We have endogenously tagged our protein of interest in U2-OS cells. Unfortunately, it is not a homozygous insertion; there is a wild-type allele remaining, as well as an allele containing an inserted A nucleotide at the sgRNA/Cas9 cut site. This is near the 3' end of the sgRNA. What are the odds that the original sgRNA will cut both? Or do we have to redesign a second guide to match the added A?
You most likely can. There are conflicting reports about tolerance of mismatches in the PAM proximal region in the literature therefore it is important to look at papers the specifically test your cell line/organism. There is one such paper for U2-OS cells (however they use 2RNAs for guide delivery rather than sgRNA but this should still be applicable to your situation):
"single-base mismatches are tolerated (resulting in detectable and significant mismatch targeting relative to the on-target activity), particularly if the mismatches occur in the PAM-distal end of the crRNA, away from the seed region, defined here as nucleotides 1–10 of the crRNA (Fig. 3A)."
Ref:Article Systematic Analysis of CRISPR-Cas9 Mismatch Tolerance Reveal...
Best of luck!
Hanna
I believe it is better to design a new sgRNA. Also if they are too similar, chances are, it would still cut both. You can come up with a screening strategy to distinguish the cut between the WT and modified one and then select single clones.
Nelson B. Cole just a quick question; did you not destroy the PAM site with the insertion that you made?
No, the PAM site is still intact; only an A was inserted at the cut site -3/-4 position. I do want to cut both alleles to insert the tag into both sites to make a completely homozygous cell line.
Ah ok then as you can see the old guide will most likely have some measure of activity even if it is not completely complementary. However it is a little bit weird that only an A got inserted and that stopped further cutting without disrupting the PAM. This may indicate that the insertion in that specific position with an A may cause the guide not to cut anymore if you are unsuccessful in your first attempts I recommend that you just repeat with a clean background that does not have a heterozygous insertion.
Best of luck!
Hanna
Good point. I think I'll design a new sgRNA for the insertion.
The issue I am having in general is that U2-OS are a lot more heterogeneous as far as gene copy number than I thought. In fact, in my case, it is easier to get homozygous tagged lines in HeLa cells than U2-OS.
I hope it works for you. Just remember some sgRNA works better than other and at this point it is still difficult to predict which ones are the better ones so I guess just redesign and try to get lucky!