Hi,
I am going to use CRISPR nCas9 to insert 80bp in a specific region of the genome (in HEK293T). I am aware that the use of ssDNA as template is recommended for insertions 100bp. However, I can't find anything about the use of ssDNA for insertion between 50-100bp. Could you share any experience you have had with this? Could you point out any website/paper where use ssDNA for insertions bigger than 50bp? I know you can also use long ssDNA for big insertions but at the moment I am not considering that option.
Thank you!!
There is a system called scarless editing using piggybac-ITR system. Marker can be cut out with transposase later, leaving only extra TTAA on site after cutting. You can contact Noahgen if you are interested in making such a donor vector.
Thank you Delin!
Is there no risk for the donor to get integrated in random TTAA chromosomal sites?
No, a donor will be something like this: arm5' for recombination:your insertion:piggybac3ITR:screening marker:piggybac5ITR: arm3 for recombination.
The bold fonts region can be cut with transiently expressed transposase and leave only ttaa. ITR is pretty only and the cutting is specifically.
It sounds very good. I will take it into account for my next gene targeting design.
Thank you again Delin!
- Badges
- Science method