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In vivo CRISPR screening protocol to identify metastasis mediators using iteratively selected mouse models
Yuqi Wang1, Mangze Hu2, Yilong Zou1
1Westlake Four-Dimensional Dynamic Metabolomics (Meta4D) Laboratory, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China.
Abstract:
In vivo CRISPR screens uncover metastasis genes in native contexts, surpassing in vitro model limitations. Here, we present a protocol to identify metastasis-driving genes in ovarian cancer using an in vivo CRISPR screening technique. Key steps include single-guide RNA (sgRNA) library design and validation, lentiviral transduction, establishment of metastatic mouse models, tissue collection, sgRNA amplification for sequencing, bioinformatics-based candidate gene identification, and functional validation. For complete details on the use and execution of this protocol, please refer to Wang et al.1.
Insights
This study details an in vivo CRISPR screening protocol to identify genes driving ovarian cancer metastasis in living organisms. This method overcomes limitations of lab-based models for discovering critical metastasis genes.
Area of Science:
- Genetics
- Oncology
- Molecular Biology
Background:
- In vitro models have limitations in fully recapitulating complex biological processes like cancer metastasis.
- Identifying genes that drive metastasis in native physiological contexts is crucial for understanding and treating advanced cancers.
Purpose of the Study:
- To present a detailed protocol for an in vivo CRISPR screening technique to identify metastasis-driving genes in ovarian cancer.
- To provide a reproducible method for researchers studying cancer progression and metastasis.
Main Methods:
- The protocol involves single-guide RNA (sgRNA) library design and validation.
- Establishment of metastatic mouse models and subsequent tissue collection are key steps.
- Bioinformatics analysis of sgRNA amplification by sequencing is used for candidate gene identification.
Main Results:
- The in vivo CRISPR screen successfully identifies potential metastasis-driving genes within the native tumor microenvironment.
- The protocol enables the discovery of genes that may be missed by traditional in vitro screening methods.
Conclusions:
- This in vivo CRISPR screening protocol offers a powerful approach to uncover novel metastasis genes in ovarian cancer.
- The methodology surpasses in vitro limitations, providing insights into metastasis in a more relevant biological context.
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