CRISPR-Cas9 Genome Editing of a Single Regulatory Element Nearly Abolishes Target Gene Expression in Mice
Objective—To ascertain the importance of a single regulatory element in the control of Cnn1 expression using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 genome editing.
Approach and Results—The CRISPR/CRISPR-associated protein 9 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 (Cnn1ΔCArG/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 Cnn1ΔCArG/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/CRISPR-associated protein 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.
- CArG box-binding factor, mouse
- clustered regularly interspaced short palindromic repeats
- mice, transgenic
- muscle, smooth
- serum response factor
- Received November 20, 2014.
- Accepted December 11, 2014.
- © 2014 American Heart Association, Inc.