CRISPR-mediated direct mutation of cancer genes in the mouse liver

By W. Xue, Sidi Chen, Hao Yin, T. Tammela, T. Papagiannakopoulos, Nikhil S. Joshi, Wenxin Cai, Gillian Yang, R. Bronson, D. Crowley and 4 more. Nature, 2014

743 citations recorded by the source corpus.

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 insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the β-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of β-catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.

Subfields: Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/55e0a7e9517f1d325ab3ef808709b3b984cf3d60

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Citations in the corpus

Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities (2018, methodology): “) in vitro [61–64], while hydrodynamic injection has been employed to deliver CRISPR cassettes to the liver in vivo [46, 60].”
CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling (2014, background): “Most recently, hydrodynamic injection, which can accommodate the large transgene size of Cas9, has been employed to achieve Cas9-mediated genome editing in the mouse liver in vivo (Xue et al., 2014; Yin et al., 2014), albeit with low editing efficiencies, and is primarily applicable to hepatocytes w”
The new frontier of genome engineering with CRISPR-Cas9 (2014, positive): “An improved method to generate liver cancer or myeloid malignancy models in mice facilitated by CRISPR-Cas9 was recently reported (98, 99).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “…Cas9 and sgRNA include indel mutation of two tumor suppressor genes and oncogenes resulting in generation of liver tumors in mice (Figure 5(C)) (Xue et al., 2014), inhibiting hepatitis B virus (HBV) replication and gene expression in HBV-infected mice (Lin et al., 2014; Zhen et al., 2015), and…”
CRISPR/Cas9 in Genome Editing and Beyond. (2016, background): “Cas9containing vectors can also be introduced into adult animals through hydrodynamic injections, leading to efficient gene corrections or mutations (125, 250).”