
The Weirdness of CRISPR - dnetesn
http://alliance.nautil.us/feature/190/the-unbearable-weirdness-of-crispr
======
jfarlow
Now that we've made economical the tools to sequence, search, and have some
understanding of an organism's genome, we're reaping fast rewards in learning
what the natural world is actually capable of in terms of astounding feats of
nano-engineering. There's a massive nanotechnology textbook in our ecosystem's
genomes that's a few orders of magnitude more impressive than our best
semiconductor patents.

The CRISPR system here was discovered in a single-celled organism that likes
brine water - a tool it used to intercept hijackers. The 'standard' Cas9 that
is being used extensively in the rest of the CRIPSR stories comes from the
bacteria that causes strep throat. Studies are now mining the genomes of
organisms found in 'mud pits' and 'pond scum' to find all sorts of smaller,
faster, more efficient variants of Cas9. And that's just for the single
purpose of editing a genome.

The capabilities of proteins found (off the top of my head) in jellyfish, in
sulfur springs, in colored algae, in electric eels, in little pond creatures,
in HIV, in waterbears, etc. are all depositing huge amounts of technology into
our collective knowledgebank. We have been given a store of 'alien'
technologies to sift through that are applicable in every condition to be
found on Earth, literally. The cures to many of our ills will not be
engineered from scratch, but rather will be adapted from existing technologies
found in extant organisms literally scattered across the globe. _This_ is how
'basic science' works. A magnanimous scientist is curious how a little cell
can survive in really salty water, and owing to his curiosity we discover a
nanotechnology that can edit an organism's genome. We humans are smart - but
not yet that smart.

(some of those protein technologies we like to talk about here on HN:
[https://serotiny.bio/notes/proteins/](https://serotiny.bio/notes/proteins/) )

~~~
SolarNet
Which is also a reason we should stop destroying the environment. There is a
wealth of knowledge out there we have yet to acquire. If you ran a deep-
learning algorithm using an entire planet for 3 billion years, the results
would be considered invaluable. Well what is natural selection and evolution
but that?

~~~
jfarlow
Yes.

If in a sci-fi novel an alien handed a character their 'magic rock' (iPhone)
and they smelted it for its gold content rather than reverse-engineered it's
wireless charging, communication, and computation capabilities you would
scream at the character for being horribly short-sighted. Such a waste of
sophisticated technology laid right in front of them for such little temporary
gain.

~~~
novalis78
Why can't we do both . At least smelting until we have enough energy to
reverse engineer and then keep smelting space rocks. I think that's actually
what we are doing, birds eye view.

~~~
dagss
Once a species is gone it is gone. Lots and lots of species disappear now and
many of them we are not even aware about, let alone any DNA samples taken or
research done (e.g. burn down rainforest). So you can't "have both".

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stepik777
BTW there is a patent pool being formed by MPEG LA to license CRISPR. It seems
to be similar to patent pools for video codecs.

* [http://www.mpegla.com/main/pid/CRISPR/default.aspx](http://www.mpegla.com/main/pid/CRISPR/default.aspx)

* [http://www.mpegla.com/Lists/MPEG%20LA%20News%20List/Attachme...](http://www.mpegla.com/Lists/MPEG%20LA%20News%20List/Attachments/102/CRISPR%20PrsRls%202016-12-06.pdf)

~~~
jfarlow
You have got to be kidding me...

...you are not. What is this insanity?

~~~
IncRnd
From the about page of mpegla.com:

 _" We are the world’s leading packager of patent pools for standards and
other technology platforms used in consumer electronics, as well as chemical,
eCommerce, education, energy, environment, healthcare and biotechnology,
manufacturing and materials, transportation and wireless technology. We
developed the pool market space. (view link) Our business model supports a
large number of patent users – creating reasonable access and profitable
opportunities for all parties."_

------
Para2016
CRISPR, Gene Drive, and IPSC biotech is incredible.

Right now I'm killing time until my residency starts and I've been reading
recent journal articles relating to each of these.

One of the weirder journal articles I read was about this one group taking
fibroblasts from XX and XO (Turner's syndrome) females and XY cells from a
male and de-differntiating them into induced pluripotent stem cells. They then
re-differentiated the cells into germ cells. Sure, that's not controversial
just yet, but then they decided to put the XX female germ cells into a teste.
The female germ cells (XX and XO) and the male XY cells all differentiated
into cells that could succesfully undergo meiosis. And these cells, could
terminally become sperm. That means an XX skin cell could be induced to become
a sperm and possibly fertilize an egg from another woman. Of course all
offspring would be XX female. This process totally bypasses males.

