CRISPR is believed to be, at its core a bacterial immune system designed to 'knock out' problem viral genes by chopping them up at specific 'remembered' loci and hoping that the lossy repair mechanisms cause mutations that prevent the gene from functioning. It also evolved to work on the fairly small genomes of single-celled organisms.
So we've known for awhile that it's going to be quite tricky to get only the exact mutations that we want, at only the exact point that we want them.
It's not magic.
It's not a computer is probably apt.
This is academic publishing at its absolute worst. Put out a sensational press release and whip up a fervour before the article is available. Those diving into the scrum to comment have not read the article.
Also, it's research by MDs. Just sayin'.
Following to reference 1:
> "The sgRNA plasmid was co-injected with the single-stranded oligodeoxynucleotide (ssODN) donor template and the Cas9 protein into FVB/N zygotes to generate eleven F0 founders.
[...]
Double-strand breaks (DSB) were detected in 7 of 11 mice
[...]
The target region was sequenced, revealing that F0 3 and 5 incorporated the donor template precisely in 35.7% and 18.8% of somatic cells, respectively (Fig. 1c), while F0 7 and 8 incorporated indels in the integration, corroborating the unexpected results in the RFLP data.
[...]
A mixture of 3 ng/mL sgRNA plasmid, 3ng/mL of Cas9 protein (NEB Ipswich, MA), and 1mM ssODN (Integrated DNA Technologies, Iowa) was injected into the pronuclei and cytoplasm of FVB/N inbred zygotes. Zygotes that survived injection were transferred into oviducts of 0.5-day post-coitum, pseudopregnant B6xCBA F1 females and carried to term. The resulting gene-corrected mice were backcrossed, initially into the FVB/N background, to determine the germ-line transmission efficiency of the repair." https://www.ncbi.nlm.nih.gov/pubmed/27203441
This is missing some crucial info isn't it? How many FVB/N zygotes were injected to generate those 11 original mice?
I find it interesting that although anyone can experiment with CRISPR in their living room using something like this http://www.the-odin.com/ that no one has just tried modifying the non-coding DNA a lot and then observed any changes (or lack thereof) in the specimen.
You need probably low 4-figures of equipment first; a -20C freezer for DNA/buffer storage, small centrifuge, thermal cycler, some way of getting genes into a target, and incubation space for whatever you're modifying. Preferably you'll also have consistent temperature control and an extremely clean environment. You also need a source of primer sequences, which are short customized sequences of DNA used to tl;dr get a lot of copies of a DNA sequence which you only have in small quantities.
Nothing too difficult, and much of that equipment can be DIY'd for the home lab. But many chemical suppliers also won't ship to residential addresses or onboard individuals as customers, so you'll need to incorporate and shop around a bit. And wet lab protocols can be very finicky; you'll probably need to run through your workflow a lot before you get to the point where you can semi-reliably go from start to finish without fucking up. Often, you won't know exactly how you fucked up, but you can't argue with a lack of results.
It's possible, I think, but it's also time-consuming and difficult.
Anyways, if you did get a reliable setup working with say, micropropagated plant specimens, there are far more interesting prizes than seeing what non-coding DNA does. Plants make all kinds of cool stuff, from scents to flavors to alkaloids. And they've demonstrated an ability to take genes from things like jellyfish for e.g. autoluminescence, too.
This almost feels like bikeshedding but...to be fair, pretty much the first thing anybody does with a new work organism in biotech is make it glow and it's been that way long before CRISPR! There's just something about it that people find irresistible.
How about dry ice from the grocery store?
It's possible that upstream UTRs impact RNA transcription rates, but I'd be surprised if UTRs mediate protein folding.
The original bacteria CRISPR can only cut off pre-defined sequences, but the artificial gene tech CRISPR can add and edit as well. I'm wondering if the later two modes are the sources of the genome damage.
I was under the impression that addition and editing capability were essentially done by injecting the sequence that is desired and performing the cut. Then you simply rely on the repair mechanisms to have a chance to put the new segment in instead of the old segment. Is that not the case?
If CRISPR isn't actually editing the DNA but rather just selecting natural mutants that happen to have the desired edit, would that cause what's seen here?
I don't know if this experiment has been done, but I actually think that there's a good chance that a bacterial cell might acquire a sequence that it itself contains - CRISPR is known to work at the population level but my understanding is that the mechanism for acquisition of new sequences is unclear.
I'll take a look for any papers on the topic and repost should I find anything.
After all a colony of bacteria is (usually) a set of identical clones. As long as a few survive, the DNA lives on.
They got 11 surviving mice in the end, but only 7 were "edited", and usually we see that ~1%-.1% cells are mutants at any given site I would expect they needed ~ 1000 zygotes.
This only really explains the NHEJ results though, not the HDR (when the repaired DNA includes an exogenous template). They report that 2 (out of the 7) mice had the sequence matching the template. However this was only in some of their cells (36% and 19%).
Two other mice had a sequence that was similar but contained mutations...
Anyway, maybe someone can email them and ask how many zygotes were used originally.
>"The fecundity of the FVB/N strain was assessed by data from nine breeding pairs, which produced 43 litters. Litter size ranged from 7 to 13, with a mean value of 9.5. (First litters were generally smaller.) This is superior to other commonly used inbred strains; for example 6.7 for C57BL/6J, 6.6 for SJL/J, 5.4 for 129/J, or 5.0 for DBA/2J (15). A typical breeding pair mated at every postpartum estrous cycle and continued breeding for at least half a year, usually longer." http://www.pnas.org/content/88/6/2065.full.pdf
>"Mice have a 4-5 day estrous cycle and ovulate on the third day. Placing the females with a male on the third day of their cycle will result in the maximum number of pregnancies." Also from the same reference (table 1), number of fertile cycles is ~26: https://www.jax.org/strain/001800
But going back,the findings were much more measured than that- they still saw off-site integration.
I'm just a normal person with nothing to do with this, and I expected such "surprises" from the beginning.
I remain convinced mankind should not mess with this.
http://www.sciencemag.org/news/2016/05/gene-editor-crispr-wo...
It's pretty clear there are some huge hurdles before it's useful, even for your average biochem laboratory. But the effort put in to overcome those hurdles is probably worth it.
