Hello,
I am trying to introduce a point mutation (A-->T) in my mammalian cell line (say 293T) and have it stably express the mutated protein. My sgRNA works fine because I get nice indels at the correct site.
However, nothing changes if I add my ssODN to the electroporation mixture, I just get indels again. I also tried adding 1µM SCR7, to no avail. I'm using a combined Cas9/sgRNA vector with puro selection, so I can select for positively transduced cells.
Moreover, the point mutation serves as a resistance factor (i.e. in another cell line), so even with low efficiency i should be able to select for the mutation, which doesen't work either.
I am using 5µl of 10µM ssODN per 1x106 cells. That translates to 40µl per 8mio. cells ind 400µl in an old BioRad electroporator. But EP itself works fine with very good efficiency.
Anyone can tell my why this doesn't work?
I have attached the exon + desired mut. + ssODN. In this ssODN teher are a few more additional point mutations to prevent re-cutting of after HDR. There are a few more mutations because I am trying a Cas9n strategy in parallel using different PAMs. Are these too many point mutations in the ssODN, maybe?
How far away from the DSB is your desired mutation? If much more than 20 nt, all bets are off.
Hi Joeseph,
Thanks for your reply. I brought the issue to the google groops from the Zhang lap and got some detailed trouble shooting. The mains issue seems to be just what you said so i designed new guides and template.
Cheers,
Thomas
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