Anyways, very interesting to me, maybe it will be to others. Here's the
journal article.

[http://www.nature.com/articles/srep06432](http://www.nature.com/articles/srep06432)

I know this article I'm posting under is about CRISPR, but I feel like
new/controversial biology research could be posted here too. Hopefully anyone
reading gets the same feeling of discovery/wonder/incredulity that I had when
reading about it.

~~~
tmoot
I didn't read the paper but it's in scientific reports (minimal and rushed
peer review, bottom of the barrel nature subjournal)...I'd be a bit skeptical.

It's not exactly anyone's first choice place to publish.

Crispr/Cas9 is not as robust as people make it out to be, but it's promising.
(I do some work with crispr/cas9)

~~~
MegaButts
> Crispr/Cas9 is not as robust as people make it out to be, but it's
> promising. (I do some work with crispr/cas9)

Please expand on this. I feel like CRISPR is being overhyped, and would love
to hear form someone educated on the subject as to what its possible
limitations are, or at least some of the known challenges ahead.

~~~
jfarlow
Cas9 is a protein with two _functions_ : 1) locate a DNA sequence that matches
the little RNA it grabs ahold of, and 2) cut that DNA at that location. The
first of those functions, it's ability to locate particular and arbitrary
sequences, is its comparative advantage against all other technologies we know
of. The second, cutting DNA, well, works I guess, but will likely be
engineered to be more useful, or turned off in order to make way for other
more useful functions.

The protein is _special_ for two different reasons: 1) it is able to locate
DNA sequences with very high precision 2) and the sequence it locates can be
swapped out as easily as changing the sequence of the RNA it grabs ahold of
(trivial to do, can be done in a day, and can cost >$1 per target sequence to
swap). Note, it is not special because it can cut DNA - there are lots of
proteins that do that, and there are lots of better ways to change or alter
DNA once you get to a particular sequence - but Cas9 was originally a self-
defense mechanism, so it's evolutionary function in strep throat bacteria was
to kill invaders by dicing up their DNA (at particular sequences that strep
throat doesn't have).

Cas9 is powerful because it could be used to direct _any_ function at a
_particular_ DNA sequence, where the sequence can be altered in the lab
quickly and cheaply. As it is a protein encoded by a particular sequence, you
can fuse it to other proteins with other functions to build a more powerful
machine. (see [1] if you want to play with those sequences yourself.) As an
experimental tool it will likely become a foundational tool used all
throughout molecular biology - and for that alone is is worth it's fame.
Thermophilic polymerase used in PCR is another such tool. As are restriction
enzymes. As is GFP. That's the scientist's perspective.

However, Cas9 also _previews_ the capability of directly and arbitrarily
editing of a genome - a holy grail of biomedical sciences. Though unengineered
Cas9 it's not great at editing a genome (we're not entirely sure why what it
does even works) - but some 2-10% of the time it can actually edit a genome
with fidelity. And that's good enough for many experiments (though not good
enough for therapies). It has off-target cuts, and when it cuts it slices all
the way through the double stranded DNA, and if it isn't properly stitched
back together you have a broken chromosome. And getting payload DNA to the
site that Cas9 cut is still really tricky. It's also a multi-part system (it
needs it's little RNA as well as the protein itself), and so it's hard to
deliver directly as a therapeutic. So the wild-type Cas9 is likely limited in
its direct therapeutic relevance in terms of pure genome editing. But it will
be used extensively for its ability to 1) further research quickly and
cheaply, 2) prototype what genomic changes would do if they were successful
(when you only need 10% efficacy to conduct a study), and 3) act as an
engineering platform upon which other functions can be placed, and its own
wild-type limitations can be overcome.

It's powerful. It's not perfect, there's lots more engineering to do with/to
it. It's not going to get to the holy grail of genome editing all on its own,
but it's a very solid platform to start building off of, as well as simply
being a solid tool that will become a workhorse of further synthetic biology.

[1] [https://serotiny.bio/](https://serotiny.bio/)

~~~
nonbel
>"Though unengineered Cas9 it's not great at editing a genome ( _we 're not
entirely sure why what it does even works_) - but some 2-10% of the time it
can actually edit a genome with fidelity."

