

CRISPR Gene Editing - PieSquared
http://andrew.gibiansky.com/blog/genetics/crispr/

======
ProAm
Great podcast on CRISPR from radiolab [1]

[1] [http://www.radiolab.org/story/antibodies-
part-1-crispr/](http://www.radiolab.org/story/antibodies-part-1-crispr/)

~~~
cheapsteak
One of the main arguments given against splicing human embryos on there was
that embryos couldn't decide for itself whether it wanted it.

I think that's kind of absurd. The decision that impacts it the most has
already been made for it - existence. If we take the Buddha's view that life
is suffering, then it has been decided that is suffers. Compared to that, what
sin is it to give it whatever advantages a few spliced genes can offer?

------
avinashv
While the author is pretty clear that "only a passing knowledge of modern
microbiology" is necessary, I think that understates some of the technical
language in here. I really laughed at the line, "Silencing a gene with
CRISPR/Cas is incredibly simple." Still, I learned a lot. This is the closest
I've come to feeling like I know what's going on in CRISPR.

CRISPR has got to be one of the most important scientific achievements of the
past few decades, right?

~~~
neuronic
Yes. The next time some "What is the most promising technology?" thread pops
up on reddit, you can safely post CRISPR.

Depending on how things go with the patent stuff and the technology itself,
sooner or later this will absolutely transform our lives. We are looking at
the incubation of a technology that may easily save millions of lives (over a
long time frame).

Potential for misuse is near infinite though - imagine a privatized CRISPR
inaccessible to the sub-$50 million/ year crowd.

The limitations of CRISPR really do appear to be few though. Lots of
techniques and methods will be developed and figured out in the next years. It
allows us near complete control over the most essential biology. And all that
in vivo.

The road ahead is rough but I am confident that CRISPR can become the magic
tool I just described. It will be black and white magic. Question is which
will dominate?

~~~
zardo
Potential for misuse is near infinite though - imagine a privatized CRISPR
inaccessible to the sub-$50 million/ year crowd.

I can imagine a lot worse than that. Imagine genetic engineering gets dirt
cheap, and that does seem to be the direction we're headed. Novel pathogens
are going to be a lot easier to design than treatments and preventative
measures to protect against them. How do you stop the proliferation of bio-
weapons? I'm thinking that would be about as easy as stopping the
proliferation of malware.

~~~
technotony
One major difference is that you have to order your DNA typically from a third
party provider and they can screen for pathogenic sequences. We could probably
stop malware if we could screen all the code before anyone was allowed to run
it. I think the current system is reasonably robust for stopping novel syn bio
pathogens.

In my mind the big risk comes with home based DNA printers. There are several
close to getting to market (eg
[http://www.kilobaser.com/](http://www.kilobaser.com/)), at that point we lose
control over what gets printed and then maybe there are concerns... though I
do think creating a pathogen is really hard and most likely to end up killing
the creator before anyone else.

~~~
mbreese
It's _very_ hard to Figure out what a random piece of DNA will do just from
the sequence. Combine that with other molecular biology techniques for
combining, slicing, and dicing DNA, and there would be very little hope to do
any sort of prospective screen.

~~~
jashephe
> It's very hard to Figure out what a random piece of DNA will do just from
> the sequence.

While it's certainly true that it's hard to determine the function of a DNA
sequence from scratch, it's considerably easier to compare that sequence (or
the sequence of the translated polypeptide) to other homologous sequences to
see if it matches something dangerous.

I previously worked in a lab that studied _Bacillus anthracis_ , and we had a
bit of trouble getting a major gene synthesis company [1] to produce a plasmid
with a variant of _atxA_ [2], and _atxA_ isn't even a toxin, it's just a
transcriptional regulator. We presumed that they just BLASTed [3] the sequence
we gave them and threw up a red flag when it matched anthracis. So this sort
of sequence-checking already occurs.

