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Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational
Oliver Hartmann1,2, Michaela Reissland1,2, Carina R Maier3
1Deregulated Protein Stability and Cancer Laboratory, Lehrstuhl für Biochemie und Molekularbiologie, Biozentrum, Universität Würzburg, Würzburg, Germany.
Abstract:
Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53 fl/fl :lsl-KRas G12D/wt . Developing tumors were indistinguishable from Trp53 fl/fl :lsl-KRas G12D/ wt -derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.
Insights
CRISPR-Cas9 gene editing technology enables rapid development of accurate non-small cell lung cancer (NSCLC) mouse models. This approach models human lung cancer mutations, improving translational research and reducing reliance on traditional mouse models.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Lung cancer is a leading cause of cancer deaths globally, with low survival rates for non-small cell lung cancer (NSCLC).
- Effective translational research requires accurate surrogate models that reflect patient-specific somatic mutations impacting treatment response.
- Current genetically engineered mouse models (GEMMs) are time-consuming to develop and modify.
Purpose of the Study:
- To establish a rapid and efficient method for creating NSCLC mouse models using CRISPR-Cas9 technology.
- To demonstrate that CRISPR-Cas9 can accurately recapitulate established NSCLC mouse models.
- To highlight the potential of CRISPR-Cas9 in advancing in vivo cancer research and translational studies.
Main Methods:
- CRISPR/Cas9 gene editing was employed to target Trp53 and KRas genes in mice, specifically aiming to replicate the Trp53fl/fl:lsl-KRasG12D/wt NSCLC model.
- Tumor development was induced either through constitutive Cas9 expression or via adeno-associated virus (AAV) delivery of CRISPR components to wild-type animals.
- Morphological, marker expression, and transcriptional profiles of CRISPR-generated tumors were compared to conventional GEMMs.
Main Results:
- CRISPR/Cas9-mediated targeting successfully recapitulated the Trp53fl/fl:lsl-KRasG12D/wt murine NSCLC model.
- Tumors generated using CRISPR exhibited indistinguishable morphology, marker expression, and transcriptional profiles compared to tumors from conventional GEMMs.
- Tumorigenesis was achieved efficiently in both Cas9-expressing and wild-type mice using AAV delivery, demonstrating broad applicability.
Conclusions:
- CRISPR/Cas9 technology offers a powerful and faster alternative to conventional GEMMs for in vivo cancer modeling.
- This approach significantly accelerates the development of clinically relevant NSCLC models for translational research.
- The CRISPR toolbox is revolutionizing in vivo cancer research, enabling swift creation of models that closely mimic human malignancies.
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