| Autor: |
Laura Schubert, Anh T. Le, Trista K. Hinz, Andre Navarro, Sarah K. Nelson-Taylor, Raphael A. Nemenoff, Lynn E. Heasley, Robert C. Doebele |
| Rok vydání: |
2023 |
| DOI: |
10.1101/2023.04.06.535912 |
| Popis: |
CRISPR/Cas9 gene editing technology is an indispensable and powerful tool in the field of cancer biology. To conduct successful CRISPR-based experiments, it is crucial that sgRNAs generate their designed alterations. Here, we describe a simple and efficient sgRNA screening method for validating sgRNAs that generate oncogenic gene rearrangements. We used IL3-independence in Ba/F3 cells as an assay to identify sgRNA pairs that generate fusion oncogenes involving theRetandNtrk1tyrosine kinases. We confirmed these rearrangements with PCR or RT-PCR as well as sequencing. Ba/F3 cells harboringRetorNtrk1rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNAs that catalyze theKif5b-RetandTrim24-Retrearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) was established from aTrim24-Retpositive tumor that exhibited highin vitrosensitivity to RET-specific TKIs. Moreover, orthotopic transplantation of TR.1 cells into the left lung yielded well-defined tumors that shrank in response to LOXO-292 treatment. The method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel murine oncogene-driven tumor models. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
|
