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Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
- ackfoo 9y agoPre-publication press release. Full text of article not available. How good was their control group? The press release says they used mice that had previously been edited with CRISPR/Cas9 and found off-target mutations, most of which were single nucleotides. Did they have a good control group so as to exclude random point mutations? Who knows? What is the theory on how CRISPR/Cas9 causes single nucleotide off-target mutations? No idea. 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'.
- amitutk 9y agoThe article is available: https://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293.html https://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293....
- nonbel 9y ago> "Co-housed FVB/NJ mice without CRISPR-mediated correction were used as the functional-deficient control. Briefly, an sgRNAexpressing plasmid had been coinjected, into FVB/NJ zygotes, with the single-stranded oligodeoxynucleotide (ssODN) donor template and Cas9 protein to generate mosaic F0 founders.1" 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 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?
- ethelward 9y agoThe more you work in the field, the more you discover how underwhelming the details in biology papers are. They give results without detailing the algorithms, hence destroying reproducibility, they hide datasets behind confidentiality, presents large-scale graphs without precise data, and so on. Generally, reproducibility in biology papers is but a far away dream.
- nonbel 9y agoIt is still awful but far better now than a few decades ago. The absolute worst is Nature/Science from ~2000. For someone trying to figure out what is going on (rather than just believe what the authors claim) most of those papers are not even worth reading.
- leemailll 9y agoThe problem with top-ranked journal more likely arises from page limitation. The amount of data needed for a paper published in such journals with so little pages and figures allowed basically means no one could have enough space to put enough material in the main content. And then who really takes a serious effort in supplementary materials? With regards to method section of paper, while it is an important part for reproducibility, it is not writing in great details in top journals. The reasons I think lie in it is mostly in the supplementary material, the multiple methods used make it crazily long, furthermore applications of the commercial kits and highly-automated machines make it not necessary for researcher to write in great details. Anyway, I agree that papers from Nature/Science are hard to read sometime, especially without reading the supplementary.
- Amygaz 9y agoAny time before the advent of "supplementary materials" pretty much meant that you had nothing more than a highlight of the method. It was terrible indeed. However, even today I find that roughly half the publications are not worth the paper they are printed on, or the bandwidth to download them.
- give_me_a_break 9y agoAhahahahahahahahahahahahaha <takes a deep breath>. Ahahahahahahahahahahahah <wipes away a tear> http://lmgtfy.com/?q=nobel+prize+winning+physicians http://lmgtfy.com/?q=nobel+prize+winning+physicians
- glup 9y ago...and in the context of an already sensationalized technique ("it's like TextEdit, but for genes!")
- j7ake 9y agoOff target effects from CRISPR is not new. The method leverages DNA repair mechanisms in the cell to do the recombination. All errors involved in those DNA repair mechanisms (there are distinct ones) would apply here。
- mee_too 9y agoBut if CRISPR cuts off a single DNA sequence, surely the repair mechanisms won't damage a random gene far away. My very limited understanding is this paper is reporting widely spread damage on the genome. 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.
- placeybordeaux 9y ago> but the artificial gene tech CRISPR can add and edit as well. 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?
- mbreese 9y agoMoreover - this is biology. Biology is messy. Mistakes are the reason why the system works (evolution). To say that off-target effects were unanticipated is nonsense. Rather - how many off target effects will there be, can they be predicted, and can their effect be predicted.
- j7ake 9y agoOff target effects from CRISPR is not new. The method leverages DNA repair mechanisms in the cell to do the recombination. All errors involved in those DNA repair mechanisms (there are distinct ones) would apply here。
- zigzigzag 9y agoI seem to recall that in prior HN stories about CRISPR some poster was saying he believed CRISPR didn't work the way people thought it worked. He said it was simply killing cells that didn't have the desired mutation and biologists weren't realising that because of design errors in the experiments (or rather, sometimes mass cell dieoffs were being reported but not dwelled upon). 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?
- mee_too 9y agoDon't think so. Originally the CRISPR mechanism was discovered in bacteria, which only have one cell. It doesn't make sense for bacteria's anti-virus system to kill it's only cell.
- 0530_micro 9y agoThis is a really interesting point. 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.
- throwaway2048 9y agoThats assuming that its selection mechanism works at an individual level, and not a population level. After all a colony of bacteria is (usually) a set of identical clones. As long as a few survive, the DNA lives on.
- nonbel 9y agoSee my other post here. They don't report the initial number of zygotes injected (why not?) so we cannot assess the "selection" explanation. 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.
- dekhn 9y agoI believe George Church published some work several years ago now using whole genome sequencing after CRISPR, saying they had neglible off-site integration or other mutations. But going back,the findings were much more measured than that- they still saw off-site integration.
- Amygaz 9y agoIt seems to me that this high profile character often goes on record mentioning things that ultimately end up being just his wishful thinking and don't have much bearing.
- dekhn 9y agoI'm inclined to agree, except he did invent much of modern sequencing. He's great at technology, but limited in actual science.
- DiNovi 9y agothe article headline kinda belies a bias before you even read it
- mrcactu5 9y agoi am sick of all the CRISPR; hype this is the cure being worse than the disease.
- sndean 9y agoNot sure about it being overhyped, but similar sentiment, stated better is in this article (from last year): http://www.sciencemag.org/news/2016/05/gene-editor-crispr-won-t-fully-fix-sick-people-anytime-soon-here-s-why 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.
- daemonk 9y agoTitle is a bit dramatic. People just need to choose their gRNAs better based on available genomes.
- leggomylibro 9y ago"Unanticipated?" 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.
- acchow 9y ago> It's not magic. It's not a computer is probably apt.
- psychometry 9y agoThe mutations were unanticipated by the computational tools designed to identify likely off-target effects. This was in the article.
- feelix 9y agoMost of the DNA that suffers random mutations is non-coding DNA. It's completely unknown what effects, if any, modifying non-coding DNA might have as its use is unknown. I find it interesting that although anyone can experiment with CRISPR in their living room using something like this http://www.the-odin.com/ 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.
- leggomylibro 9y agoIt's still fairly hard to do in a living room, if you want to do anything novel. 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.
- peter303 9y agoThere is the bio-maker movement, sometimes called biolabs, that puts fairly elaborate biotech in the hands of unaffiliated researchers and hobbyists. I wouldn't be surprised to see homebrew CRISPR at science fairs in the near future. https://www.meetup.com/denverbiolabs/ https://www.meetup.com/denverbiolabs/
- systematical 9y agoI couldn't be the only one worried about an "I Am Legend" type problem with this stuff. Obviously, that movie is an extreme, but the underlying problem of unintended mutation exists.
- 88e282102ae2e5b 9y agoAll known CRISPR systems work only under very specific conditions. You could drink a vial of Cas9 and it wouldn't do anything to you because it couldn't enter your cells.
- BrandonMarc 9y agoUnanticipated 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.
- randyrand 9y agoThe rest of us will enjoy leaving you behind =)
- BrandonMarc 9y agoHammond, is that you? Jurassic Park was a cautionary tale about hubris. People with lots of money and smarts were convinced they could control nature.