Crispr’s Next Big Debate: How Messy Is Too Messy?
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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.
― 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.
- 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.
AIUI Cas9 matches 20-base-pair sequences, in something the size of the human genome, wouldn't you expect more than one such exact match, anyway?
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...
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.
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".
Then, some years down the line, we'll make use of the benefits of whatever technology the Chinese develop. It would be evil not to, given how it could spare people the suffering of genetic diseases, or create carbon-capturing crops, or whatever.
Why exactly do you want to invade China?
http://blogs.sciencemag.org/pipeline/archives/2017/05/31/tro...
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.
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.
I guess his domain knowledge allowed him to foresee challenges the rest of us are now becoming aware of.
I asked: "How many FVB/N zygotes were injected to generate those 11 original mice?"