A functional sgRNA-CRISPR screening method for generating murine RET and NTRK1 rearranged oncogenes.

Autor:	Schubert L; Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA., Le AT; Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA., Hinz TK; Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA., Navarro AC; Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA., Nelson-Taylor SK; Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA., Nemenoff RA; Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA., Heasley LE; Craniofacial Biology, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA.; Eastern Colorado VA Healthcare System, Rocky Mountain Regional VA Medical Center, Aurora, CO80045, USA., Doebele RC; Departments of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO80045, USA.
Jazyk:	angličtina
Zdroj:	Biology open [Biol Open] 2023 Aug 15; Vol. 12 (8). Date of Electronic Publication: 2023 Aug 15.
DOI:	10.1242/bio.059994
Abstrakt:	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. Competing Interests: Competing interests R.C.D. is an employee and shareholder of Rain Oncology Inc and has received licensing fees from Takeda, ThermoFisher, Voronoi, Loxo, Histocyte, and Black Diamond. A.T.L. receives licensing fees from Abbott Molecular. No disclosures were reported by the other authors. (© 2023. Published by The Company of Biologists Ltd.)
Databáze:	MEDLINE
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