HDR with ssDNA, LentiCRISPRv2, and HEK293T

I was hoping if someone could help me with a simple question. I really tried finding a clear answer to this online but I don't see a defined solution anywhere. This is my question:

I am transfecting HEK293T cells with LentiCRISPRv2 + ssDNA via Lipofectamine 2000 in a 12 well plate. I added puromycin after 24 hours of selection. Now I'm hoping that after single cell isolation, I'd be able to find at least 1 cell with the right mutation. However, where I read I find that Lipofectamine is not good for doing this (people use nucleofection instead). But a lot of protocols use lipofectamine. My question is can lipofectamine work even if the efficiency is low? And if the efficiency is low can i just select more cells to increase my probability of finding one single cell with the right mutation?


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Brown Valkyrieover 3 years ago

Lipofectamine 2000 should be OK, may be you need to give more time for selection and enrichment. But, since 293T cells are to some extent delicate, we use PEI transfection. You can try both.

If they did not work, then lentivirus delivery would be the

Black Pegasusover 3 years ago

Many thanks for your reply! But is there a study that used Lipo2K to cotransfect LentiCRISPRv2 + ssODN? I ask because I'd like to get some idea about how many cells to isolate into single colonies.

Black Pegasusover 3 years ago

Just to follow up - I did the ssODN (~100 bp) transfection with lipofectamine and I got one successful knockin. The other one failed. It seems to be that lipo2k can work but the probability of success is low. I would recommend electroporation.

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