CRISPR Gene Editing
andrew.gibiansky.com
andrew.gibiansky.com
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?
CRISPR has got to be one of the most important scientific achievements of the past few decades, right?
[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.
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)
At the end of the day, they are just tools, and tools however technically shiny, don't by themselves deliver better end products.
In the case of restriction enzymes, though, they've been around in continuous use since something like the 70s, they're very well characterized, NEB has had a continuous research program where they've been optimized out the wazoo.
Technically speaking they are fundamentally simpler than CRISPR (one component, vs. 2). They also are more generally useful when your genetic manipulation is done outside the cell. So there's a clear tooling difference in CRISPR/RE. Most people who use CRISPR, will use REs in the process of make the DNA piece they're putting in alongside the CRISPR. You'd be a fool to use CRISPR in E. Coli or Yeast.
So 'clan'ing ain't gonna really happen here.
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?
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.
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/), 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.
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.
You have never had control. Perhaps instead you are more worried about our biological weaknesses; there are many! People die all the time from various diseases and even aging. I suggest fixing this before you outlaw DNA manipulation. All of life is a manipulation of DNA in one way or another, and is in fact essential to the maintenance of life... But vulnerabilities should be patched, not swept under the carpet.
From the RadioLab podcast linked above (excellent, btw), far more frightening in the long run: introduce genetic changes to the reproductive cells.
... which then automatically become part of any offspring.
... which are then automatically inherited by any subsequent generations.
And, new species.
Fully agree, though I see the potential for good to be more.
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!
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...
> 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.”
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.
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.
https://en.wikipedia.org/wiki/Zinc_finger_nuclease
https://en.wikipedia.org/wiki/Transcription_activator-like_e...
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).
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...
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.
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.
The entire process of reading the DNA code and turning it into proteins is just amazing.
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.
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.
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/
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?
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.