[1] [https://www.dna20.com/](https://www.dna20.com/)

[2]
[http://www.ncbi.nlm.nih.gov/pubmed/8577251](http://www.ncbi.nlm.nih.gov/pubmed/8577251)

[3] [http://blast.ncbi.nlm.nih.gov/](http://blast.ncbi.nlm.nih.gov/)

------
panic
_(Unfortunately, it seems like there is some debate over who invented CRISPR
and should be awarded the patent for it.)_

Maybe the bacterium which first expressed a CRISPR sequence should be awarded
the patent. We're just using the tools that nature invented for us!

~~~
tstactplsignore
Truly- When asked why he did not patent the polio vaccine, Jonas Salk
responded by asking if you could patent the sun- it seems as though if the sun
were discovered today, we'd have a legal battle over all of the attempts to do
so.

~~~
kirsebaer
> “When Jonas Salk asked rhetorically “Would you patent the sun?” during his
> famous television interview with Edward R. Murrow, he did not mention that
> the lawyers from the National Foundation for Infantile Paralysis had looked
> into patenting the Salk Vaccine and concluded that it could not be patented
> because of prior art – that it would not be considered a patentable
> invention by standards of the day.

[http://www.biotech-now.org/public-policy/patently-
biotech/20...](http://www.biotech-now.org/public-policy/patently-
biotech/2012/01/the-real-reason-why-salk-refused-to-patent-the-polio-vaccine-
a-myth-in-the-making)

> In the decades since, a great myth has grown to dominate the popular
> imagination. Its name is “The Conquest of Polio,” and Salk is its hero....
> This retelling of the history of polio, however, is largely a distortion.
> The full, true story is far more complex. Its hero is Albert Sabin – for if
> any one man conquered polio, it was Sabin, who developed the oral attenuated
> live-virus vaccine. While Salk’s vaccine did slow down the incidence of
> polio among middle-class Americans, its cost and its requirement of three
> injections and a booster meant that for years the disease continued to
> affect the poor and others lacking access to proper medical care. It was
> only after Sabin’s oral vaccine, which was cheap, effective, and easy to
> administer, was licensed for production in 1962 that polio could be fully
> controlled in the United States.

[http://www.technologyreview.com/review/404390/the-myth-of-
jo...](http://www.technologyreview.com/review/404390/the-myth-of-jonas-salk/)

~~~
gwern
[http://www.slate.com/articles/technology/history_of_innovati...](http://www.slate.com/articles/technology/history_of_innovation/2014/04/the_real_reasons_jonas_salk_didn_t_patent_the_polio_vaccine.html)

> There is an important footnote regarding Salk’s statement that “there is no
> patent.” Prior to Murrow’s interview with Salk, lawyers for the National
> Foundation for Infantile Paralysis did look into the possibility of
> patenting the vaccine, according to documents that Jane Smith uncovered
> during her dive into the organization’s archives. The attorneys concluded
> that the vaccine didn’t meet the novelty requirements for a patent, and the
> application would fail. This legal analysis is sometimes used to suggest
> that Salk was being somewhat dishonest—there was no patent only because he
> and the foundation couldn’t get one. That’s unfair. Before deciding to forgo
> a patent application, the organization had already committed to give the
> formulation and production processes for the vaccine to several
> pharmaceutical companies for free. No one knows why the lawyers considered a
> patent application, but it seems likely that they would only have used it to
> prevent companies from making unlicensed, low-quality versions of the
> vaccine. There is no indication that the foundation intended to profit from
> a patent on the polio vaccine.

> The decision not to patent the vaccine made perfect economic sense under the
> circumstances. “The National Foundation for Infantile Paralysis was a
> nonprofit, centralized research and development operation,” says Robert
> Cook-Deegan, who studies intellectual property and genomics at Duke
> University. “They didn’t need an incentive structure.”

------
dperfect
Can someone help me understand the main differences between CRISPR and
traditional genetic engineering that has been done for many years now? My
understanding is that we've had technologies to selectively modify DNA for
some time, but perhaps it hasn't been as targeted or reliable as CRISPR?

One thing that stands out to me (especially from the radiolab episode) is that
it sounds like CRISPR isn't just gene editing in the sense of engineering
something in a lab; it's gene editing in an _already living_ organism. If DNA
is anything like an organism's "source code", once the code is "shipped"
(organism is conceived), traditionally we tend to think of that code as being
locked/frozen. It sounds like CRISPR is akin to modifying the code _live_ \-
"in production", so to speak. Is that a fair analogy?

Edit: to explain, when I say "in an already living organism", I'm referring
mostly to a developed, multi-celled organism. I understand that traditional
techniques also use living cells, but the radiolab episode makes it sound as
if a full-grown adult human may someday get a live "DNA upgrade" \- at least
to applicable portions of the body - via CRISPR, e.g. to remove a genetic
predisposition for developing a particular disease. To me, that would be
substantially different (in practical application) from genetically
engineering something like a gamete or a single-celled bacteria.

