The PARPi olaparib is widely used for the treatment of patients with breast cancer (26) and its efficacy has been demonstrated on breast cancer cell line such as MCF7 (27). Thus, the MCF7 breast cancer cell-line was used as control. Pathogenic or deleterious mutations in BRCA1 or BRCA2 are good response predictors to olaparib. However, only 4 to 7% of patients with pancreatic cancer harbor a germline BRCA mutation (22), which ultimately represents few patients. In this study, we were able to successfully knockdown BRCA1 or BRCA2 using the CRISPR/Cas9-mediated KI technology in two PDAC cell lines (Capan-2 and T3M4). We first optimized and controlled the effective delivery of our CRISPR/Cas9 system in breast cancer MCF7 cells, this technology being already used on these cells (28) and as expected the olaparib sensitivity was higher for MCF7 BRCA KD cells compared to MCF7 WT cells.
Our CRISPR/Cas9 system is an all-in-one HDR complex, comprising Cas9-RNP (recombinant Cas9 protein complexed with two sgRNAs transcribed in vitro) and donor template DNA (ssODNs) with phosphorothioate modifications at extremities. This all-in-one strategy increases the HDR efficiency due to the presence of donor template at the time of DSB generation (29). Utilization of ssODNs for donor template with phosphorothioate modifications at extremities also improves the efficiency of HDR. The design of our ssDNA donors proposed by tools was symmetric but it has been shown that asymmetric donor design may favored for HDR (30). It would be interesting to experiment more if an asymmetric donor design can improve HDR efficiency in this study. The donor design is thus critical for the success of CRISPR/Cas9 experiment. For our all sgRNAs, the DSBs induced by Cas9 and the donor template insertion-site are separated by less than ten nucleotides and all presented a PAM motif, as recommended (31). The nature of nucleotides at position -4 from the PAM sequence also influences the editing precision. In our study, the two crRNAs for the BRCA1 mutation harbored a “T” in position -4 and for the BRCA2 mutation a “C” and “A” and the presence of a “T” or “A” at position -4 predict efficient insertions at the regions of interest. In contrast, a “G” is synonymous with a more imprecise target (32), which concerns none of our sgRNAs. Some chemicals as nocodazole or ABT751 allowing cell cycle synchronization can be used to increase HDR, which is restricted to the late S and G2 phases. However, these molecules have a potential toxicity in vivo (29). Hence, we chose not to use them during the CRISPR/Cas9-system optimization on cell lines in order to transpose the protocol more quickly to in vivo.
The number of cells with the on-target mutations is overall higher for T3M4 cells than for Capan-2 cells (> 50% and < 50% respectively) despite an identical CRISPR/Cas9 system for all cell lines. Besides the transfection capacity specific to each cell type, this difference can be explained by the fact that HDR activity is restricted to late S and G2 phases of cell cycle. Indeed, the Capan-2 cell doubling time is three times longer than the T3M4 cell doubling time, thus cell cycles were staggered and not at the same phase at the CRISPR/Cas9 transfection-time. Furthermore, T3M4 cells harbor the TP53 mutation c.215C>G p.Pro72Arg. TP53 plays a significant role in the cell cycle checkpoint control of G1/S phases and the Arg72 form induces lower G1 arrest than the Pro72 form (33), which can also explain the difference observed between Capan-2 and T3M4 cells. In an ideal setting to increase the transfection efficiency, the CRISPR/Cas9 transfection and preparation protocol should be developed and adapted for each cell lines, but we chose to use only one to simplify and attempt to do so universalize.
We observed a decrease of BRCA mutated cells according with cells passaging or after thawing for all models. On one side, cells which were not transfected with the CRISPR/Cas9 survived more longer than BRCA KD cells, due to the consequences of genetic instability generated by the loss of BRCA1/2 (blocking cell proliferation or apoptosis) (34). This finally leads to a pool population only composed of no BRCA KD clones. Another phenomenon has also been observed in vivo inducing resistance to treatment: patients may present a BRCA recovery upon progression while at diagnosis they owned a somatic disruption of BRCA1/2 (35). Chemotherapy regimens composed of DNA damaging agents or PARPi can also lead to secondary mutations restoring BRCA1/2 or induce a selective pressure on BRCA1/2 restored cells (36). These kinds of reversion mutations could be prevented by a pharmacological inhibition of the DNA end-joining repair pathways, as suggested by Tobalina et al. (37), although drug development is still ongoing (38). Therefore, the CRISPR/Cas9 transfection can be optimized to obtain better clonal evolution with 100% of cells exhibiting a biallelic deletion of BRCA1/2 and resulting in a total-loss-of-function mutation before treatment with PARPi. On the other side, the non-persistence of BRCA mutations over time or after thawing of our cells may occur due to a transient expression of our RNP complex, a DNA-free CRISPR/Cas9 mediated gene editing without genome integration. Utilization of plasmid as delivery method for CRISPR/Cas9 might allow to override this phenomenon due to a better stability. However, transfection of plasmid often leads to random genome integration and host immunogenic activation in vivo, and generate potentially more off-target effects than RNP complex due to a persistent expression of Cas9 in cells (39).
The main limitation of CRISPR/Cas9 technology is the high probability of off-target effects with a frequency > 50% (40). However, reducing off-targets effects while maintaining editing efficacy remains a challenge. Several strategies for limiting off-targets effects are now proposed, such as optimization of Cas9, sgRNAs, and guides designs (41). CRISPR/Cas9 design tools make it possible to obtain the best designs and can highlight hypothetical off-target sites. In our study, no off-target effect was predicted with the CRISPR LIFEPIPE® tool. In contrast, the CrispRGold tool predicted various off-target effects for each sgRNAs but mainly are intergenic or intronic. In cultured cells, this category of off-targets are not to be taken into account, at least for the functional studies (42). Utilization of RNP complex for the delivery of Cas9 protein and gRNAs, as in our study, appears to be an excellent delivery method for the CRISPR/Cas9 system with decreased off-target effects and high on-target mutations (40,41).
Olaparib, a PARPi, is known to be effective on cancer cells which presented a deficiency in HRR, including BRCA mutated cells. Indeed, PARPi induce accumulation of SSBs which lead to DSBs, resulting to an accumulation of DNA damage in HRD tumors, and therefore tumor-cell death (43). Therapeutic efficacy of olaparib has now been proven as maintenance treatment in metastatic pancreatic cancer which harbored a germline BRCA mutation (8). As expected, in our study, all BRCA KD cell lines presented an increased sensitivity at olaparib. IC50 values of BRCA KD cells were significantly lower than IC50 values of WT cells (P < 0.01), especially for the two PDAC cell lines. Our in vitro results are also in agreement with clinical results. Apoptosis analysis results of the present study have shown an increased apoptosis of pancreatic cells after treatment with 40µM of olaparib, and these differences were found to be statistically significant for BRCA KD cells (P < 0.01). In fact olaparib is known to induce apoptosis of cancer cells (44,45). In this respect, our in vitro results suggest that BRCA KD cells may have clinically relevant response to olaparib treatment.