Hello,
I want to insert a gene into my cells using sgRNA, Cas9, and an HDR template. The gene that I will insert into has its own promoter. Because I will cut the gene downstream of its start codon, will my inserted gene have additional nucleotides from the upstream sequence of the sgRNA-targeted gene? How do I overcome this issue? Should I put a stop codon upstream of my the ATG of my inserted gene? Should I include a strong constitutive promoter upstream of my inserted gene?
Thank you!
Hello David,
Yes, it will be good to insert stop codon in frame with the targetted gene upstream of ATG of gene (insert).
It sounds like you wish to insert a new coding region into an endogenous gene and you intend to do this downstream of the endogenous ATG so that the endogenous promoter will be used? If that is the case, it would be best NOT to insert downstream of the ATG but to try and insert into the 5' UTR. Including a stop codon upstream of your introduced ATG will likely reduce translation because translation will initiate at the endogenous ATG if it is still present. There is no guarantee that translation will re-initiate at the second ATG or that your introduced coding region will be in frame with the endogenous ATG.
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