Can I use CRISPR gene editing in primary cells?
One of the factors that influences your ability to do gene editing in primary cells is how long the cells can be cultured for. If for example you are aiming to derive a clonal population of targeted cells, then a life of ten passages is going to make this extremely challenging, as clones will need to be expanded from the single cell and screened for successful editing. The other option therefore is to work with primary cells in the pool, but again this comes with its own challenges relating to the efficiency with which editing can be achieved in that pool. Efficiency will be influenced by the following factors: Proportion of cells that are transfected/infected with CRISPR/Cas9 reagentsProportion of those cells that are subsequently modified at target locus by Cas9 disruptionProportion of those cells that contain desired modification at target locus (i. e. a frameshift causing mutation for a knockout)Proportion of those cells that contain desired modification in all copies of the gene. And so within the pool there will only be a proportion of cells that have for example, a gene knockout. Given the short time lines, and the challenges with efficiency, if a CRISPR strategy is still desired then using a viral vector such as lentivirus, that stably integrates into the genome and constitutively expresses the guide RNA and Cas9, is almost certainly going to be the most appropriate route. If cells can be analysed individually then CRISPR may still represent an improvement in situations where RNAi can only achieve incomplete knockdown.