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Updated: Feb 24, 2026
In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity
Published on: November 2, 2020
AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma
Ryan D Chow1,2,3, Christopher D Guzman1,2,4,5,6, Guangchuan Wang1,2
1Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA.
Abstract:
A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as B2m-Nf1, Mll3-Nf1 and Zc3h13-Rb1, which were subsequently validated using AAV minipools. Distinct from Nf1-mutant tumors, Rb1-mutant tumors are undifferentiated and aberrantly express homeobox gene clusters. The addition of Zc3h13 or Pten mutations altered the gene expression profiles of Rb1 mutants, rendering them more resistant to temozolomide. Our study provides a functional landscape of gliomagenesis suppressors in vivo.
Insights
Researchers developed a novel genetic CRISPR screen in mice to study glioblastoma. This approach identified key gene mutations driving tumor development and revealed how specific mutations impact tumor characteristics and drug resistance.
Area of Science:
- Neuro-oncology
- Genetics
- Molecular Biology
Background:
- Understanding genetic factors in glioblastoma is crucial for developing effective treatments.
- Glioblastoma multiforme (GBM) remains a highly aggressive brain tumor with limited therapeutic options.
- Existing models often fail to fully recapitulate human GBM complexity.
Purpose of the Study:
- To develop and utilize an autochthonous genetic CRISPR screen in vivo for glioblastoma research.
- To identify genetic drivers and suppressors of glioblastoma pathogenesis.
- To investigate the functional consequences of genetic alterations in glioblastoma.
Main Methods:
- Adeno-associated virus (AAV)-mediated delivery of a CRISPR library targeting cancer genes.
- Stereotaxic injection into the brains of conditional-Cas9 mice to generate autochthonous tumors.
- Next-generation sequencing (capture sequencing) for mutational profiling.
- Co-mutation analysis and validation using AAV minipools.
Main Results:
- The developed mouse model successfully recapitulated human glioblastoma, exhibiting diverse mutational profiles.
- Mouse mutation frequencies correlated with those observed in human glioblastoma patient cohorts.
- Identified co-occurring driver gene combinations (e.g., B2m-Nf1, Mll3-Nf1, Zc3h13-Rb1).
- Rb1-mutant tumors showed distinct undifferentiated phenotypes and aberrant homeobox gene expression.
- Co-mutations involving Zc3h13 or Pten altered gene expression and increased temozolomide resistance in Rb1-mutant tumors.
Conclusions:
- This study establishes a powerful in vivo CRISPR screening platform for glioblastoma research.
- The findings provide a functional landscape of gliomagenesis suppressors and identify novel driver combinations.
- The results offer insights into the genetic basis of glioblastoma heterogeneity and therapeutic resistance.
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