I’m performing a CRISPR knock-in to introduce a FLAG tag at the C-terminus of a gene, just before the stop codon. Due to guide RNA design constraints, my guide cuts approximately 80 bp upstream of the stop codon.

My plan is to introduce:

• the 80 bp sequence (which will be replaced),
• followed by the FLAG tag,
• then a stop codon,
• and use IRES-puromycin for selection.

I’m concerned about whether this design might disrupt proper transcription termination or mRNA stability, since I am inserting a stop codon earlier than the original one and altering the native 3′ UTR context.

My questions: