CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice - Brief report

Yu Han, Orazio J. Slivano, Christine K. Christie, Albert W. Cheng, Joseph M. Miano

Research output: Contribution to journalArticle

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) 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 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.

Original languageEnglish (US)
Pages (from-to)312-315
Number of pages4
JournalArteriosclerosis, thrombosis, and vascular biology
Volume35
Issue number2
DOIs
StatePublished - Feb 2 2015
Externally publishedYes

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CRISPR-Associated Proteins
Clustered Regularly Interspaced Short Palindromic Repeats
Smooth Muscle Myocytes
Gene Expression
Serum Response Factor
Conserved Sequence
Chromatin Immunoprecipitation
Fluorescence Microscopy
Point Mutation
Confocal Microscopy
Genes
Reverse Transcription
Western Blotting
Genome
Polymerase Chain Reaction
Messenger RNA
DNA
Gene Editing
Proteins

Keywords

  • CArG box
  • CRISPR
  • mice
  • muscle, smooth
  • serum response factor
  • transgenic

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine

Cite this

CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice - Brief report. / Han, Yu; Slivano, Orazio J.; Christie, Christine K.; Cheng, Albert W.; Miano, Joseph M.

In: Arteriosclerosis, thrombosis, and vascular biology, Vol. 35, No. 2, 02.02.2015, p. 312-315.

Research output: Contribution to journalArticle

Han, Yu ; Slivano, Orazio J. ; Christie, Christine K. ; Cheng, Albert W. ; Miano, Joseph M. / CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice - Brief report. In: Arteriosclerosis, thrombosis, and vascular biology. 2015 ; Vol. 35, No. 2. pp. 312-315.
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