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

https://www.statnews.com/2018/07/16/crispr-potential-dna-damage-underestimated/

by valeg • 8 years ago

288 49 8 years ago

ejstronge2• 8 years 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

nonbel14• 8 years 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

The more cells that die, the higher percent of "edited" cells later.

ejstronge2• 8 years 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-...

azeotropic9• 8 years ago

Extended data figure 1h is testing the optimum pulse settings for the electroporation machine. There is a well-known trade-off between efficiency and viability (too small a pulse and no DNA is transferred into cells, too strong a pulse and the cells are destroyed). The efficiency increase at lower viability cannot be attributed to pre-existing mutants, because they're assaying for the presence of a GFP transgene inserted by homology-directed repair, not indels in general.

aaavl28212• 8 years 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

21• 8 years ago

currently using crispr to treat humans

Correct me if I'm wrong, but my impression was that crispr is used for treatment only in cases where there is nothing else left and the alternative is sure death

aaavl28212• 8 years ago

CRISPR is not used for any treatments yet because we dont know if any crispr based treatments 1) will work in humans or 2) are safe. there are no FDA approved crispr based treatments. there is virtually no human data on whether crispr treatments are safe in humans.

in cases where a patient has exhausted all treatment options, they can choose to enroll in a clinical study of an unapproved medicine. there are generally dozens if not hundreds of clinical studies for a given cancer type. if a patient is eligible for trials, these days they have plenty of options (however many pts are not eligible for studies). crispr based treatments would be one of many options

the 5 cancer studies of crispr treatments cited in the paper are all chinese studies, and all of them are basically crispr versions of therapies that work. for example, three of them use crispr to remove a gene that makes a protein called pd1. there are approved drugs that treat cancer by blocking PD1, and the safety of these is much better understood than crispr therapies.

for the patients in the crispr studies in china, there are drugs that essentially target the same disease related protein that are known to be fairly safe. so the patients could get a drug that probably works roughly as well as the crispr treatments but with less unknowns about safety

entee• 8 years ago

It's also likely that treatments where you can actually screen for mutations extensively will be more successful. The issue here is you get a lot of mutations that aren't desirable, if you can grow lots of different cultures and ID the ones that are OK, maybe that can work.

DINKDINK• 8 years ago

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

XalvinX4• 8 years ago

Even something such as ginseng that has a 5000+ year history of use with no real known side effects?

steve_adams_86• 8 years 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.

sgt101• 8 years ago

Asbestos was in use for 3900 years (roughly :o) ) before it was noticed that it can be dangerous (we now treat it as utterly deadly in the UK at least, I wonder if that is proportionate though - I am not slightly qualified to say!)

XalvinX4• 8 years 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• 8 years 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• 8 years ago

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

aurizon• 8 years 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.

narrator2• 8 years 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• 8 years ago

You should probably read this thread: https://news.ycombinator.com/item?id=11560943

narrator2• 8 years 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.

a_bonobo• 8 years 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-scissor...

It's from Gaetan Burgio, who runs his own research group using CRISPR

KenanSulayman• 8 years ago

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

your-nanny2• 8 years 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

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

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