Implement USER cloning

[dgruano]

We need USER cloning for this, so @manulera and I thought it would be better to implement it in pydna. I have a minimal working example and will write some tests before opening a PR. Feel free to check it out in my fork.

[hiyama341]

Hi, we actually use it in teemi and you can see the implementation here: And the function names:

• USER_enzyme()
• nicking_enzyme()

Also there is also a working example here where we build a USER plasmid. Just for reference.

Hope it helps. If you need some ideas please reach out. :)

[hiyama341]

Hi again @dgruano and @manulera,

I just wanted to mention this tool that a lot of people use: https://services.healthtech.dtu.dk/services/AMUSER-1.0/

Maybe we can get inspired by their implementation :)

[dgruano]

Hey @hiyama341 thanks for passing by! I checked your implementation in teemi and found it pretty straightforward. For now, I based mine on the Cas9 object, but not sure it's the best way to go. Also, I wanted to make the search for Us a bit more complex, as USER relies on the proper detachment of the oligo upstream of the cut: if we don't limit the distance of the U to the 5' end of the molecule, cloning may never happen (i think).

I'll certainly give a look at the web tool and at the nicking enzyme, as this is something I have not implemented. Thanks again!!

[dgruano]

Actually, the end result of a USER + AP liase reaction vs a Nicking enzyme is very similar in the edges of linear sequences: detachment of the upstream oligo due to instability and creation of a ssDNA overhang. Would it be worth it to base USER on a nicking enzyme?

[hiyama341]

Hi again, So normally you use the nicking enzyme along with PacI to open the USER cassette so it is ready for integration of the PCR-amplified products (see below).