~~~
eggie
Mostly, CRISPR/Cas9 reduces the cost of getting a custom endonuclease
(molecular scissors that cut DNA at particular sequences). It is several
orders of magnitude cheaper than alternatives, and it is also incredibly quick
to set up! This makes it much easier to try more experiments. Also dCas9
(partly or wholly disabled Cas9) can be used to make the system the basis for
multiplex gene targeting experiments that can be used to induce entirely new
regulatory networks in one step! Wow!

So there is a lot of good but don't forget your question: haven't we had this
for a long time? Yes, the techniques are fundamentally the same as others
which have been used for a long time. Endonuclease and homologous repair are
standard tools in genome engineering. It just costs much less to design custom
endonuclease now. It seems like there is a bit too much hype about CRISPR/Cas9
techniques as genome engineering tools--- we are engineering genomes in
exactly the same way as before. The scissors have changed but the glue is
still endogenous to the organisms that we are engineering.

To my knowledge there has only been one case in which DNA was shipped as code
to be the genome of a dead cell. Maybe someday we will be able to write large
genomes. Until then nearly all the editing we do will be in living organisms,
as it has been forever (even before CRISPR/Cas9).

~~~
DaveWalk
Well said. I'll add some scientific esoterica, because it parallels software a
little: we've even had custom endonuclease services for a while (TALENS[1]),
which serve a very similar function. But they were hard to generate and
difficult to work with. Companies like Invitrogen even sold a TALEN-making
service, costing in the dozens-of-thousands of dollars to generate a TALEN for
preclinical drug discovery use.

Then CRISPR came along. It was like the open-sourced, better-performing
alternative to the cumbersome, proprietary software. Switching was a no-
brainer, and it has handily become the future, if not the mainstream already.

[1][https://en.wikipedia.org/wiki/Transcription_activator-
like_e...](https://en.wikipedia.org/wiki/Transcription_activator-
like_effector_nuclease)

~~~
dekhn
Right. My own PhD work was on designing custom transcription factors to bind
to specific sequences (not even with TALENs- it tried to do full molecular
dynamics to predict the binding constant for multiple different DNA sequences,
which was absurdly expensive). It would have to be re-engineered once for each
sequence; with CRISPR, you just provide a matching template sequence.

------
brock_r
Will anyone here be _really_ surprised if it turns out ribosomes are
duplicating machines designed by some alien race?

The entire process of reading the DNA code and turning it into proteins is
just amazing.

~~~
Balgair
I understand the downvotes, but the wonder here with biology that the OP has
is well placed. I came into bio from DoD/Physics and am constantly astounded
by what nature has made. I mean, it's been ~4 billion years and the generation
to generation time is ~20 minutes (~1.1E14 generations total), so I think we
all can expect a fair bit from nature, but still, she is really clever.

I do think about this as well. The complexity, the reliability, the ability
for nature to do what she does even in the face of all the thermal noise and
viruses and enviroment, it really seems like there must be someone making it
happen. Alas, no though! As far as we can tell, it's all just evolution and
chance on a planet wide stage with microscopic actors. If anything, I think
this makes nature even more exciting and awesome (in the true sense of the
word). That she did so much with so little is stupefying to me.

------
iaw
This actually raised an interesting thought to mind about the long-term
ethical implications of lowering the barrier to entry for genetic engineering.
What happens when anyone with a little know-how and $10K can use the
techniques?

~~~
kanzure
> This actually raised an interesting thought to mind about the long-term
> ethical implications of lowering the barrier to entry for genetic
> engineering. What happens when anyone with a little know-how and $10K can
> use the techniques?

