are a few other shortcomings of the research into "crispr induced lesions", the article lists them in detail the scientists suspect that prior research has significantly underestimated the level of genomic alteration induced by crispr, and that some of this alteration could cause disease like cancer the authors first tried to delete a gene with crispr by targeting the protein coding part of the gene, and also non protein coding parts. the crispr targeting the protein coding part of the gene showed significnat knockdown of the gene (over 97%). however, so did the crispr targeting the non protein coding part of the gene (up to 20% of cells did not express the gene). some crispr constructs targeted sites that were over 2,000 base pairs from the nearest protein coding site, and 5-7% of those cells did not express the gene. they determined that loss of the gene was due to loss of the protein coding region, not just regulatory elements in the non protein coding part. they did similar experiments in other cell lines and with other genes so this suggests that researchers may have wildly underestimated the amount of genetic chaos induced by crispr. it is more possible than we thought that people receiving crispr therapies may also have pathogenic crispr induced dna damage the paper references six studies, all in China, currently using crispr to treat humans. 3 of the studies use crispr to "knock out" PD1, a molecule that blocks the immune system from attacking cancer, 2 use CRISPR to make cells express CD19 to attack cancerous blood cells, and one study tests an HIV therapy. anti PD1 antibodies and anti CD19 cell therapies using other gene editing techniques are approved and highly effective. anti pd1 antibodies are expected to generate over $20B in sales in the next few years. so these treatments have strong scientific basis supporting their potential effectiveness, but we may have drasticlly underestimated the risks
DINKDINK 8y ago
No treatment can ever be proven to be safe. It can only be shown that you have yet to observe harm.
- XalvinX 8y ago
  Even something such as ginseng that has a 5000+ year history of use with no real known side effects?
  - steve_adams_86 8y ago
    I suppose some people would argue that 'no known side effects' extends to 'no known effects' as well in this case. Scientific evidence that it does anything at all in humans appears to be non-existent to very weak at best.
    - XalvinX 8y ago
      Well, you can believe what you may, of course. But at least no one is dying from it...you can't say that about very many modern medicines, even Tylenol kills a lot of people. Probably half of the world's peoples have had good effects from ginseng, whether that is "placebo" (as scientist and western doctors might say...) or based on chemical compounds that haven't been extracted yet (they are still extracting them from another plant medicine in use for over 5000 years called Cannabis...scientists have no idea about most of the 500 or so in that plant either), or based on some kind of spiritual element as is said about Ayahuasca and Peyote is really all besides the point. Ginseng has cured many people and kept many more from ever getting ill in the first place. Explain it however you want.
      - chillacy 8y ago
        I have family who work in TCM, and I think their guidance is pretty decent: if it's serious go see a doctor, get medicine, get better. If it's not serious (you're okay with living with it), go with the herbs, because the downsides are much smaller (and the effect sizes too), but that's okay because it's not a serious disease. But I must stress this: if you have cancer, don't eat herbs and pray, go to a damn doctor.
XalvinX 8y ago
IMO it will be hundreds or thousands of years (if ever) until humans will fully understand the intricacies of DNA...the complexities are just enormous.
- fifnir 8y ago
  We went from not knowing how hereditary information is passed on to actually manipulating it in humans in the span of a few decades, don't be so pessimistic !
mirimir 8y ago
This isn't exactly surprising, in that CRISPR is a bacterial anti-bacteriophage defense system.
- aurizon 8y ago
  Yes, it it seems the bacteria jam it anywhere in their DNA (perhaps in many places at the same time) in the faint hope it will work to jam the machinery of the virus/phage it can work on in their successors. It looks as if a degree of specificity needs to engineered in to increase it's precision, like zinc fingers or something comparable.
narrator 8y ago
I ran into Liz Parrish at a longevity convention last year and she looked pretty damned good for being 2 years into CRISPRing herself. I cannot begin to fathom how wildly disruptive widespread modification of somatic cells would be to medicine and the drug industry. They'll just have to do it in China first I guess.
- baxtr 8y ago
  You should probably read this thread: https://news.ycombinator.com/item?id=11560943 https://news.ycombinator.com/item?id=11560943
  - narrator 8y ago
    Even if she lived to 150, a skeptic would just say it's a fluke and we have to wait 110 years for a large cohort double blind placebo controlled multi-center study of 40 year olds to finish at $800,000 a procedure before we can say that the intervention worked. It wouldn't get past the IRB either. Longevity science, even if it works, will be impossible to prove to skeptics for purely practical reasons.
deleted 8y ago
[deleted]
extralego 8y ago
This article is wreckless, i.e. really bad for business. If you support this article, you seriously should take a moment and reconsider the value of business and the freedom that the market has brought to your loved ones. This article is socialist propoganda.
a_bonobo 8y ago
Here's another good writeup (imho better than OP's) on this study with some potential ways forward: https://theconversation.com/crispr-cas9-gene-editing-scissors-are-less-accurate-than-we-thought-but-there-are-fixes-100007 https://theconversation.com/crispr-cas9-gene-editing-scissor... It's from Gaetan Burgio, who runs his own research group using CRISPR
KenanSulayman 8y ago
Another interpretation could be that the study found just how much our DNA deteriorates over time when replicated..
your-nanny 8y ago
>Academic scientists were less dismissive of the new study, in Nature Biotechnology. One leading CRISPR developer called it “well-done and credible,” “a cautionary note to the [genome-editing] community,” and consistent with other research showing that the DNA cuts that CRISPR makes, called double-stranded breaks, “can induce the types of genomic DNA rearrangements and deletions they report.” He asked not to be identified so as not to jeopardize business relationships with genome-editing companies. no. no. no. no. that is not good for scientific integrity

