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Multiplexed in vivo base editing identifies functional gene-variant-context interactions.
Jonuelle Acosta1,2, Grace A Johnson1,2, Samuel I Gould1,2
1Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA.
Biorxiv : the Preprint Server for Biology
|March 10, 2025
Summary
This study introduces a new in vivo platform using base editing to analyze cancer-associated genetic variants in mice. The findings reveal the crucial role of the in vivo environment and highlight limitations of standard CRISPR-Cas9 approaches for variant functional analysis.
Area of Science:
- Genomics
- Cancer Biology
- Genetic Engineering
Background:
- Human genome sequencing identifies numerous genetic variants linked to cancer predisposition, progression, and treatment outcomes.
- Understanding the functional impact and relative importance of these variants in their native context is crucial for clinical applications.
- Current methods for studying genetic variants in vivo are limited, hindering comprehensive analysis of their physiological roles.
Purpose of the Study:
- To develop a multiplexed in vivo platform for systematic functional analysis of endogenous genetic variants in cancer.
- To investigate the impact of the physiological in vivo environment and cellular organotropism on gene-variant phenotypes.
- To compare the efficacy of base editing with standard CRISPR-Cas9 nuclease approaches for variant analysis.
Main Methods:
- Integration of cross-species base editing sensor libraries with syngeneic cancer mouse models.
- Development of a multiplexed in vivo platform for high-throughput screening of genetic variants.
- Engineering and phenotyping of 7,783 human cancer-associated mutations across 489 genes using 13,840 guide RNAs.
Main Results:
- Construction of a comprehensive compendium of gene-mutation-context interactions in vivo.
- Demonstration that the in vivo environment and organotropism significantly influence gene-variant phenotypes.
- Identification of mutations and their in vivo effects missed by standard CRISPR-Cas9 approaches, revealing potential site-specific mechanisms.
Conclusions:
- The developed in vivo platform enables systematic functional interrogation of genetic variants in their native physiological context.
- The study underscores the importance of the in vivo microenvironment in determining the phenotypic consequences of genetic variations.
- This versatile platform offers a powerful tool for investigating genetic variation in cancer and other diseases, potentially uncovering new therapeutic strategies.
