[Obi_Juan_Kenobi]

This is a baseless critique.

No one has to address alternative hypotheses that don't make any sense. Are you unaware that Sanger Sequencing exists, and the actual lesions can be read? Or that heterologous genes are being introduced with CRISPR methods? Neither of these common results can be explained by the spontaneous insertion of hundreds of nucleotides that happen to precisely match the sequence of the construct being inserted.

Again, this is utter nonsense and demonstrates a complete absence of basic understanding in molecular biology.

[nonbel]

>"Are you unaware that Sanger Sequencing exists, and the actual lesions can be read? Or that heterologous genes are being introduced with CRISPR methods? Neither of these common results can be explained by the spontaneous insertion of hundreds of nucleotides that happen to precisely match the sequence of the construct being inserted."

The first is just as consistent with the selection mechanism, because low levels of baseline mutants ARE reported (see the Schumann et al and Hendel et al papers I linked to in this thread for examples).

The second would indeed be difficult to explain with a selection mechanism, unfortunately I have not seen that actually published. Instead, the primers used can just as well be amplifying the template and/or the sequence of the inserted cassette is not shown (eg Figure 2D and S1 here: http://www.sciencemag.org/content/348/6233/442 )

If you have a reference to a specific paper I would appreciate it.

[daemonk]

What do you mean by the primers can just well be amplifying the template? How does that explain knock-ins without an actual insertion? And there are plenty of knock-in CRISPR papers out there. Just literally search for "CRISPR knock-in".

[nonbel]

Let me ask this. Say you have sequence A that is not supposed to exist before your treatment and sequence B that you have added to the environment in large amounts. Is it safe to use primers where one matches exactly to sequence B and the other is this similar?

CTCATTAGGCACCCCAGGCTTTACA

CTCAGT------CCCAGGCTTTACA

[daemonk]

Are you suggesting that they are just detecting the un-incorporated foreign DNA after CRISPR? I think the fact it has been shown that the knocked-in DNA is inherited to the progeny is strong enough evidence that the DNA was actually inserted.

Unless you want to argue that the un-incorporated DNA was also transmitted to the next generation, which honestly, is extremely unlikely.

[nonbel]

That could possibly explain some results, but not those involving transmission. If the knocked-in DNA is transmitted to the next generation then I'd think it must have gotten incorporated somewhere, however, this need not be at the intended site if the primers are amplifying the template.

Then again, supposedly shingles is caused by extragenomic Varicella-zoster DNA that is somehow stable for decades and can be passed on during pregnancy. I'm not sure I believe that though, and of course that is viral DNA.

Anyway, in that Ruan et al (2015) they claim to have detected exactly the expected sequence across the junction in at least a few cells. I can't think of any explanation for that data other than CRISPR working as advertised. However, they don't report in what percent of the cells this was observed.

Edit: I mean supposedly those exact sequences shown in figure S2 never physically existed before and now they do, exactly as predicted by the theory. That is strong evidence.

[nonbel]

Ok, I did that search and here is the first paper I found: http://www.nature.com/articles/srep14253

EDIT: Let me look again at this paper later.

[nonbel]

Here is another. At first you may think they show successful insertion of GFP, but it turns out no! Instead all that needed to happen was mutation of an early stop codon: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712628/

That is why I am asking others to provide their own references. The papers I have read do not seem capable of distinguishing between modification vs selection after careful inspection.