i am sick of all the CRISPR; hype this is the cure being worse than the disease.
Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
11–20 of 66 posts
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#12Pre-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…
The article is available: https://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293....
> "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
This is missing some crucial info isn't it? How many FVB/N zygotes were injected to generate those 11 original mice?
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#13I 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 D…
Don'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.
This 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.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#14I 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 D…
See 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.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#15i am sick of all the CRISPR; hype this is the cure being worse than the disease.
Not 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-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.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#16Title is a bit dramatic. People just need to choose their gRNAs better based on available genomes.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#17"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.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#18I 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 D…
What are the chances that a specific mutation exists once you modify more than 4 or 5 bits of DNA? It's like 1 / (number of base pairs ^ 4) or something. The chances are almost nil.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#19I 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 D…
What are the chances that a specific mutation exists once you modify more than 4 or 5 bits of DNA? It's like 1 / (number of base pairs ^ 4) or something. The chances are almost nil.
That isn't how NHEJ works. All they see is some random indel at the site. So all you have to explain is the presence of any mutation at the target site.
Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise
#20Earlier quoted context omitted.
The article is available: https://www.nature.com/nmeth/journal/v14/n6/full/nmeth.4293....
> "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…
The 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.