A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes
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Published on: November 2, 2020
Laura Schubert1, Anh T Le1, Trista K Hinz2
1Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA.
Abstract:
CRISPR/Cas9 gene editing represents a powerful tool for investigating fusion oncogenes in cancer biology. Successful experiments require that sgRNAs correctly associate with their target sequence and initiate double stranded breaks which are subsequently repaired by endogenous DNA repair systems yielding fusion chromosomes. Simple tests to ensure sgRNAs are functional are not generally available and often require single cell cloning to identify successful CRISPR-editing events. Here, we describe a novel method relying on acquisition of IL3-independence in Ba/F3 cells to identify sgRNA pairs that generate oncogenic gene rearrangements of the Ret and Ntrk1 tyrosine kinases. The rearrangements were confirmed with PCR, RT-PCR and sequencing and Ba/F3 cells harboring Ret or Ntrk1 rearrangements acquired sensitivity to RET and TRK inhibitors, respectively. Adenoviruses encoding Cas9 and sgRNA pairs inducing the Kif5b-Ret and Trim24-Ret rearrangements were intratracheally instilled into mice and yielded lung adenocarcinomas. A cell line (TR.1) established from a Trim24-Ret positive tumor exhibited high in vitro sensitivity to the RET inhibitors LOXO-292 and BLU-667 and orthotopic TR.1 cell-derived tumors underwent marked shrinkage upon LOXO-292 treatment. Thus, the method offers an efficient means to validate sgRNAs that successfully target their intended loci for the generation of novel, syngeneic murine oncogene-driven tumor models.
Insights
A new method uses IL3-independence in Ba/F3 cells to validate CRISPR/Cas9 gene editing sgRNAs for creating cancer gene fusions. This approach efficiently generates oncogenic rearrangements and novel mouse tumor models for drug sensitivity testing.
Area of Science:
- Cancer Biology
- Gene Editing
- Oncology
Background:
- CRISPR/Cas9 gene editing is crucial for studying cancer-associated fusion oncogenes.
- Validating sgRNA functionality for generating specific gene rearrangements is challenging and often requires laborious single-cell cloning.
- Developing efficient methods to confirm sgRNA targeting is essential for advancing cancer research.
Purpose of the Study:
- To introduce a novel, efficient method for validating single-guide RNA (sgRNA) pairs used in CRISPR/Cas9 gene editing.
- To establish a system for identifying sgRNAs that successfully generate oncogenic gene rearrangements in tyrosine kinases like Ret and Ntrk1.
- To create syngeneic murine tumor models for evaluating targeted cancer therapies.
Main Methods:
- Utilized IL3-independence in Ba/F3 cells as a screening system to identify functional sgRNA pairs.
- Generated Kif5b-Ret and Trim24-Ret gene rearrangements in Ba/F3 cells.
- Confirmed rearrangements via PCR, RT-PCR, and sequencing.
- Induced lung adenocarcinomas in mice using adenoviruses encoding Cas9 and specific sgRNA pairs.
- Established and characterized a Ret-rearranged cell line (TR.1) for drug sensitivity studies.
Main Results:
- Successfully identified sgRNA pairs that induce IL3-independence by creating Ret and Ntrk1 oncogenic rearrangements.
- Ba/F3 cells with these rearrangements showed sensitivity to specific tyrosine kinase inhibitors.
- Intratracheal instillation of engineered adenoviruses led to the development of lung adenocarcinomas in mice.
- The TR.1 cell line derived from a Trim24-Ret tumor demonstrated significant sensitivity to RET inhibitors (LOXO-292, BLU-667) both in vitro and in vivo.
- Orthotopic tumors derived from TR.1 cells showed marked shrinkage upon treatment with LOXO-292.
Conclusions:
- The described IL3-independence-based method provides an efficient means to validate sgRNAs for generating oncogenic gene fusions.
- This approach facilitates the rapid creation of novel, syngeneic murine models driven by specific oncogenic rearrangements.
- These validated models are valuable tools for preclinical evaluation of targeted therapies against specific driver mutations in cancer.
