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Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

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Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#31

Post by leggomylibro »

peter303 wrote: post #30
leggomylibro wrote: post #27

Earlier quoted context omitted.

It'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…

There 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/

That's a good point; community labs and bio makerspaces have great potential to help make this more accessible to us lay people!

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#32

Post by nonbel »

nonbel wrote: post #14
zigzigzag wrote: post #6

I 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 exoge…

It looks like this strain of mice can produce ~10 pups (a lower bound since not all zygotes will make it to birth) every 5 days for 26 cycles. So, one breeding pair could produce at least ~260 zygotes in 4 months. From this, I'd say it is plausible (economically) that the number of zygotes injected for this study was in the thousands:

>"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

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#33

Post by acchow »

leggomylibro wrote: post #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 g…

> It's not magic.

It's not a computer is probably apt.

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#34

Post by mbreese »

j7ake wrote: post #4

Off 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。

Moreover - 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.

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#35

Post by randyrand »

BrandonMarc wrote: post #26

Unanticipated 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.

The rest of us will enjoy leaving you behind =)

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#36

Post by psychometry »

leggomylibro wrote: post #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 g…

The mutations were unanticipated by the computational tools designed to identify likely off-target effects. This was in the article.

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#37

Post by Amygaz »

nonbel wrote: post #22
ethelward wrote: post #20

Earlier quoted context omitted.

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.

It 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.

Any 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.

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#38

Post by Blackthorn »

leggomylibro wrote: post #27
feelix wrote: post #21

Most 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/ that no one has just tried modifying the non-coding DNA a lot and then observed any changes (or lack thereof…

It'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…

> 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.

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#39

Post by Amygaz »

dekhn wrote: post #7

I 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.

It 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.

Re: Crack in CRISPR Facade After Unanticipated in Vivo Mutations Arise

#40

Post by nonbel »

Amygaz wrote: post #37
nonbel wrote: post #22

Earlier quoted context omitted.

It 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.

Any 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.

Check out pre-1940 papers (the year may be later depending on exact sub field). I've seen that they used to make it a point to include all the info (including raw data) to the point it was practical. Somewhere along the line the attitudes went wrong, I blame NHST personally.
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