288 points by valeg 8y ago

And noone was shocked

Note that this study covers 'on-target' effects. As often covered in the news, CRISPR-mediated gene editing can be used to edit a DNA sequence to another, desired sequence. In these applications, it's very important that no other mutations besides the desired mutation are introduced. Such additional mutations would be 'off-target' mutations. In a second kind of experimental approach, researchers can use CRISPR technology to make arbitrary mutations at a desired site in the genome. This approach cares less about the resulting damage; the goal is simply to destroy the existing gene. The referenced article[1] seems to characterize the types of mutations that arise when people use CRISPR to ablate a gene (i.e., the second approach). It would be interesting to see if the range of DNA damage is restricted when CRISPR is instead used to change the sequence of a targeted DNA segment into another, desired sequence. [1] https://www.nature.com/articles/Nbt.4192 https://www.nature.com/articles/Nbt.4192

nonbel 8y ago
The first thing to realize is that the crispr/cas9 strategy only produces damaged DNA (double stranded breaks, DSBs). It is not supposed to repair the DNA or do anything else. You have five main responses to this: 1) Necrosis/Lysis (cell dies due to malfunction) 2) Apotosis (cell kills itself) 3) Senescence (cell doesn't die but stops dividing) 4) Non-homologous end joining (NHEJ, the free ends of the damaged DNA are just pasted back together by the cells repair machinery) 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used by the cells repair machinery to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) The second thing to realize is that cells containing the targeted DNA sequence will be more likely to have damaged DNA than those without. So once a cell is "mutated" (either because it just existed beforehand, or because it survived the crispr/cas9 treatment), it will have a survival advantage. The third thing to realize is that often these cells they use can divide multiple times per day, I've seen as high as 12 times a day for activated T-cells. That means in two days a single mutant cell could possibly produce 2^24 = 16,777,216 offspring cells containing the mutation. Anyway, I think the efficiency of this procedure may have been far overstated. There may be only a very few cells in each experiment that actually get edited, but a few days later when they check, those (already mutated) cells dominate the population due to their resistance to the crispr/cas9 treatment. Eg, look at extended data fig 1h from this recent paper: https://www.nature.com/articles/s41586-018-0326-5/figures/5 https://www.nature.com/articles/s41586-018-0326-5/figures/5 The more cells that die, the higher percent of "edited" cells later.
- ejstronge 8y ago
  > 5) Homology-directed repair (HDR, other DNA around that has a similar sequence to the free ends gets used to paste something in between; eg you have two of each chromosome so use the corresponding sequence on the other one to fill in the gap) I wanted to elide the details, but this is what I was referring to when I discussed CRISPR applications that try to change a nucleotide sequence. This of course leaves discussions of base-editing aside[1] 1. http://www.sciencemag.org/news/2017/10/novel-crispr-derived-base-editors-surgically-alter-dna-or-rna-offering-new-ways-fix http://www.sciencemag.org/news/2017/10/novel-crispr-derived-...
  - nonbel 8y ago
    Yes, you mentioned the two possibilities that everyone likes to focus on, but not the others which (I suspect) are much more common. EDIT: I really mean that I think in some of these experiments, where they only try to cause NHEJ, they just already had mutants in the population and selected for them. Its possible not a single cell was successfully edited but you still have so many cells with indels at the targeted sequence afterward.
