Han Y, Slivano OJ, Christie CK, Cheng AW, Miano JM.
Arterioscler Thromb Vasc Biol. 35(2):312-315
Publication year: 2014


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.


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.


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.