Publication Date:
2014-08-15
Description:
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 beta-catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of beta-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.〈br /〉〈br /〉〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199937/" target="_blank"〉〈img src="https://static.pubmed.gov/portal/portal3rc.fcgi/4089621/img/3977009" border="0"〉〈/a〉 〈a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199937/" target="_blank"〉This paper as free author manuscript - peer-reviewed and accepted for publication〈/a〉〈br /〉〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Xue, Wen -- Chen, Sidi -- Yin, Hao -- Tammela, Tuomas -- Papagiannakopoulos, Thales -- Joshi, Nikhil S -- Cai, Wenxin -- Yang, Gillian -- Bronson, Roderick -- Crowley, Denise G -- Zhang, Feng -- Anderson, Daniel G -- Sharp, Phillip A -- Jacks, Tyler -- 1K99CA169512/CA/NCI NIH HHS/ -- 2-P01-CA42063/CA/NCI NIH HHS/ -- 5-U54-CA151884-04/CA/NCI NIH HHS/ -- DP1 MH100706/MH/NIMH NIH HHS/ -- K99 CA169512/CA/NCI NIH HHS/ -- P30 CA014051/CA/NCI NIH HHS/ -- P30-CA14051/CA/NCI NIH HHS/ -- R00 CA169512/CA/NCI NIH HHS/ -- R01 DK097768/DK/NIDDK NIH HHS/ -- R01-CA115527/CA/NCI NIH HHS/ -- R01-CA132091/CA/NCI NIH HHS/ -- R01-CA133404/CA/NCI NIH HHS/ -- R01-EB000244/EB/NIBIB NIH HHS/ -- Howard Hughes Medical Institute/ -- England -- Nature. 2014 Oct 16;514(7522):380-4. doi: 10.1038/nature13589. Epub 2014 Aug 6.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2]. ; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA. ; Tufts University and Harvard Medical School, Boston, Massachusetts 02115, USA. ; Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts 02142, USA. ; 1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2] Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [3] Harvard-MIT Division of Health Sciences &Technology, Cambridge, Massachusetts 02139, USA [4] Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA. ; 1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA. ; 1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [2] Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA [3] Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25119044" target="_blank"〉PubMed〈/a〉
Keywords:
Animals
;
Base Sequence
;
*CRISPR-Cas Systems
;
Cell Transformation, Neoplastic/genetics
;
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
;
Female
;
*Genes, Tumor Suppressor
;
Genes, p53/genetics
;
Genetic Engineering/*methods
;
Hepatocytes/metabolism/pathology
;
Lipid Metabolism
;
Liver/cytology/*metabolism/pathology
;
Liver Neoplasms/genetics/metabolism/pathology
;
Mice
;
Molecular Sequence Data
;
Mutagenesis/*genetics
;
Mutation/*genetics
;
Oncogenes/*genetics
;
PTEN Phosphohydrolase/genetics
;
Phosphorylation
;
Proto-Oncogene Proteins c-akt/metabolism
;
beta Catenin/genetics
Print ISSN:
0028-0836
Electronic ISSN:
1476-4687
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
,
Natural Sciences in General
,
Physics
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