[tmoot]

I didn't read the paper but it's in scientific reports (minimal and rushed peer review, bottom of the barrel nature subjournal)...I'd be a bit skeptical.

It's not exactly anyone's first choice place to publish.

Crispr/Cas9 is not as robust as people make it out to be, but it's promising. (I do some work with crispr/cas9)

[Para2016]

It's not the only paper I read regarding IPSC and germ cell induction. The Yamanaka paper has been out since 2007, this kind of work is well established. But, this paper is the only one I can find that has researchers attempting to make sperm from a female. There are other papers regarding iPSCs induced to become female germ cells to oocytes and then IVF producing viable offspring, in mouse, pig, and monkey.

[MegaButts]

> Crispr/Cas9 is not as robust as people make it out to be, but it's promising. (I do some work with crispr/cas9)

Please expand on this. I feel like CRISPR is being overhyped, and would love to hear form someone educated on the subject as to what its possible limitations are, or at least some of the known challenges ahead.

[jfarlow]

Cas9 is a protein with two functions: 1) locate a DNA sequence that matches the little RNA it grabs ahold of, and 2) cut that DNA at that location. The first of those functions, it's ability to locate particular and arbitrary sequences, is its comparative advantage against all other technologies we know of. The second, cutting DNA, well, works I guess, but will likely be engineered to be more useful, or turned off in order to make way for other more useful functions.

The protein is special for two different reasons: 1) it is able to locate DNA sequences with very high precision 2) and the sequence it locates can be swapped out as easily as changing the sequence of the RNA it grabs ahold of (trivial to do, can be done in a day, and can cost >$1 per target sequence to swap). Note, it is not special because it can cut DNA - there are lots of proteins that do that, and there are lots of better ways to change or alter DNA once you get to a particular sequence - but Cas9 was originally a self-defense mechanism, so it's evolutionary function in strep throat bacteria was to kill invaders by dicing up their DNA (at particular sequences that strep throat doesn't have).

Cas9 is powerful because it could be used to direct any function at a particular DNA sequence, where the sequence can be altered in the lab quickly and cheaply. As it is a protein encoded by a particular sequence, you can fuse it to other proteins with other functions to build a more powerful machine. (see [1] if you want to play with those sequences yourself.) As an experimental tool it will likely become a foundational tool used all throughout molecular biology - and for that alone is is worth it's fame. Thermophilic polymerase used in PCR is another such tool. As are restriction enzymes. As is GFP. That's the scientist's perspective.

However, Cas9 also previews the capability of directly and arbitrarily editing of a genome - a holy grail of biomedical sciences. Though unengineered Cas9 it's not great at editing a genome (we're not entirely sure why what it does even works) - but some 2-10% of the time it can actually edit a genome with fidelity. And that's good enough for many experiments (though not good enough for therapies). It has off-target cuts, and when it cuts it slices all the way through the double stranded DNA, and if it isn't properly stitched back together you have a broken chromosome. And getting payload DNA to the site that Cas9 cut is still really tricky. It's also a multi-part system (it needs it's little RNA as well as the protein itself), and so it's hard to deliver directly as a therapeutic. So the wild-type Cas9 is likely limited in its direct therapeutic relevance in terms of pure genome editing. But it will be used extensively for its ability to 1) further research quickly and cheaply, 2) prototype what genomic changes would do if they were successful (when you only need 10% efficacy to conduct a study), and 3) act as an engineering platform upon which other functions can be placed, and its own wild-type limitations can be overcome.

It's powerful. It's not perfect, there's lots more engineering to do with/to it. It's not going to get to the holy grail of genome editing all on its own, but it's a very solid platform to start building off of, as well as simply being a solid tool that will become a workhorse of further synthetic biology.

[1] https://serotiny.bio/

[Obi_Juan_Kenobi]

What organisms are you working with? I've known several groups that have had problems recovering heterozygotes because CRISPR-Cas transformation is so efficient in plants.

If it was just 2-10 percent, everyone would still be using Agro.

[nonbel]

>"Though unengineered Cas9 it's not great at editing a genome (we're not entirely sure why what it does even works) - but some 2-10% of the time it can actually edit a genome with fidelity."

Can you expand on the part I italicized, preferably also linking to some journal articles?

[jfarlow]

Cas9 finds, then cuts both strands of DNA. Now you have to flat edges of a braid you must rejoin. This is called Non-homologous end-joining of DNA (NHEJ). The recognition, repair, fidelity, and correct repair during NHEJ is not well understood. Cas9 does no joining, no ligation, no pasting, it only does the snipping. The joining is done 'on it's own' by the cell at some rate, sometimes correctly, sometimes even uptaking an insert into the process. So insofar as Cas9 is doing any 'editing', the repair process is entirely an accidental after-effect of Cas9's targeted DNA breakage.

Here is CRISPR breaking DNA and creating the insult: https://www.ncbi.nlm.nih.gov/pubmed/27866150

Some recent work on understanding how those double-stranded breaks actually repair themselves: https://www.ncbi.nlm.nih.gov/pubmed/27924007

Some basic research in Yeast: https://www.ncbi.nlm.nih.gov/pubmed/27915381

Double-stranded break repair in breast cancer: https://www.ncbi.nlm.nih.gov/pubmed/28053956

[nonbel]

I quickly looked through your links. The first article you link seems to lack any control group (which we need to assess the proportion of pre-existing mutant cells), the second is primary research but does not mention CRISPR-Cas9, and the other two are review articles that don't contain any quantitative data.

As mentioned, I only looked quickly, but I do not think any will be helpful on the point I am bringing up. Let me know if I missed it.

[AlexCoventry]

If you want to know why people are assuming NHEJ occurs at a double-stranded break, you might look at the dozen or so references in the first paragraph of https://www.ncbi.nlm.nih.gov/pmc/articles/PMC231204/pdf/1621...

[nonbel]

Right, so you are just saying no one really understands how NHEJ works at this point.

Have you considered that mechanism (NHEJ) is not even necessary to explain the rise in proportion of mutant cells after the CRISPR-Cas9 treatment? Because while it may happen to some extent, I do not think that is the simplest explanation for what I have seen reported.

I've been thinking this for awhile now, but never bothered to really write it up and keep track... you can check the discussion here (there are some other posts about CRISPR-Cas9 strewn about that thread as well): https://news.ycombinator.com/item?id=14014098

I have really only read papers other people bring up in conversations online or that get media coverage I come across on this topic. But every single paper I looked into has reported results that fit my explanation just as well as , or even better than, the NHEJ one, and this has been a couple years now...

[ajuc]

So, editing code with Cas9 is like sculping with sniper gun :)

[Ultimatt]

As far as investor understanding goes the hype is probably under selling the importance of CRISPR. Because it makes lots of functionality reliable, cheap and much simpler. Even if it did nothing novel at all that would be important enough.