The barrier to entry for genetic engineering is already zero; but it has much
less sexy names like "washing your hands" and "sex". DNA synthesis is just a
matter of being more specific and deliberate about which biological organisms
you keep around. Anyone is capable of selectively breeding bacteria, fungi,
molds, or anything else. I think the reverse(?) question needs to be asked as
well, which is what are the ethical implications of trying to restrict the
ability to make DNA?

Here's some infotainment I guess:
[http://diyhpl.us/wiki/diybio/faq/news/](http://diyhpl.us/wiki/diybio/faq/news/)

~~~
iaw
I should have been more specific, when I said 'genetic engineering' I was
referring to using techniques like CRISPR to perform gene modifications in
animals and humans.

No one with the resources required currently would surreptitiously test a new
gene on another human, what happens when the resource and knowledge barrier
drop?

------
dnautics
I think this explanation is missing the real explanation of why CRISPR is
useful.

I want to take a crack at it. Let's say we want to change in the genome the
sequence (where each of the 'letters' represents a somewhat long stretch of
base pairs):

ABCDE to ABC'DE

you would normally create the sequence

BC'D

in vitro and put it into the cells. The organisms contain mechanisms to match
the B & D sections and thus 'swap out' the C section for the C' section.

Note that C could be "" which would make the process a straight insertion. C'
could be "" which would make the process a straight deletion. C and C' could
be a single base pair, which would mimic a point mutation, etc...

However, you don't have TOTAL control over this process, it's stochastic, and
doesn't have 100% efficiency. So you have to do something clever to make sure
you have what you want. Typically that involves inserting resistance to a
chemical factor (e.g. antibiotic). So for insertions (if you don't mind a
dirty insertion) it's fine, but for other transformations like mutations and
deletions, you might have to be clever, and say, do C -> C' -> C'' where the
C' includes the selection factor. And C'' is chosen either because it lacks a
toxic factor that we put in alongside C' or by doing a reverse selection where
we pick clones and test to see if they die (and keep some of the originals in
case they pass the test).

This process generally works quite well in most microbes with small genomes
(E. coli requires a tweak to the process). It is basically effortless with
yeast.

With higher eukaryotes it's not quite so simple. A competing process is
inserting the BC'D sequence _elsewhere in the genome_. It's not entirely clear
why this is such a huge problem, but likely it's because of the increasing
complexity and size of the genome. If C' contains a selectable marker, it
becomes difficult to distinguish between what you want (ABC'DE) and just BC'D
somewhere random in your genome. Both are resistant. And the process becomes
bogged down by the need to isolate single cells, propagate them, and check to
see if your strain has the substitution you want (relative easy, just a PCR
reaction) and no other substitutions elsewhere in the genome (haaaaaaard).

The CRISPR advantage is that just before you add BC'D to your cell you create
a scission somewhere in C so you're left with ABc//cDE - and what this does is
triggers the cell repair system to search for B & D sequences to hook into.
Naturally it will find BC'D. Well, if it doesnt, usually a fragmented
chromosome will also result in death of the cell, so you're virtually
guaranteed that the surviving cells have ABC'DE. With this, the rate of
successful targetting so exceeds the rate of random insertion that the
necessity to check is basically eliminated (or at least you don't have to
search through so many clones to pull out a total success).

The net effect is that for many higher organisms genetic manipulation becomes
much much much easier. YMM(still)V with some plants which have high level of
repeats within the genome.

------
bcheung
I've been studying biochem as a hobby and have been hearing CRISPR off and on
but never really heard a good explanation until now. I don't fully understand
everything that was said but at least I have a general picture of going on.
Thanks for writing this.

------
jbattle
is there any practical approach on the horizon (or here) that allows
scientists to apply crispr throughout all the cells in a living organism? I
get how this works with a single cell, but typically that's only useful for
either very very very small or very very very young individuals (right?)

~~~
dnautics
No. For permanent genomic changes you have to select what you want. For
precise edits, there's usually a counterselection (so it takes two hits). The
efficiency of the process is still low. You have to be willing to throw away a
lot of cells to get precisely the correct one. One way to think of it is a big
component of what CRISPR does is to make it easier to find the good edits
(although it does also make good edits more likely).

------
NN88
This will win the Nobel

