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.

NKCC and KCC transporters mediate coupled transport of Na++K++Cl− and K++Cl− across the plasma membrane, thus regulating cell Cl− concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.

CLC family proteins translocate chloride ions across cell membranes to maintain the membrane potential, regulate the transepithelial Cl− transport, and control the intravesicular pH among different organelles. CLC-7/Ostm1 is an electrogenic Cl−/H+ antiporter that mainly resides in lysosomes and osteoclast ruffled membranes. Mutations in human CLC-7/Ostm1 lead to lysosomal storage disorders and severe osteopetrosis. Here, we present the cryo–electron microscopy (cryo-EM) structure of the human CLC-7/Ostm1 complex and reveal that the highly glycosylated Ostm1 functions like a lid positioned above CLC-7 and interacts extensively with CLC-7 within the membrane. Our complex structure reveals a functionally crucial domain interface between the amino terminus, TMD, and CBS domains of CLC-7. Structural analyses and electrophysiology studies suggest that the domain interaction interfaces affect the slow gating kinetics of CLC-7/Ostm1. Thus, our study deepens understanding of CLC-7/Ostm1 transporter and provides insights into the molecular basis of the disease-related mutations.