CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice--brief report
- PMID: 25538209
- PMCID: PMC4304932
- DOI: 10.1161/ATVBAHA.114.305017
CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice--brief report
Abstract
Objective: To ascertain the importance of a single regulatory element in the control of Cnn1 expression using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing.
Approach and results: The CRISPR/Cas9 system was used to produce 3 of 18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell-restricted Cnn1 gene. Each founder was bred for germline transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1(ΔCArG/ΔCArG)) mice. Quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal immunofluorescence microscopy showed dramatic reductions in Cnn1 mRNA and CNN1 protein expression in Cnn1(ΔCArG/ΔCArG) mice with no change in other smooth muscle cell-restricted genes and little evidence of off-target edits elsewhere in the genome. In vivo chromatin immunoprecipitation assay revealed a sharp decrease in binding of serum response factor to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall.
Conclusions: CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in smooth muscle cell DNA synthesis. This facile genome editing system paves the way for a new generation of studies designed to test the importance of individual regulatory elements in living animals, including regulatory variants in conserved sequence blocks linked to human disease.
Keywords: CArG box; CRISPR; mice, transgenic; muscle, smooth; serum response factor.
© 2014 American Heart Association, Inc.
Figures
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Comment in
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Genome editing of a CArG element in the mouse genome establishes its role in gene expression.Arterioscler Thromb Vasc Biol. 2015 Mar;35(3):496-7. doi: 10.1161/ATVBAHA.115.305175. Arterioscler Thromb Vasc Biol. 2015. PMID: 25717176 No abstract available.
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