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RE: Lab Diaries #2 - Making My Own Transgenic Cell Line
I guess you can get multiple independent lines to correct for non specific effects. Have you considered using CRISPR Cas9 to target your gene to a specific locus? I totally understand about not mentioning the name of your over-expressed gene. Good luck with your research!
CRISPR/Cas9 is used for targeted genome editing in such way that you perform knockout of the gene of interest. It cannot be used to "direct" lentiviral integration to the specific site (at least I haven't heard of that).
Here is a nice review on some of the CRISPR applications and tools
Thanks for the review, but I wasn't able to find application of CRISPR/Cas9 where you would make the cut and integrate your gene at specific site in genome by using only CRISPR/Cas9. If you would be so kind to point out on the exact paragraph which describes such application, because I would really like to learn something new! :)
Yes its called Homology Directed Repair (HDR) and its in Figure 1B of that review but here is a figure from NEB
![](https://steemitimages.com/640x0/https://www.neb.com/-/media/nebus/page-images/applications/cloning-and-mapping/ge_cas9forge_0616.png?h=400&w=300&la=en&hash=6F0DEA73A9CBBD4D6ACFBA0221026B45F3EDA126)
![](https://steemitimages.com/DQmVkhNinsvnqKRAYgpYXVfnrCcoWomQ1tDbeaQbX1HHgNB/image.png)
showing Non Homologous End Joining (NHEJ) for knock outs and HDR for "knocking in" any piece of DNA you can dream of (the donor DNA with homology arms in figure below).
Also have a look at this video as you might be interested in using HDR in non dividing cells:
Great, I actually didn't know about this particular application of CRISPR! However, after searching through PubMed for publications/applications, I saw that method is still in its early phase of development, and that it still doesn't exhibit great efficiency. But thanks anyway for sharing this, I will certainly follow further development of this method!
I am almost certain that whatever your end result you wanted using your lentiviral integration you can do with CRISPR Cas9 technology, but the beauty of this is that the integration is not random. CRISPR is not just for knock outs of genes. For example in your research if you want a strong promoter driving your gene of interested (like your lentivirus vector) you just make the construct and direct CRISPR Cas9 where to make the cut and integration site of your gene of interest.