I want to knockout a gen and I would like to know how to select the clones without the protein. There is a antibiotic sistem in the plasmid but that does not provide that the mutation take place in every cell if I am using a pool of them. Thank you!!
Right. These viruses allow you to select for cells expressing the Cas9, but not for ones having an insertion. To select for an insertion directly, you want to insert a resistance marker in the gene to be mutated, and not wherever the Cas9 happens to land. Check out for example the HDR plasmids from SCBT that are intended to accomplish this.
If your GOI is expressed in your target cell line already (because some people first modify one easy to transfect/harvest cell type and afterwards transdifferentiate it to another), you can create a "Donor" sequence like this:
5'-HA_SA_P2A_Marker_2xpA_3'-HA when targeting intronic sequence with already at least one coding exon before.
5'-HA_SA_Marker_2xpA_3'-HA when targeting 5'-UTR (Don't forget start codon for Marker)
5'-HA_P2A_Marker_2xpA_3'-HA when targeting exonic coding sequences
HA: Homology Arms
SA: Splice Acceptor
P2A: Porcine Teschoivirus 2A peptide
Marker: Can also be fluorecent proteins (especially when you have FACS sorting systems in your facility) and not only selection markers. I prefer using fluorescent proteins since selection often leads to multiple non-specific integrations especially when you use too much antibiotics.
2xpA: 2x strong pA signal to really terminate the transcript at this point (e.g. SV40 late pA + bGH pA), because you might have very few times a read-through
Pay attention that all is in-frame which is important when using SA and P2A sequences. Also be aware of isoforms and alternative TSS.
/EDIT
I prefer to method targeting an intron inlcuding loxP sites flanking the "SA_P2A_Marker_2xpA" region because then you can show elegantly that you can rescue the phenotype or whatever by cutting this "stop-cassette" out with Cre and showing the reviewers that your actually phenotype is because of your knoch-in...
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