    - azeotropic 8y ago
      Indel mutation rates aren't high enough, and the number of cells screened for mutations is too low for this explanation to suffice.
      - nonbel 8y ago
        Do you have a paper in mind? From what I've seen 0.01% - 10% of controls have indels at any given site (obviously this depends on treatment, cell type, etc). You could call that noise of course, at the very least they can't detect rates below those using current methods. I'll go find some refs on it if you ask.
        azeotropic 8y ago
        You must be misunderstanding what you've read. There's no way cells survive with indels at 10% of their base positions, that's not possible.

aaavl2821 8y ago

CRISPR is a revolutionary research tool that enables researchers to efficiently study the effect of changing genes, commonly by "knocking them out". CRISPR basically cuts a specific piece of DNA from the genome, so you can study how a cell / animal / disease model is different with and without the gene -- testing causality of gene and phenotype when CRISPR knocks out a gene, the cell tries to repair the DNA using its natural dna repair mechanisms. this can lead to unexpected insertions or deletions of DNA. researchers are widely aware of this risk and have studied it however, this paper exposes shortcomings of prior assessments of this risk. specifically, prior studies thought that the unexpected insertions or deltions of DNA were usually small, less than 20 nucleotides. there were observations of larger "indels" but these were thought to be rare. there are a few other shortcomings of the research into "crispr induced lesions", the article lists them in detail the scientists suspect that prior research has significantly underestimated the level of genomic alteration induced by crispr, and that some of this alteration could cause disease like cancer the authors first tried to delete a gene with crispr by targeting the protein coding part of the gene, and also non protein coding parts. the crispr targeting the protein coding part of the gene showed significnat knockdown of the gene (over 97%). however, so did the crispr targeting the non protein coding part of the gene (up to 20% of cells did not express the gene). some crispr constructs targeted sites that were over 2,000 base pairs from the nearest protein coding site, and 5-7% of those cells did not express the gene. they determined that loss of the gene was due to loss of the protein coding region, not just regulatory elements in the non protein coding part. they did similar experiments in other cell lines and with other genes so this suggests that researchers may have wildly underestimated the amount of genetic chaos induced by crispr. it is more possible than we thought that people receiving crispr therapies may also have pathogenic crispr induced dna damage the paper references six studies, all in China, currently using crispr to treat humans. 3 of the studies use crispr to "knock out" PD1, a molecule that blocks the immune system from attacking cancer, 2 use CRISPR to make cells express CD19 to attack cancerous blood cells, and one study tests an HIV therapy. anti PD1 antibodies and anti CD19 cell therapies using other gene editing techniques are approved and highly effective. anti pd1 antibodies are expected to generate over $20B in sales in the next few years. so these treatments have strong scientific basis supporting their potential effectiveness, but we may have drasticlly underestimated the risks

DINKDINK 8y ago

No treatment can ever be proven to be safe. It can only be shown that you have yet to observe harm.

