[iso1337]

Their claims around Type IIS assembly are also suspect. eg in Golden Gate assembly, you choose Type IIS that reach over and cut, so the restriction site is absent from the final assembled product.

"Additionally, because the final product does not have a Type IIS restriction enzyme recognition site, the correctly-ligated product cannot be cut again by the restriction enzyme, meaning the reaction is essentially irreversible"

https://en.wikipedia.org/wiki/Golden_Gate_Cloning ----

The choice of focusing on a particular RE pair also smells of p-hacking. Their claim that BsaI/BsmBI makes for easy mixing/matching genomes doesn't make sense in this day and age, when you can use other techniques to make hybrids more effectively (eg, you are not restricted to the natural location of those restriction enzyme sites)

[throwawaymaths]

The argument is about the negative space of the RE. Regardless of what the article says, To do golden gate/Gibson/etc. best practice is to cut the template first (with RE) then assemble against the open ends, so to do this you must ablate the existing re sites. The alternative is to linearize by round the horn pcr. At 33kb, it's not impossible but why bother with the pain when it's much easier to snip.

[tripletao]

Their description of the assembly strategy as "Golden Gate" seems like incorrect terminology. The WIV has at least published papers using BsaI and BsmBI, though.

https://twitter.com/jbkinney/status/1583267221047869441

https://twitter.com/jbkinney/status/1583248052969562112

https://journals.plos.org/plospathogens/article?id=10.1371/j...

EDIT: Kinney also asserts here that they left the sites in a final assembled genome (i.e., the genome of a replication-competent virus). I'm still trying to figure out if that's true, though.

[emsign]

Just because there are more modern techniques doesn't mean there haven't been older ones used, or techniques used incorrectly.