Hello, I am trying to use CRISPR/Cas9 for the knock-in of a transgene using HDR by targeting the safe harbour locus AAVS1 in human iPSC.
Is orientation of the transgene important for its expression? How do I decide the best orientation? Is it best to insert the transgene in the same orientation as found endogenously in its native locus? Or is it better to follow the orientation of the harbour gene? If in my cassette my GOI is expressed in one orientation and the reporter and resistance gene in the other, would this affect expression of the cassette?
Many thanks!
This question could get even more complicated: In some situations you could be dealing with a Complementary Protein pair.
Dear Dario,
The DNA cassettes should be in following order from 5' to 3' direction 5' - promoter - Gene of Interest+STOP codon - poly A tail - 3' . I think, if you integrate your transgene on any of the strands in 5' to 3' direction, it is likely to function because in genome, endogenous genes exist on both the strands of DNA. Moreover, you can also check that how genes near your region of interest are oriented. If I were you, I will the keep the transgene in the same orientation as found endogenously in its native locus to avoid dealing with unneeded variables.
Yes, it is a good idea to keep your GOI in one orientation and the reporter and resistance gene in the other. I think, it should work well.
Best regards,
Kshitiz
You can check this paper where they integrated a cassette in both sense and antisense orientation at the AAVS1 locus
Article Site-specific integration and tailoring of cassette design f...
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