This system uses two lentiviral vectors. We made the LentiCRISPRv2-loxP-sgRNA-Cas9-loxP vector (Fig. 1a) by modifying the LentiCRISPRv2-mCherry vector (Addgene plasmid # 99154 from Agata Smogorzewska), which expresses a single sgRNA driven by a U6 promoter, with the Cas9 and mCherry genes driven by an EF1a promoter. Cre mediated recombination of the two inserted loxP sites allows deletion of the sgRNA, Cas9, and mCherry coding regions. Figure 1b shows the Tet-On 3G Cre EGFP lentiviral vector in which expression of Cre can be induced by addition of Doxycycline (Dox).
An experimental schema for the generation of Bcl2−/−Bak1−/− clonal lines of WEHI7 thymoma cells is depicted in Fig. 1c. We first established the WEHI7 iCre cell line by transfecting wild type (WT) WEHI7 cells with the Dox inducible lentiviral Tet-On 3G Cre expression vector, and sorted EGFP positive cells using a FACSAria Fusion flow cytometer.
To test the efficacy of this system for generation of multiple sequential CRISPR/Cas9 knockouts in WEHI7 cells, we chose Bcl2 and Bak1 because sgRNAs against these genes have been proven to work well in WEHI7 cells 15.
First, the Lenti-loxP-Bcl2 sgRNA-loxP vector was transfected into the WEHI7 iCre cell line, and after 48 hrs culture, individual mCherry positive cells were sorted into a 96 well plate by FACS single cell sorting. Once the mCherry positive lines had expanded, the levels of BCL2 protein in the Lenti-Bcl2 sgRNA-loxP transfected lines was evaluated by Western blot. In 16 lines tested, we obtained 12 independent clones that in which BCL2 protein was undetectable (Fig. 2a).
We then treated the Bcl2−/− WEHI7 iCre cells with Dox for 48 hrs, and analysed them by flow cytometry. As shown in Fig. 3a, after 48 hrs of induction of Cre, almost all of the cells became mCherry negative. In one of the WEHI7 iCre Bcl2−/− lines, some mCherry negative cells appeared without the addition of Dox, consistent with some leakiness in the expression of Cre recombinase 16, 17.
After 48 hrs treatment with Dox, mCherry negative WEHI7 iCre Bcl2−/− cells were sorted by flow cytometry. We then transfected these cells with the Lenti-loxP-Bak1 sgRNA-loxP vector, and after 2 days culture, we isolated mCherry positive cells by FACS single cell sorting. After expansion of these cells, Western blot analysis revealed that of 20 independent clonal lines tested, 9 lacked detectable BAK1 (in addition to lacking BCL2) (Fig. 2b). Thus, we successfully generated WEHI7 cell lines with double knock-out of Bcl2 and Bak1 genes (Fig. 2c).
To confirm that mCherry fluorescence could be removed in the WEHI7 iCre Bcl2−/− Bak1−/− clones upon Cre activation, we treated the cells with Dox for 2 days and examined them by flow cytometry. As anticipated, treatment with Dox caused loss of mCherry fluorescence in all the three independent iCre Bcl2−/− Bak1−/− clones (Fig. 3b).
In summary, we present here an easy method for generation of multiple sequential CRISPR/Cas9 knockouts in cell lines. This system allows the same vector to be re-used without the need for additional selection markers. Because the sgRNA, Cas9 and mCherry fluorescent protein coding regions were flanked by the two loxP sites, once the targeting mutation has been achieved, they can be removed by inducing expression of Cre recombinase. This enables the cells to be re-infected with the same vector bearing further sgRNAs, such that those transfected can be sorted by expression of mCherry once again. In addition, this system prevents prolonged expression of Cas9 and the sgRNA, reducing the likelihood of off-target effects, because these sgRNAs and Cas9 coding regions can be removed by activating expression of Cre recombinase.