Somatic CRISPR/Cas9-mediated tumour suppressor disruption enables versatile brain tumour modelling (original) (raw)
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AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma
Nature neuroscience, 2017
A causative understanding of genetic factors that regulate glioblastoma pathogenesis is of central importance. Here we developed an adeno-associated virus-mediated, autochthonous genetic CRISPR screen in glioblastoma. Stereotaxic delivery of a virus library targeting genes commonly mutated in human cancers into the brains of conditional-Cas9 mice resulted in tumors that recapitulate human glioblastoma. Capture sequencing revealed diverse mutational profiles across tumors. The mutation frequencies in mice correlated with those in two independent patient cohorts. Co-mutation analysis identified co-occurring driver combinations such as 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 stu...
A CRISPR/Cas9 Functional Screen Identifies Rare Tumor Suppressors
Scientific Reports, 2016
An enormous amount of tumor sequencing data has been generated through large scale sequencing efforts. The functional consequences of the majority of mutations identified by such projects remain an open, unexplored question. This problem is particularly complicated in the case of rare mutations where frequency of occurrence alone or prediction of functional consequences are insufficient to distinguish driver from passenger or bystander mutations. We combine genome editing technology with a powerful mouse cancer model to uncover previously unsuspected rare oncogenic mutations in Burkitt’s lymphoma. We identify two candidate tumor suppressors whose loss cooperate with MYC over-expression to accelerate lymphomagenesis. Our results highlight the utility of in vivo CRISPR/Cas9 screens combined with powerful mouse models to identify and validate rare oncogenic modifier events from tumor mutational data.
CRISPR-mediated direct mutation of cancer genes in the mouse liver
Nature, 2014
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed inse...
Frontiers of CRISPR-Cas9 for Cancer Research and Therapy
Journal of Exploratory Research in Pharmacology, 2021
In recent years, gene editing technologies have made significant progress in understanding gene function and regulation. The Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system has emerged as a versatile tool for gene editing and genome engineering. In the last few years, CRISPR-Cas9 technology has been widely applied to cancer research, mainly to understand the mechanisms of oncogenesis, drug-target identification, and the development of various cell-based therapies. When combined with genome sequence information, this technology has also shown promise to cure heritable genetic disorders. This review summarizes some of the recent developments and preclinical applications of CRISPR-Cas9 technology in cancer research and therapy. We will discuss how CRISPR based approaches have been used as a tool to identify cancer-specific vulnerabilities and potential applications in cancer therapy.
CRISPR-Cas9 therapies in experimental mouse models of cancer
Future oncology (London, England), 2018
The CRISPR-Cas9, a part of the defence mechanism from bacteria, has rapidly become the simplest, fastest and the most precise genome-editing tool available. The therapeutic applications of CRISPR are boundless: correction of mutations in several disorders, inactivation of oncogenes and viral oncoproteins, and activation of tumor suppressor genes. In this review, we expose recent advances concerning animal models of cancer that use CRISPR-Cas9, addressing also the current efforts to develop CRISPR-Cas9-based therapies, focusing on proof-of-concept studies. Finally, the review exposes some of the main challenges that this genome-editing tool faces. The key issue remains: does CRISPR-Cas9 have real potential for therapeutic application or will it just remain a wonderful research tool?
Genes
The tumor microenvironment plays important roles in cancer biology, but genetic backgrounds of mouse models can complicate interpretation of tumor phenotypes. A deeper understanding of strain-dependent influences on the tumor microenvironment of genetically-identical tumors is critical to exploring genotype–phenotype relationships, but these interactions can be difficult to identify using traditional Cre/loxP approaches. Here, we use somatic CRISPR/Cas9 tumorigenesis approaches to determine the impact of mouse background on the biology of genetically-identical malignant peripheral nerve sheath tumors (MPNSTs) in four commonly-used inbred strains. To our knowledge, this is the first study to systematically evaluate the impact of host strain on CRISPR/Cas9-generated mouse models. Our data identify multiple strain-dependent phenotypes, including changes in tumor onset and the immune microenvironment. While BALB/c mice develop MPNSTs earlier than other strains, similar tumor onset is ob...
Science advances, 2018
Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adeno-associated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice. All mice that received the AAV-mTSG library developed liver cancer and died within 4 months. We used molecular inversion probe sequencing of the sgRNA target sites to chart the mutational landscape of these tumors, revealing the functional consequence ...
Identification of oncogenic driver mutations by genome-wide CRISPR-Cas9 dropout screening
BMC genomics, 2016
Genome-wide CRISPR-Cas9 dropout screens can identify genes whose knockout affects cell viability. Recent CRISPR screens detected thousands of essential genes required for cellular survival and key cellular processes; however discovering novel lineage-specific genetic dependencies from the many hits still remains a challenge. To assess whether CRISPR-Cas9 dropout screens can help identify cancer dependencies, we screened two human cancer cell lines carrying known and distinct oncogenic mutations using a genome-wide sgRNA library. We found that the gRNA targeting the driver mutation EGFR was one of the highest-ranking candidates in the EGFR-mutant HCC-827 lung adenocarcinoma cell line. Likewise, sgRNAs for NRAS and MAP2K1 (MEK1), a downstream kinase of mutant NRAS, were identified among the top hits in the NRAS-mutant neuroblastoma cell line CHP-212. Depletion of these genes targeted by the sgRNAs strongly correlated with the sensitivity to specific kinase inhibitors of the EGFR or RA...
CRISPR-Cas9: a promising genetic engineering approach in cancer research
Therapeutic advances in medical oncology, 2018
Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions inandenvironments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional ...
CRISPR Cas System: an efficient tool for cancer modelling
Journal of the Pakistan Medical Association
The Clustered Regularly Interspaced Short Palindromic Repeats–Cas-9 (CRISPR-Cas9) system has been a revolutionising tool in the field of molecular genetics, which provides a versatile range of editing potentials. Researchers can produce breaks or alter genomes with ease using the system. Cancer is one of the multi-gene diseases whose genes need to be studied in detail. The CRISPR-Cas9 technology may also provide a promising potential in the field of cancer genetics. The current narrative review comprised 50 research articles which were keenly analysed and the applications and outcomes of CRISPR-Cas9 system in cancer genetics were comprehensively and critically discussed. It was concluded that application of the system had great potential to help understand cancer biology of various types and could be used for its genetic modelling. However, much work is still needed to be done to apply the technology for understanding...Continuous...