Can you expand on the part I italicized, preferably also linking to some
journal articles?

~~~
jfarlow
Cas9 finds, then cuts both strands of DNA. Now you have to flat edges of a
braid you must rejoin. This is called Non-homologous end-joining of DNA
(NHEJ). The recognition, repair, fidelity, and correct repair during NHEJ is
not well understood. Cas9 does no joining, no ligation, no pasting, it only
does the snipping. The joining is done 'on it's own' by the cell at some rate,
sometimes correctly, sometimes even uptaking an insert into the process. So
insofar as Cas9 is doing any 'editing', the repair process is entirely an
accidental after-effect of Cas9's targeted DNA breakage.

Here is CRISPR breaking DNA and creating the insult:
[https://www.ncbi.nlm.nih.gov/pubmed/27866150](https://www.ncbi.nlm.nih.gov/pubmed/27866150)

Some recent work on understanding how those double-stranded breaks actually
repair themselves:
[https://www.ncbi.nlm.nih.gov/pubmed/27924007](https://www.ncbi.nlm.nih.gov/pubmed/27924007)

Some basic research in Yeast:
[https://www.ncbi.nlm.nih.gov/pubmed/27915381](https://www.ncbi.nlm.nih.gov/pubmed/27915381)

Double-stranded break repair in breast cancer:
[https://www.ncbi.nlm.nih.gov/pubmed/28053956](https://www.ncbi.nlm.nih.gov/pubmed/28053956)

~~~
nonbel
I quickly looked through your links. The first article you link seems to lack
any control group (which we need to assess the proportion of pre-existing
mutant cells), the second is primary research but does not mention CRISPR-
Cas9, and the other two are review articles that don't contain any
quantitative data.

As mentioned, I only looked quickly, but I do not think any will be helpful on
the point I am bringing up. Let me know if I missed it.

~~~
AlexCoventry
If you want to know why people are assuming NHEJ occurs at a double-stranded
break, you might look at the dozen or so references in the first paragraph of
[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC231204/pdf/1621...](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC231204/pdf/162164.pdf)

~~~
nonbel
Frequency of survival is shown in table 2... it ranges from 5/1,000 to
1/100,000. So cell death is a 200-100,000 times more common result than NHEJ
using those methods. I have no idea how well that would correspond to the
conditions we are interested here, but see no way this paper can be taken to
support NHEJ is a more likely result than cell death.

It also says: "Yeasts, like other eucaryotes, exhibit cell cycle arrest in
response to DNA damage (17)."

------
somberi
I learnt a bit from listening to this Podcast from Radiolab.
[http://www.radiolab.org/story/update-
crispr/](http://www.radiolab.org/story/update-crispr/)

~~~
archon810
That's what I immediately thought of as well. Fascinating episode, I highly
recommend listening to it.

------
phkahler
Do humans have the same low level immune system bacteria have? It seems like
passing immunity between generations would be extremely unlikely, but it could
still be useful for offering some immunity to viruses that one has already
had.

Would it be possible to introduce a genetic template from a viral disease into
an embryo to produce a human essentially immune to that virus?

~~~
Ultimatt
CRISPR is a solution to just retroviruses. Bacteria through phages actually
come under more attack than humans in this way. If you've got a single cell
for the next generation you care a lot about this. Humans any old crap can
happen to litterally trillions of cells without affecting the germ line.
CRISPR itself has capacity to introduce damage to the genome.

So its a balance between error correction and error introduction. In Bacteria
its worth it, in humans not so much, especially as we have a bunch of other
subtler more complex ways of handling viruses _before_ they get all the way to
the nucleus
[https://en.wikipedia.org/wiki/RNA_interference](https://en.wikipedia.org/wiki/RNA_interference)
Bacteria dont have this luxury as their DNA lacks this extra barrier. The
biggest difference is simply cells dont matter in a human and can just kill
themselves if all else fails.

The rest of the "innate" immune system has similar proteins involved nearly
everywhere e.g. [https://en.wikipedia.org/wiki/Toll-
like_receptor](https://en.wikipedia.org/wiki/Toll-like_receptor) These are the
components involved in cell surface recognition of sugars. But much more
similar between all animals than animals and bacteria.

Answer to your second question is yes you could add a CRISPR construct with
templates for known disease and add artificial immunity. However, finessing
this so that CRISPR doesnt cause lots of damage from randomly cutting your DNA
throught your life leading to a higher risk of systemic cancers... is a big
hurdle.

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carapace
Small sans-serif body text means you hate your readers.

