
Crispr’s Next Big Debate: How Messy Is Too Messy? - digital55
https://www.wired.com/2017/06/crispr-mutations/
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jfarlow
This recent note in an "obscure but high-profile journal" (what?!) is entirely
a technical observation. I fear that the CRISPR phenomenon has a lot of
people, even scientists, who know just enough about the topic to want to
affect stock prices. And this too seems to be the Wired article's
interpretation even after its own semi-clickbaity headline: "But most
scientists, while skeptical of the results, were more disappointed in the way
the paper was blown out of proportion."

The promise of CRISPR was _not_ that it was to be the singular tool that would
change how genetics and biology behaved. Rather the Cas9 protein could be used
as a cheap and fast technique to colocalize with specific DNA sequences. And
that is fantastic, useful, and worth all of the praise the tool has gotten.
But that's it.

That Cas9 happened to also actually allow a modification at specific sites
using its inherent DNA cleavage capability was a great little bonus - and that
it could be cleverly controlled to use that capability to actually, today,
cure some diseases, was amazing.

There is very little in biology that is binary in nature - it takes a lot of
energy to maintain such an entropic dam between two states. So a protein that
cuts at ATTGCTTGTA with 80%/hr/molecule efficiency, will also cut ATTGGTTGTA
with some non-0% efficiency as well. Every scientist who works with Cas9
(should) already know this. I don't think Cas9 is fantastic for it's ability
to cleave DNA - we've had restriction enzymes for a long time - but rather for
it's ability to colocalize with arbitrary DNA sequences. And with that
colocalization we can now bring to bear all the rest of the fantastic tools we
already have in synthetic molecular biology. And that is the interesting part
of our future - in which Cas9 is but a single (very useful) tool in our kit.

~~~
aargh_aargh
“If it squirms, it's biology. If it stinks, it's chemistry. If it doesn't
work, it's physics. And if you can't understand it, it's mathamatics.”

― Magnus Pyke

Anyway, thinking about your example it occured to me that someone using Cas9
should be able to look at the sequenced genome and identify all sequences
sufficiently similar to the target sequence that could be unintentionally
affected by Cas9. There's one idea for an experiment. As for mitigation, they
could try to find another specimen (fat chance) or species which does not have
such similar sequences. Of course, mutations will still happen for a myriad of
reasons other than Cas9, as they always do.

~~~
jfarlow
Sure - those are good ideas, and they do (did) such searches prior to
determining which sequence to look for in the genome. Just a few caveats off
the top of my head though:

\- When you are sequencing a genome how do you know whether or not the
windowed sequence you have is found only once in the genome? A solvable
problem for most sequences, but still not a trivial caveat.

\- Organisms _utilize_ genomic duplication in order to create
backups/redundancies/variants of their most critical systems. These backups
eventually 'drift' from their parent, but how much drift is required to know
whether or not it will be accidentally 'found' by Cas9.

\- Cas9, like squirmy biology, is time dependent. If you give it 100 years it
will likely cut most any sequence at some point during the period. So how do
you measure/account for the literal number of Cas9 proteins, much less the
amount of time they remain in a given nucleus.

\- DNA is relatively hardy as far as biological molecules go, but there are
all sorts of different kinds of chemical errors (much less intentional
modifications) associated with DNA that could make a given sequence more/less
likely to be misread by Cas9.

\- Are all your gRNAs _actually_ the exact sequence you think they are? RNA is
much less stable than the hardy DNA mentioned above. What if some of your RNA
is being modified before it even gets to go homing around for its
complementary sequence.

etc. etc. etc.

------
sigmar
To be clear- the fact that gene therapy is "messy" has been one of the biggest
issues holding it back for the past 20 years. This isn't a new concern based
on "one study," as Wired seems to frame it. Crispr is just the latest and most
promising method and it should be framed in that context. One step forward,
not a silver bullet.

