Use of antibiotic resistance markers with CRISPR is an effective tool for cells that proliferate. However, for primary cells and non-dividing cells, all-in-one sgRNA-Cas9 lentiviral vector that contains a fluorescence marker (GFP-CRISPR) could be a better option.
Our standard, Single-Vector CRISPR-Cas9 System contains a puromycin selection that can be used to isolate transduced cells. In cases where you want to knockout a gene in cytostatic populations, substitution of the puromycin selection for a fluorescent marker enables selection using FACS. Hence, a fluorescent marker such as GFP also allows easy visualization and can expedite your workflow.
Ideally, the most effective approach would be to include both the fluorescent and antibiotic genes in the vector, as we normally do in our shRNA constructs, this design is not an option in our lentiviral based single-vector CRISPR system due to size constraints imposed by the large 4 kb Cas9 nuclease gene, which is required for CRISPR, to be effectively packaged into lentiviral particles. As a result, only one selection marker can be included in Cas9-containing lentiviral vectors.
For genome-wide knockout screens, we recommend using a two-vector CRISPR. With this configuration, both the fluorescent and antibiotic markers are included.
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