Hello!
I was reading up on the using the CRISPR-Cas9 system to introduce a point mutation in a protein I work with, and I had a couple of queries:
- Do I need to have two plasmids - one with the guide RNA and the Cas9 and the other with the donor template?
- Following transfection, how do I screen for the transfected cells from the non transfected ones? I heard that there's a transient expression of GFP in order to initially identify and separate the transfected cells from the non-transfected ones, but how do the cells get rid of the GFP after?
- Also, for additional screening of the transfected cells, what would be a good technique?
- I talked with a technical specialist at Sigma for designing a donor plasmid, but it seems that I need to make one since they don't synthesize the donor plasmids along with the Cas9 plasmids. Do the vectors have to be the same for the donor and the cas9 plasmids? What would be the vector of choice for the same?
Thank you for your time!
Best,
Ashutosh
1.There are some vectors where both Cas9 and gRNA are expressed from the same plasmid.
2. You can co-transfect with a plasmid carrying GFP (some gRNA and/or Cas9 vector may even have a florescent reporter on them) and FACS sort your transfected cells. Alternatively you can add a vector carrying a selection and do a quick treatment in order to kill off untransfected cells. As you don't select for them and they don't replicate the GFP or selection vectors will get diluted and lost as cells divide.
3. Once you're cells are forming colonies you would need to preform a surveyor assay to detect mutations, we're currently use the Guide-It mutation detection kit from Clontech but there are other kits on the market all working pretty much the same way. If you introduce a specific mutation rather then just forming indels than you can try to make it create a new restriction site which you can use for screening.
4. By donor I assume you mean a sequence that you want to introduce into the genome, if so you don't need a vector. I just synthesize a long oligo with about 40-50nt overlap to the genome on each side and the specific change in the middle.
https://www.addgene.org/crispr/guide/
Dear Ashutosh,
there are many companies who can produce your targeting/donor vector of choice. The problem is to find tose cells with the mutation w/o selection. Maybe you could use a targeting vector including a positive selection which can be removed later (loxP flanked, the loxP site left over after Cre mediated deletion should be in an intron) like people use it in ES cell culture.
kai
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