CRISPR-Cas9 Engineered Isogenic Luciferase-expressing Cell Lines as In Vitro and In Vivo Models for Cancer Research

The CRISPR-Cas9 system provides a robust gene-editing tool for basic research in biology and for the development of disease models for translational research. The objective of this study is to use advanced technologies including CRISPR-Cas9 and bioluminescence to generate novel human cell lines for use as both in vitro and in vivo models in cancer research. Approximately 50% of melanoma patients have the BRAF^V600Emutation and often become resistant to current BRAF inhibitors after several months of treatment. KRAS^G13Dis an acquired mutation associated with resistance to these inhibitors. In this study, CRISPR-Cas9 was used to knock-in the KRAS^G13D point mutation into the A375 malignant melanoma cell line, which also contains the targetable BRAF^V600E mutation. The resulting KRAS^G13D mutant isogenic line A375, which has been validated at the genomic, transcript, and protein bio-functional levels, exhibits significant resistance to the BRAF inhibitors dabrafenib and vemurafenib when studied both in traditional 2-D and 3-D cell culture. Based on the in vitro model described above, we developed additional models for use in live-animal bioluminescence imaging by introducing a stable luciferase reporter into the isogenic A375 and KRAS^G13D A375 cell lines. Both the relative and absolute bioluminescence signals within the cells were quantified and found to emit 4.9 x 10⁵photons/cell/sec (A375) and 3.5 x 10⁵ photons/cell/sec (KRAS^G13D A375). A subcutaneous xenograft model was utilized in this study and the in vivo live bioluminescence signal was quantified using the Xenogen IVIS™ imaging system to correlate tumor growth with luciferase expression. Both A375-Luc2 and KRAS^G13D A375-Luc2 grew as subcutaneous tumors with increasing levels of bioluminescence when injected into nude mice. In addition, a portfolio of 5 human isogenic luciferase reporter cell line pairs and 18 human and mouse luciferase reporter cell lines were developed for the study of various cancer types. In conclusion, the combination of two technologies CRISPR-Cas9 technology and stable luciferase expression allows for the generation of isogenic luciferase-expressing cell lines, which are valuable tools for elucidating mechanisms involved in tumorigenesis and for studying drug responses in vitro and in vivo.