XalvinX 8y ago
Even something such as ginseng that has a 5000+ year history of use with no real known side effects?
- steve_adams_86 8y ago
  I suppose some people would argue that 'no known side effects' extends to 'no known effects' as well in this case. Scientific evidence that it does anything at all in humans appears to be non-existent to very weak at best.
  - XalvinX 8y ago
    Well, you can believe what you may, of course. But at least no one is dying from it...you can't say that about very many modern medicines, even Tylenol kills a lot of people. Probably half of the world's peoples have had good effects from ginseng, whether that is "placebo" (as scientist and western doctors might say...) or based on chemical compounds that haven't been extracted yet (they are still extracting them from another plant medicine in use for over 5000 years called Cannabis...scientists have no idea about most of the 500 or so in that plant either), or based on some kind of spiritual element as is said about Ayahuasca and Peyote is really all besides the point. Ginseng has cured many people and kept many more from ever getting ill in the first place. Explain it however you want.
    - chillacy 8y ago
      I have family who work in TCM, and I think their guidance is pretty decent: if it's serious go see a doctor, get medicine, get better. If it's not serious (you're okay with living with it), go with the herbs, because the downsides are much smaller (and the effect sizes too), but that's okay because it's not a serious disease. But I must stress this: if you have cancer, don't eat herbs and pray, go to a damn doctor.

XalvinX 8y ago

IMO it will be hundreds or thousands of years (if ever) until humans will fully understand the intricacies of DNA...the complexities are just enormous.

fifnir 8y ago
We went from not knowing how hereditary information is passed on to actually manipulating it in humans in the span of a few decades, don't be so pessimistic !

mirimir 8y ago

This isn't exactly surprising, in that CRISPR is a bacterial anti-bacteriophage defense system.

aurizon 8y ago
Yes, it it seems the bacteria jam it anywhere in their DNA (perhaps in many places at the same time) in the faint hope it will work to jam the machinery of the virus/phage it can work on in their successors. It looks as if a degree of specificity needs to engineered in to increase it's precision, like zinc fingers or something comparable.

narrator 8y ago

I ran into Liz Parrish at a longevity convention last year and she looked pretty damned good for being 2 years into CRISPRing herself. I cannot begin to fathom how wildly disruptive widespread modification of somatic cells would be to medicine and the drug industry. They'll just have to do it in China first I guess.

baxtr 8y ago
You should probably read this thread: https://news.ycombinator.com/item?id=11560943 https://news.ycombinator.com/item?id=11560943
- narrator 8y ago
  Even if she lived to 150, a skeptic would just say it's a fluke and we have to wait 110 years for a large cohort double blind placebo controlled multi-center study of 40 year olds to finish at $800,000 a procedure before we can say that the intervention worked. It wouldn't get past the IRB either. Longevity science, even if it works, will be impossible to prove to skeptics for purely practical reasons.

deleted 8y ago

[deleted]

extralego 8y ago

This article is wreckless, i.e. really bad for business. If you support this article, you seriously should take a moment and reconsider the value of business and the freedom that the market has brought to your loved ones. This article is socialist propoganda.

a_bonobo 8y ago

Here's another good writeup (imho better than OP's) on this study with some potential ways forward: https://theconversation.com/crispr-cas9-gene-editing-scissors-are-less-accurate-than-we-thought-but-there-are-fixes-100007 https://theconversation.com/crispr-cas9-gene-editing-scissor... It's from Gaetan Burgio, who runs his own research group using CRISPR

KenanSulayman 8y ago

Another interpretation could be that the study found just how much our DNA deteriorates over time when replicated..

your-nanny 8y ago

>Academic scientists were less dismissive of the new study, in Nature Biotechnology. One leading CRISPR developer called it “well-done and credible,” “a cautionary note to the [genome-editing] community,” and consistent with other research showing that the DNA cuts that CRISPR makes, called double-stranded breaks, “can induce the types of genomic DNA rearrangements and deletions they report.” He asked not to be identified so as not to jeopardize business relationships with genome-editing companies. no. no. no. no. that is not good for scientific integrity