Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling

Abstract

In vivo functional investigation of oncogenes using somatic gene transfer has been successfully exploited to validate their role in tumorigenesis. For tumour suppressor genes this has proven more challenging due to technical aspects. To provide a flexible and effective method for investigating somatic loss-of-function alterations and their influence on tumorigenesis, we have established CRISPR/Cas9-mediated somatic gene disruption, allowing for in vivo targeting of TSGs. Here we demonstrate the utility of this approach by deleting single (Ptch1) or multiple genes (Trp53, Pten, Nf1) in the mouse brain, resulting in the development of medulloblastoma and glioblastoma, respectively. Using whole-genome sequencing (WGS) we characterized the medulloblastoma-driving Ptch1 deletions in detail and show that no off-targets were detected in these tumours. This method provides a fast and convenient system for validating the emerging wealth of novel candidate tumour suppressor genes and the generation of faithful animal models of human cancer.

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland

“…The advent of CRISPR/Cas9 technologies allows for rapid somatic gene editing in nearly any cell type to study the effects of gene perturbation in situ. Several studies have already shown that tumor initiation and development in various tissues, including the liver, lung, pancreas, and brain, can be modeled in vivo by using CRISPR/Cas9-based somatic gene editing (Platt et al 2014;Sánchez-Rivera et al 2014;Xue et al 2014;Chiou et al 2015;Weber et al 2015;Zuckermann et al 2015). As a proof of concept, we performed intraductal injections with pSECC-sgPten lentiviral vectors in Cdh1 F/F female mice to simultaneously ablate Ecadherin expression and disrupt the TSG Pten, a negative regulator of PI3K/AKT signaling.…”

“…For example, liver-specific gene disruption was achieved by transient delivery of components of the CRISPR/Cas9 system in the tail veins of mice, leading to hepatocellular carcinoma Weber et al 2015). Similar approaches have been used to deliver targeted oncogenic mutations to the lung (Platt et al 2014;Sánchez-Rivera et al 2014), brain (Zuckermann et al 2015), and pancreas (Chiou et al 2015).…”

piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice

“…1). In the future, combined with other innovative delivery methods, such as nanoparticles and electroporation (12,24), the extreme simplicity of PB-CRISPR libraries should greatly enhance the already powerful CRISPR weaponry.…”

“…However, because of their low efficiency, these two methods have not been widely used. Recently, CRISPR/Cas9 has been developed as an efficient mutagenesis tool (7)(8)(9) and was quickly adapted as a technique for in vivo tumor induction and validation of cancer genes (10)(11)(12)(13)(14)(15). By transplanting CRISPR librarytransduced cancer cells into immunocompromised mice, several genes involved in growth and metastasis of human lung cancer were identified (16).…”

Models in Translational Oncology: A Public Resource Database for Preclinical Cancer Research

“…Recently, a completely new strategy for rapid gene targeting was introduced, which is CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated gene 9 encoding a nuclease)-meditated genome editing (27,28), allowing even the one-step generation of mice with mutations in multiple genes (29)(30)(31). This technical breakthrough also for the field of cancer modeling allows the introduction of sitespecific modifications into the genomes of cells and organisms, irrespective of the species.…”