title: 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.
  
  
  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.
authors.affiliation: From the Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester Medical Center, Rochester, NY (Y.H., O.J.S., C.K.C., J.M.M.); and Jackson Laboratories, Bar Harbor, ME (A.W.C.).
authors.forename: Yu
authors.initials: Y
authors.lastname: Han
authors.affiliation: From the Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester Medical Center, Rochester, NY (Y.H., O.J.S., C.K.C., J.M.M.); and Jackson Laboratories, Bar Harbor, ME (A.W.C.).
authors.forename: Orazio J
authors.initials: OJ
authors.lastname: Slivano
authors.affiliation: From the Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester Medical Center, Rochester, NY (Y.H., O.J.S., C.K.C., J.M.M.); and Jackson Laboratories, Bar Harbor, ME (A.W.C.).
authors.forename: Christine K
authors.initials: CK
authors.lastname: Christie
authors.affiliation: From the Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester Medical Center, Rochester, NY (Y.H., O.J.S., C.K.C., J.M.M.); and Jackson Laboratories, Bar Harbor, ME (A.W.C.).
authors.forename: Albert W
authors.initials: AW
authors.lastname: Cheng
authors.affiliation: From the Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester Medical Center, Rochester, NY (Y.H., O.J.S., C.K.C., J.M.M.); and Jackson Laboratories, Bar Harbor, ME (A.W.C.). j.m.miano@rochester.edu.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Miano
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D049109:Cell Proliferation; D015536:Down-Regulation; D006720:Homozygote; D007438:Introns; D019394:Ki-67 Antigen / Q000378:metabolism; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000473:pathology; D010641:Phenotype; D017354:Point Mutation; D012333:RNA, Messenger / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*; D000096985:Calponins
chemical_list: D002135:Calcium-Binding Proteins; D019394:Ki-67 Antigen; D008840:Microfilament Proteins; D012333:RNA, Messenger; D000096985:Calponins
keywords: CArG box; CRISPR; mice, transgenic; muscle, smooth; serum response factor