One example of this concern stopping research in 2002, but there were some in
the late 90s too iirc:
[https://www.nature.com/nature/journal/v420/n6912/full/420116...](https://www.nature.com/nature/journal/v420/n6912/full/420116a.html)

~~~
adventured
I'd argue that it's closer to five steps forward than one, on a ten step
scale.

CRISPR is a gigantic leap forward in simplifying and expediting gene editing
across the board. It has also significantly reduced the cost. The tools, such
as SHERLOCK from Broad, that are already cropping up around CRISPR are well
beyond what was previously possible.

China as one example has found it so easy and inexpensive to work with,
they're likely to rapidly leap ahead of the West in gene editing due to their
willingness to allow for higher rates of risk. They'll have perhaps dozens of
human trials under way by the end of 2017 / mid 2018. Just on the back of
CRISPR they're going to go from lagging far behind in biotech, to being a
global leader within two decades. Technologically they're going to skip the
decades of traditional pharma / biotech build-up, as with jumping to
cellphones and bypassing landlines.

~~~
mschuster91
> due to their willingness to allow for higher rates of risk

You're making this sound more harmless than it is. I'd rather say "due to
their willingness to reject and destroy all previously common-held ethical
boundaries of science".

~~~
astrodev
Are you referring to anything specific?

~~~
kurthr
I'd throw out testing on prisoners as a possibility.

------
entee
One of the best critiques of the Nature Methods article was in the Wired
piece, pulling it out here for quick reference:

[http://blogs.sciencemag.org/pipeline/archives/2017/05/31/tro...](http://blogs.sciencemag.org/pipeline/archives/2017/05/31/trouble-
with-crispr-maybe-but-maybe-not)

Derek Lowe is an awesome writer about these kinds of issues. He highlights
some of the best of criticisms out there, notably that it's not clear whether
the increased rate of apparent mutation and the striking amount of mutations
shared between the two treated animals was due to the "experimental" animals
being siblings, but the "control" animal being more distantly related. This
article isn't the final word by far, and I think the authors didn't claim it
to be either. They wanted to put a concern out there for the scientific
community to investigate, as well they should, and the community will figure
this out.

Remember though, CRISPR is by definition a mutagen. It does create rather
substantial disruption in DNA structure (double stranded breaks), and relies
on intrinsic DNA repair processes to affect the desired end result. This is
likely to be a somewhat noisy process, and we shouldn't be terribly surprised
if unexpected changes do occur. Indeed the scientific community is well aware
of these limitations, and I think the more popular press shouldn't sell CRISPR
as equivalent to programming DNA in the same way we write computer programs.

------
Obi_Juan_Kenobi
Off-targets is something you always look for. In a research setting, you'll
usually be back-crossing to the parental line to clean up the background
anytime you transform something.

My experience is in plants, so my point of reference is agrobacertium
tumefaciens transformation (agro). To say that CRISPR is cleaner is a massive
understatement. It takes a little doing, though, with some labs optimizing for
particular species. Some of these optimized results have been astounding,
often with no detectable off-site modifications, and 100% efficiency (no need
to wait for the T2s for homozygotes)!

I'm not worried. I suppose it's good to point out that it's not ready for
full-scale gene-therapy yet, but I'm not aware of anyone saying that it is.

------
bitwize
I had a roommate once who was a molecular biologist. I told him I was excited
about the CRISPR developments and his response was "Yeah, man. Fucking
CRISPR." \-- in a tone of voice which I read as "I'll wait until the hypetrain
passes on this one before sharing your enthusiasm."

I guess his domain knowledge allowed him to foresee challenges the rest of us
are now becoming aware of.

------
nonbel
Earlier discussion here:
[https://news.ycombinator.com/item?id=14446204](https://news.ycombinator.com/item?id=14446204)

I asked: "How many FVB/N zygotes were injected to generate those 11 original
mice?"

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daemonk
This is a great blog post response to the article:
[https://medium.com/@GaetanBurgio/should-we-be-worried-
about-...](https://medium.com/@GaetanBurgio/should-we-be-worried-about-crispr-
cas9-off-target-effects-57dafaf0bd53)

------
goatlover
Maybe the Engineers in Promethus/Alien Covenant were using CRISPR to make that
black Xenomorphic goo.

------
DonHopkins
The fact that its name is missing the penultimate "e" should have clued people
in that it was an over-hyped up sham.

~~~
sigmar
It is an acronym proposed in 2001: Clustered regularly interspaced short
palindromic repeats

