
Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise - ChronoBiologist
http://www.genengnews.com/gen-news-highlights/crack-in-crispr-fa-ade-after-unanticipated-in-vivo-mutations-arise/81254419?utm_medium=newsletter&utm_source=GEN+Daily+News+Highlights&utm_content=02&utm_campaign=GEN+Daily+News+Highlights_20170530
======
leggomylibro
"Unanticipated?"

CRISPR is believed to be, at its core a bacterial immune system designed to
'knock out' problem viral genes by chopping them up at specific 'remembered'
loci and hoping that the lossy repair mechanisms cause mutations that prevent
the gene from functioning. It also evolved to work on the fairly small genomes
of single-celled organisms.

So we've known for awhile that it's going to be quite tricky to get only the
exact mutations that we want, at only the exact point that we want them.

It's not _magic_.

~~~
psychometry
The mutations were unanticipated by the computational tools designed to
identify likely off-target effects. This was in the article.

------
ackfoo
Pre-publication press release. Full text of article not available. How good
was their control group? The press release says they used mice that had
previously been edited with CRISPR/Cas9 and found off-target mutations, most
of which were single nucleotides. Did they have a good control group so as to
exclude random point mutations? Who knows? What is the theory on how
CRISPR/Cas9 causes single nucleotide off-target mutations? No idea.

This is academic publishing at its absolute worst. Put out a sensational press
release and whip up a fervour before the article is available. Those diving
into the scrum to comment have not read the article.

Also, it's research by MDs. Just sayin'.

~~~
amitutk
The article is available:
[https://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293....](https://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293.html)

~~~
nonbel
> "Co-housed FVB/NJ mice without CRISPR-mediated correction were used as the
> functional-deficient control. Briefly, an sgRNAexpressing plasmid had been
> coinjected, into FVB/NJ zygotes, with the single-stranded
> oligodeoxynucleotide (ssODN) donor template and Cas9 protein to generate
> mosaic F0 founders.1"

Following to reference 1:

> "The sgRNA plasmid was co-injected with the single-stranded
> oligodeoxynucleotide (ssODN) donor template and the Cas9 protein into _FVB
> /N zygotes to generate eleven F0 founders._

[...]

Double-strand breaks (DSB) were detected in 7 of 11 mice

[...]

The target region was sequenced, revealing that F0 3 and 5 incorporated the
donor template precisely in 35.7% and 18.8% of somatic cells, respectively
(Fig. 1c), while F0 7 and 8 incorporated indels in the integration,
corroborating the unexpected results in the RFLP data.

[...]

A mixture of 3 ng/mL sgRNA plasmid, 3ng/mL of Cas9 protein (NEB Ipswich, MA),
and 1mM ssODN (Integrated DNA Technologies, Iowa) was injected into the
pronuclei and cytoplasm of FVB/N inbred zygotes. _Zygotes that survived
injection_ were transferred into oviducts of 0.5-day post-coitum,
pseudopregnant B6xCBA F1 females and carried to term. The resulting gene-
corrected mice were backcrossed, initially into the FVB/N background, to
determine the germ-line transmission efficiency of the repair."
[https://www.ncbi.nlm.nih.gov/pubmed/27203441](https://www.ncbi.nlm.nih.gov/pubmed/27203441)

This is missing some crucial info isn't it? How many FVB/N zygotes were
injected to generate those 11 original mice?

~~~
ethelward
The more you work in the field, the more you discover how underwhelming the
details in biology papers are. They give results without detailing the
algorithms, hence destroying reproducibility, they hide datasets behind
confidentiality, presents large-scale graphs without precise data, and so on.

Generally, reproducibility in biology papers is but a far away dream.

~~~
nonbel
It is still awful but far better now than a few decades ago. The absolute
worst is Nature/Science from ~2000. For someone trying to figure out what is
going on (rather than just believe what the authors claim) most of those
papers are not even worth reading.

~~~
Amygaz
Any time before the advent of "supplementary materials" pretty much meant that
you had nothing more than a highlight of the method. It was terrible indeed.

However, even today I find that roughly half the publications are not worth
the paper they are printed on, or the bandwidth to download them.

~~~
nonbel
Check out pre-1940 papers (the year may be later depending on exact sub
field). I've seen that they used to make it a point to include all the info
(including raw data) to the point it was practical. Somewhere along the line
the attitudes went wrong, I blame NHST personally.

~~~
eicossa
NHST ? What's that ?

~~~
nonbel
I explained it and provided some references in an earlier post here:
[https://news.ycombinator.com/item?id=13483055](https://news.ycombinator.com/item?id=13483055)

Here is another reference you could check:
[http://andrewgelman.com/2016/02/04/the-notorious-n-h-s-t-
pre...](http://andrewgelman.com/2016/02/04/the-notorious-n-h-s-t-presents-mo-
p-values-mo-problems/)

------
feelix
Most of the DNA that suffers random mutations is non-coding DNA. It's
completely unknown what effects, if any, modifying non-coding DNA might have
as its use is unknown.

I find it interesting that although anyone can experiment with CRISPR in their
living room using something like this [http://www.the-
odin.com/](http://www.the-odin.com/) that no one has just tried modifying the
non-coding DNA a lot and then observed any changes (or lack thereof) in the
specimen.

~~~
leggomylibro
It's still fairly hard to do in a living room, if you want to do anything
novel.

You need probably low 4-figures of equipment first; a -20C freezer for
DNA/buffer storage, small centrifuge, thermal cycler, some way of getting
genes into a target, and incubation space for whatever you're modifying.
Preferably you'll also have consistent temperature control and an extremely
clean environment. You also need a source of primer sequences, which are short
customized sequences of DNA used to tl;dr get a lot of copies of a DNA
sequence which you only have in small quantities.

