
Potential DNA damage from CRISPR has been ‘seriously underestimated’ study finds - valeg
https://www.statnews.com/2018/07/16/crispr-potential-dna-damage-underestimated/
======
ejstronge
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
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
> 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-...](http://www.sciencemag.org/news/2017/10/novel-crispr-derived-base-
editors-surgically-alter-dna-or-rna-offering-new-ways-fix)

~~~
nonbel
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
Indel mutation rates aren't high enough, and the number of cells screened for
mutations is too low for this explanation to suffice.

~~~
nonbel
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
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.

~~~
nonbel
Thats not what I said. I said some significant percent of cells (I remember as
high as 10% from one paper, but usually its closer to 0.1%...) seem to have
indels at any given locus. Either that or the methods used to measure this are
too noisy to detect mutants that exist at those rates.

~~~
azeotropic
How is the per-base indel rate not 10% if 10% of cells carry an indel at any
given locus?

~~~
nonbel

      Reference: 
      GAATTC
    
      Variants:
      Cell 1: TAATTC
      Cell 2: GTATTC
      Cell 3: GATTTC
      Cell 4: GAAGTC
      Cell 5: GAATGC
      Cell 6: GAATTT
      Cell 7: GAATTC
      Cell 8: GAATTC
    

7/8 cells contain the reference nucleotide at any given site. Ie, 12.5% of
cells contain an indel wherever you look.

For six cells, 1/6 nucleotides differ from the reference sequence (16.7% per
base indel rate). For two cells, there is no difference (0 % per base indel
rate).

Even though 12.5% of cells contain an indel at any given site, no single cell
contains 12.5% indels. There is no reason the two values should be the same.

~~~
azeotropic
You clearly have no idea what you are talking about.

1) There are no indels in your example, only nucleotide substitutions.

2) There are six substitutions in 48 bases, for a per-base substitution rate
of 12.5%. The fact that none of the individual cells contains 12.5%
substitutions is totally irrelevant.

~~~
nonbel
>"There are no indels in your example, only nucleotide substitutions."

It works perfectly fine as an example. A single nucleotide change could be due
to an indel:

 _" Indel is a molecular biology term for an insertion or deletion of bases in
the genome of an organism. It is classified among small genetic variations,
measuring from 1 to 10 000 base pairs in length,[1][2][3][4][5][6][7]"_
[https://en.wikipedia.org/wiki/Indel](https://en.wikipedia.org/wiki/Indel)

>"There are six substitutions in 48 bases, for a per-base substitution rate of
12.5%. The fact that none of the individual cells contains 12.5% substitutions
is totally irrelevant."

In the experiments they are counting how many cells contain a mutant (eg via
GFP expression or not). Thus the number you want is percent of cells that
contain a mutation at a given site.

You are the one calculating some other number... Remember what we are
discussing? Its percent of cells with a given mutation and mutations per cell,
the numbers I calculated:

>"There's no way cells survive with indels at 10% of their base positions,
that's not possible."

>"You clearly have no idea what you are talking about."

There seems to be some heavy dunning-kruger going on with your post.

~~~
azeotropic
I asserted that saying that 10% of cells have an indel at any given site is
equivalent to saying the per-base indel mutation rate is 10%.

You posted an example purporting to disprove this, but instead, illustrated
that you don't know what an indel is, and that the two statements are indeed
equivalent -- at any given site, 12.5% of cells are mutant, and that the per-
base mutation rate is 12.5%.

The only one with Dunning-Kruger is you.

~~~
nonbel
Here is what I am responding to (also keep in mind I was using indel to mean
"indels and substitutions", ie whatever may be counted as an error during
NHEJ):

> "There's no way cells survive with indels at 10% of their base positions,
> that's not possible."

> "How is the per-base indel rate not 10% if 10% of cells carry an indel at
> any given locus?"

I figured you were referring to per base within each cell. Your overall "per
base" rate is getting the average (across cells) number of mutated sites. The
survival of a cell is determined by its own genome, not the average of the
population its been grouped into.

However, it is the case that if x% of cells have a mutation at any given site
then there must be at least some cells in this group that contain x% or
greater mutated sites. Is that what you meant? Because that's not a bad point.
I don't know where that 10% value came from and have no attachment to it, just
assume that one is measurement noise for now, but even cells with 0.1% of
sites mutated seems like a lot.

There doesn't seem to be many whole genome single cell sequencing results
available yet, which is what I think is needed here. Here is one that reports
~ 15k SNVs, 50-100 "micro-CNVs" (ie 10-100 kb copy number variants, fig S4),
and 290 indels:

> _" We call an SNV if there is a called NR allele and: (a) the total read
> depth in the bulk sample is above 15; (b) no bulk read has the NR allele;
> (c) if two SNVs are within 100bp from each other, both are discarded. With
> this procedure, we called 15,940 SNVs on the autosome of sample BJ1...We
> called 294 putative INDELs from the BJ1-BJ2 pair, the negative control"_
> [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538131/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538131/)

So taking the smallest and biggest numbers of possible here gives:

    
    
      100%*(15e3 + 50*10e3   + 290*1)/3e9    = 0.02% of bases affected
      100%*(15e3 + 100*100e3 + 290*10e3)/3e9 = 0.4%  of bases affected
    

This seems to ignore any very small or large mutations that may affect a given
site, and not all the mutations will simulate the effect of NHEJ... but this
makes me think it isnt impossible for 0.001-1% to happen, especially in vitro.

------
your-nanny
>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

------
a_bonobo
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...](https://theconversation.com/crispr-cas9-gene-editing-scissors-are-
less-accurate-than-we-thought-but-there-are-fixes-100007)

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

------
aaavl2821
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
> 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

~~~
aaavl2821
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

~~~
verulito
Well, there's that crazy guy who's trying to make it available to the public
for at home experimentation. He already tested it on himself, trying to get
himself bigger muscles as I recall. One of the comments I saw from a
microbiologist included the words "the most cancery cancer that ever
cancered".

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

~~~
aurizon
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.

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

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

~~~
steve_adams_86
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
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
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.

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

------
XalvinX
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
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 !

------
narrator
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
You should probably read this thread:
[https://news.ycombinator.com/item?id=11560943](https://news.ycombinator.com/item?id=11560943)

~~~
narrator
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.

