Highlights d In vivo and in vitro genome-scale CD8 T cell CRISPR screen in immunotherapy contexts d Dhx37 knockout in CD8 T cells enhances adoptive transfer efficacy d Dhx37 modulates CD8 T cell activation, cytokine production, and cytotoxicity d DHX37 interacts with PDCD11 and influences NF-kB activity

Material Supplementary 6.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.100064

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.

Identification of T cell targets to improve immunotherapies is of prime interest. To facilitate largescale CRISPR screens directly in T cells in vivo, here, we developed a hybrid genetic screening system with adeno associated virus (AAV) and the Sleeping Beauty (SB) transposon, where the transposon is nested in the viral vector. The approach enables efficient gene editing in primary murine T cells and genomic integration of the sgRNA cassette for screen readout. We performed Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Enhancing production efficiency, stability, effector function, and other desired features is of prime interest for chimeric antigen receptor engineered T cells (CAR-Ts). Here, we developed a new system for efficient generation of CAR-T with significantly enhanced features by streamlined genome engineering. Leveraging tracrRNA-independent CRISPR/Cpf1 systems with adeno-associated virus (AAV), building a stable CAR-T with homology-directed repair (HDR) knockin and checkpoint knockout (KIKO CAR-T) was achieved at high efficiency in one step. The modularity of the AAV-Cpf1 KIKO system enables flexible and highly efficient generation of double knockin of two different CARs in the same T cell. Compared to Cas9-based methods, the AAV-Cpf1 system generates double knockin CAR-Ts more efficiently. Dual-targeting CD22-specific AAV-Cpf1 KIKO CAR-T cells have potency comparable to Cas9 CAR-Ts in cytokine production and cancer cell killing, while expressing lower levels of exhaustion markers. This versatile system opens new capabilities of T cell immune engineering with simplicity and precision.