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0 pointststactplsignore10y ago0 comments

I'm not an expert in this area, but I still don't understand how you manage to reconcile site specific transgene insertion. In these studies reporter genes are clearly inserted and heritable. How is there anything more to the discussion?

Like, as reported by Doudna:

http://science.sciencemag.org/content/337/6096/816.short

"We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage."

Which part of that mechanism do you doubt? It sounds like you doubt the dsDNA nuclease activity of Cas9. Why not just order a plasmid, some Cas9 + gdna, put them together and sanger sequence your products? If Cas9 isn't a site specific guided endonuclease you could prove it for $200.

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nonbel10y ago

>"It sounds like you doubt the dsDNA nuclease activity of Cas9."

Not at all. This would be why the treatment is toxic and suppressive of proliferation.

At this point, I still think the presence of indels at the site (ie the proposed NHEJ mechanism) is just as easily explained by selection for pre-existing mutants. The experiments involving insertion of DNA (ie the proposed HDR mechanism) are better, but lack controls for "off-target" PCR amplification when showing the gels. IE we need to know how often the template itself will be amplified under their primers/conditions, both free and if it gets incorporated in some random location.

When segments across the insertion junction are amplified, sequenced, and reported, I find this convincing as it is a precise prediction that matches the data and I can think of no other explanation. The other experiments are pretty much redundant and add nothing. However, the reports I have seen contain little methodological or quantitative information regarding these sequences which does make me remain skeptical, especially about claims of efficiency. Those claims seem to always be determined using the former experiments that can be explained in other ways.

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