“CRISPR” validation of recessive brain cancer genes in vivo

摘要: Profound understanding of the molecular events driving oncogenic transformation is essential for development improved therapies against cancer. While accumulating next generation sequencing data provide a plethora candidate genes various cancer entities [1], validating potential these proved to be more challenging. Autochthonous mouse models emerged as gold standard confirming gene's pathogenetic relevance, since murine feature high homology genomic structure and broad conservation gene function when compared with human. The genetically engineered (GEM) line, however, time consuming process. Especially, analysis multiple simultaneous alterations, which an aspect current research, extensive breeding GEMs often not feasible. The genetic manipulation somatic cells in situ holds great advantages terms speed flexibility. delivery oncogenes using e.g. viral vectors had long been established, knockout specific tumor suppressor has accomplished site-specific endonucleases including zinc-finger transcription activator-like effector nucleases only recently. They have shown their induce double strand breaks, possibly causing mutation by error prone non-homologous end joining. Recently, CRISPR/Cas9 system excelled providing possibility construct efficient fast convenient manner [2]. It that CRISPR via lentivirus [3] or hydrodynamic [4] generates precise alterations organs can used transformation. Modelling brain tumors utilizing technology, remained established. In our recent work, we aimed at extending methodology CRISPR/Cas9-mediated disruption utero electroporation vivo chemical transfection [5]. These techniques are less restricted transgene size than vectors, do cause additional mutations insertion adapted target variety tissues. Using approaches, delivered targeting particular thereby induced types tumors. Somatic deletion Ptch1 cerebella SHH-subgroup medulloblastoma penetrance short latency. resembled germline GEM models, ablated cerebellum. Whole genome facilitated unbiased search off targets. Utilizing this approach, did detect any recurrently mutated other CRISPR/Cas9-induced medulloblastomas. Additionally, Nf1, Pten Trp53 forebrain efficiently glioblastoma, demonstrating approach also suited simultaneously. Hence, approaches prove feasibility functionally recessive flexible manner. Furthermore, paves way refined animal driven resembling what observed patients. The future field applications. First screens pooled lentiviral libraries malignant metastasis recently conducted [6]. Further higher level multiplexing guided likely utilized leveraging transfer approaches. With latest modifications system, gain-of-function may analyzed inducing homology-directed repair, large deletions activation transcription. conceivable it soon will become possible model most kinds endogenously mouse, allowing highly sophisticated models. Together accelerated validation, greatly improve preclinical steps novel targeted therapies.