"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.

Pre-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'.

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) in the specimen.

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。

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 DNA but rather just selecting natural mutants that happen to have the desired edit, would that cause what's seen here?

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.

the article headline kinda belies a bias before you even read it

Title is a bit dramatic. People just need to choose their gRNAs better based on available genomes.

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.

I 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.

i am sick of all the CRISPR; hype this is the cure being worse than the disease.

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 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.

Its been a few years since I left the genetics world behind, and I was just a sysadmin, but even then I'm pretty sure they had determined non coding regions to play a vital role in protein formation/folding.

Because to come to any real conclusions, we'd have to iteratively modify non-coding regions one at a time, and the resources to accomplish that are far beyond the average garage-geneticists's reach. And, sadly, there isn't a lot of funding for academic research into regions that aren't believed to have a significant impact.

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.

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.

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.