AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma

A causative understanding of genetic factors that regulate glioblastoma (GBM) pathogenesis is of central importance. Here, we developed an adeno-associated virus (AAV)-mediated autochthonous CRISPR screen in GBM. Stereotaxic delivery of an AAV library targeting genes commonly mutated in human cancers into the brains of conditional Cas9 mice resulted in tumors that recapitulate human GBM. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlate with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as Mll2, 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.