One key advantage of the CRISPR/Cas9 system in comparison with other gene editing approaches lies in its potential for multiplexing. Here, we describe an elaborate procedure that allows the assembly of multiple gRNA expression cassettes into a vector of choice within a single step, termed ASAP(Adaptable System for Assembly of multiplexed Plasmids)-cloning. We demonstrate the utility of ASAP-cloning for multiple CRISPR-mediated applications, including efficient multiplex gene editing, robust transcription activation and convenient analysis of Cas9 activity in the presence of multiple gRNAs.

The qkI gene encodes a family of RNA binding proteins alternatively spliced at its 3' end, giving rise to three major spliced isoforms: QKI-5, QKI-6 and QKI-7. Their expression is tightly regulated during brain development with nuclear QKI-5 being the most abundant during embryogenesis followed by QKI-6 and QKI-7 that peak during myelination. Previously, we generated a mouse conditional qkI allele where exon 2 is excised using Olig2-Cre resulting in QKI-deficient oligodendrocytes (OLs). These mice have dysmyelination and die at the third post-natal week. Herein, we performed a transcriptomic analysis of P14 mouse brains of QKI-proficient (QKI FL/FL;- ) and QKI-deficient (QKI FL/FL;Olig2-Cre ) OLs. QKI deficiency results in major global changes of gene expression and RNA processing with >1,800 differentially expressed genes with the top categories being axon ensheathment and myelination. Specific downregulated genes included major myelin proteins, suggesting that the QKI proteins are key regulators of RNA metabolism in OLs. We also identify 810 alternatively spliced genes including known QKI targets, MBP and Nfasc. Interestingly, we observe in QKI FL/FL;Olig2-Cre a switch in exon 2-deficient qkI mRNAs favoring the expression of the qkI-5 rather than the qkI-6 and qkI-7. These findings define QKI as regulators of alternative splicing in OLs including self-splicing.

In this study, we provide a comprehensive clinical and molecular biological characterization of radiation-induced gliomas (RIG), including a risk assessment for developing gliomas. A cohort of 12 patients who developed RIG 9.5 years (3-31 years) after previous cranial radiotherapy for brain tumors or T-cell acute lymphoblastic leukemia was established. The derived risk of RIG development based on our consecutive cohort of 371 irradiated patients was 1.6% at 10 years and 3.02% at 15 years. Patients with RIG glioma had a dismal prognosis with a median survival of 7.3 months. We described radiology features that might indicate the suspicion of RIG rather than the primary tumor recurrence. Typical molecular features identified by molecular biology examination included the absence of Histon3 mutation, methylation profile of pedHGG-RTK1 and the presence of recurrent PDGFRA amplification and CDKN2A/B deletion. Of the two long-term surviving patients, one had gliomatosis cerebri, and the other had pleomorphic xanthoastrocytoma with BRAF V600E mutation. In summary, our experience highlights the need for tissue diagnostics to allow detailed molecular biological characterization of the tumor, differentiation of the secondary tumor from the recurrence of the primary disease and potentially finding a therapeutic target.