My experimental design:
Make stable cell-line that has over-expression of mouse gene X (MGX) in HEK293 cell-line. In HEK 293 MGX knock-out human gene X (HGX) using CRISPR/Cas.
Problem: MGX and HGX have highly conserved mRNA, and there is not a region with 12 non-conserved sites.
Questions:
1.) What is the minimal amount of nucleotides in the guide-RNA target that will induce a frame-shift? The introns are not well conserved and if I can target these to create HGX KO that would be helpful.
2.) Does anyone have experience with over-expressing a protein using silent mutations in nucleotide sequence?
Any other suggestions/help is appreciated.
Thanks
Thanks very much for the response. My only concern at this point is I am not sure if my cells will survive when I knock-out HGX long enough for me to sort and then knock-in MGX. I think I am going to try this first and hope that HGX is not essential for cell survival/proliferation. Very helpful insight regarding exon/intron junction targeting and information regarding the transitory expression of Cas9.
Hi Julie,
If it's acceptable you could consider introducing silent mutations into the sgRNA target site in your MGX sequence (ideally in the PAM site) - that way you would only be hitting the HGX.
Chris
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