Nothing too difficult, and much of that equipment can be DIY'd for the home
lab. But many chemical suppliers also won't ship to residential addresses or
onboard individuals as customers, so you'll need to incorporate and shop
around a bit. And wet lab protocols can be very finicky; you'll probably need
to run through your workflow a _lot_ before you get to the point where you can
semi-reliably go from start to finish without fucking up. Often, you won't
know exactly how you fucked up, but you can't argue with a lack of results.

It's possible, I think, but it's also time-consuming and difficult.

Anyways, if you did get a reliable setup working with say, micropropagated
plant specimens, there are far more interesting prizes than seeing what non-
coding DNA does. Plants make all kinds of cool stuff, from scents to flavors
to alkaloids. And they've demonstrated an ability to take genes from things
like jellyfish for e.g. autoluminescence, too.

~~~
madengr
Could the genes that produce THC be spliced into common lawn grass?

~~~
leggomylibro
Yes, and they're all characterized. It would be difficult, though; you're
looking at about a half-dozen genes for the intermediary steps from something
common like acetyl-coa.

But with just THC, it probably wouldn't mimic the effects of cannabis very
well. There wouldn't be any CBD, terpenes, etc etc.

Still, you could do it. It'd probably be easiest with something like tobacco
which is well-understood and already has a system for sequestering cytotoxins.

Doubt you'd get something with appreciable yield stably transfected before
wider legalization hits, though. And that would be some _expensive_ pot.

~~~
twic
DMT would probably be the easiest. It's only three steps from tryptophan, and
I believe ask the enzymes are pretty simple.

------
j7ake
Off target effects from CRISPR is not new. The method leverages DNA repair
mechanisms in the cell to do the recombination. All errors involved in those
DNA repair mechanisms (there are distinct ones) would apply here。

~~~
mee_too
But if CRISPR cuts off a single DNA sequence, surely the repair mechanisms
won't damage a random gene far away. My very limited understanding is this
paper is reporting widely spread damage on the genome.

The original bacteria CRISPR can only cut off pre-defined sequences, but the
artificial gene tech CRISPR can add and edit as well. I'm wondering if the
later two modes are the sources of the genome damage.

~~~
placeybordeaux
> but the artificial gene tech CRISPR can add and edit as well.

I was under the impression that addition and editing capability were
essentially done by injecting the sequence that is desired and performing the
cut. Then you simply rely on the repair mechanisms to have a chance to put the
new segment in instead of the old segment. Is that not the case?

------
zigzigzag
I seem to recall that in prior HN stories about CRISPR some poster was saying
he believed CRISPR didn't work the way people thought it worked. He said it
was simply killing cells that didn't have the desired mutation and biologists
weren't realising that because of design errors in the experiments (or rather,
sometimes mass cell dieoffs were being reported but not dwelled upon).

If CRISPR isn't actually editing the DNA but rather just selecting natural
mutants that happen to have the desired edit, would that cause what's seen
here?

~~~
mee_too
Don't think so. Originally the CRISPR mechanism was discovered in bacteria,
which only have one cell. It doesn't make sense for bacteria's anti-virus
system to kill it's only cell.

~~~
0530_micro
This is a really interesting point.

I don't know if this experiment has been done, but I actually think that
there's a good chance that a bacterial cell might acquire a sequence that it
itself contains - CRISPR is known to work at the population level but my
understanding is that the mechanism for acquisition of new sequences is
unclear.

I'll take a look for any papers on the topic and repost should I find
anything.

------
dekhn
I believe George Church published some work several years ago now using whole
genome sequencing after CRISPR, saying they had neglible off-site integration
or other mutations.

But going back,the findings were much more measured than that- they still saw
off-site integration.

~~~
Amygaz
It seems to me that this high profile character often goes on record
mentioning things that ultimately end up being just his wishful thinking and
don't have much bearing.

~~~
dekhn
I'm inclined to agree, except he did invent much of modern sequencing. He's
great at technology, but limited in actual science.

------
DiNovi
the article headline kinda belies a bias before you even read it

------
daemonk
Title is a bit dramatic. People just need to choose their gRNAs better based
on available genomes.

------
BrandonMarc
Unanticipated

I'm just a normal person with nothing to do with this, and I expected such
"surprises" from the beginning.

I remain convinced mankind should not mess with this.

~~~
randyrand
The rest of us will enjoy leaving you behind =)

~~~
BrandonMarc
Hammond, is that you? Jurassic Park was a cautionary tale about hubris. People
with lots of money and smarts were convinced they could control nature.

------
systematical
I couldn't be the only one worried about an "I Am Legend" type problem with
this stuff. Obviously, that movie is an extreme, but the underlying problem of
unintended mutation exists.

~~~
88e282102ae2e5b
All known CRISPR systems work only under very specific conditions. You could
drink a vial of Cas9 and it wouldn't do anything to you because it couldn't
enter your cells.

------
mrcactu5
i am sick of all the CRISPR; hype this is the cure being worse than the
disease.

~~~
sndean
Not sure about it being overhyped, but similar sentiment, stated better is in
this article (from last year):

[http://www.sciencemag.org/news/2016/05/gene-editor-crispr-
wo...](http://www.sciencemag.org/news/2016/05/gene-editor-crispr-won-t-fully-
fix-sick-people-anytime-soon-here-s-why)

It's pretty clear there are some huge hurdles before it's useful, even for
your average biochem laboratory. But the effort put in to overcome those
hurdles is probably worth it.

