CRISPR Gene Editing

http://andrew.gibiansky.com/blog/genetics/crispr/

by PieSquared • 11 years ago

108 43 11 years ago

andrew.gibiansky.com

ProAm• 11 years ago

Great podcast on CRISPR from radiolab [1]

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

cheapsteak• 11 years ago

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?

avinashv2• 11 years ago

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?

DaveWalk2• 11 years ago

Absolutely. The movement from discovery (from the unsexiest of all fields, bacteriology!) to a reliable tool is unprecedented[1] in the scientific realm. For my money it is easily on track for a Nobel Prize: it allows mankind to examine with precision unknown just years before.

[1] I see a parallel to short hairpin RNA gene silencing (shRNA, a.k.a. RNA interference, RNAi). A breakthrough discovery, at use at the bench in less then a decade, and an easy clinch for the Nobel Prize. CRISPR has gone even faster.

dnautics5• 11 years ago

The movement from discovery (from the unsexiest of all fields, bacteriology!) to a reliable tool is unprecedented.

Uhm, restriction enzymes?

for the uninitiated: https://en.wikipedia.org/wiki/Restriction_enzyme

(basically, restriction enzymes are what CRISPR is basically set to replace for complex systems/organisms where restriction enzymes are too weak; although for simple systems restriction enzymes are waaay simpler)

neuronic• 11 years ago

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?

zardo2• 11 years ago

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.

avinashv2• 11 years ago

Potential for misuse is near infinite though

Fully agree, though I see the potential for good to be more.

dperfect• 11 years ago

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.

cgearhart• 11 years ago

CRISPR performs pattern matching on the DNA sequence immediately preceding the cut location where new sequences are added. While we have had CTRL-X and CTRL-V for awhile (in the other gene modification techniques you alluded to) CRISPR provides a cursor that allows us to precisely control where the CTRL-V takes place.

As for updating "live" code...that's a flawed, but not completely wrong analogy. Other techniques do rely on modifying the genome before "production", and in that sense CRISPR does enable us to edit DNA in cases that would have been impractical before, but it still basically requires performing the modification on each cell individually -- so there are still practical limits on deploying the technique.

eggie4• 11 years ago

Precise targeting of genomic cuts has been possible for some time, but more expensive:

https://en.wikipedia.org/wiki/Zinc_finger_nuclease

https://en.wikipedia.org/wiki/Transcription_activator-like_e...

eggie4• 11 years ago

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).

eggie4• 11 years ago

https://en.m.wikipedia.org/wiki/Mycoplasma_laboratorium

DaveWalk2• 11 years ago

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...

searine• 11 years ago

Its makes many more organisms tractable, more accurately.

Previously, transgenics was tough mostly because of the difficulty in inserting sequence in the right place doing the right thing. It would take decades to develop the specific organism-specific tools to really change DNA.

it's gene editing in an already living organism.

True. The thing about older transgenics, is that it was like using a shotgun to build a birdhouse. It was messy and you broke a lot of things to get the one gene where you wanted it. So you had to do a lot of transformations to get it right, and afterwords there was a lot of genetic cleaning up to do.

With CRISPR you can be extremely specific with your targeting, which means you can do things like "gene therapy", which is "patching" DNA in a live organism.

dnautics5• 11 years ago

Well, for most microorganisms, traditional genetic engineering is perfectly fine. You just create long homology ends and transform the cell and it works peachy keen. (Ironically, the one organism for which this spectacularly fails is E. coli, requiring the lambda-red system).

For 'higher eukarya' the big problem is you can still do the long homology ends, but a competing process is random insertion. Basically (if my understanding is correct), CRISPR reduces the competing process and makes specific insertion of DNA the dominant result. Sometimes, though, having multiple random insertion is not a huge problem.

brock_r• 11 years ago

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.

dekhn2• 11 years ago

I would be surprised (I consider terrestrial random origin to be more likely).

Ribosomes just turn RNA into protein. Don't forget about DNA and RNA polymerase, which are both very interesting as well.

It's really mechanical, but in a way that appeared to happen randomly by evolution, not through design.

Balgair• 11 years ago

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.

jbattle• 11 years ago

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?)

zardo2• 11 years ago

You don't necessarily need to hit every cell in an organism to produce useful therapies. Most cells aren't going to be expressing the gene that's causing a disease, only cells in the affected tissue or organ need to be treated. In some cases, you may not even need to get all of the affected cells, just repairing a subset could improve outcomes. (1/8 of a functioning pancreas is a lot better than 0/8)

dnautics5• 11 years ago

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).

bcheung• 11 years ago

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.

NN88• 11 years ago

This will win the Nobel

panic• 11 years ago

(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• 11 years ago

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• 11 years ago

“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...

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...

dnautics5• 11 years ago

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.

iaw2• 11 years ago

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?

kanzure2• 11 years ago

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/

iaw2• 11 years ago

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?

george88b• 11 years ago

There is a startup that runs genetic engineering experiments for you "in the cloud": https://www.arcturus.io/

CRISPR Gene Editing

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