CRISPR-Cas9 genome editing of a single regulatory element nearly abolishes target gene expression in mice--brief report - PubMed (original) (raw)

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

Yu Han et al. Arterioscler Thromb Vasc Biol. 2015 Feb.

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.

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Figures

Figure 1

Figure 1. 3cCRISPR genome editing of a CArG box

(A) Strategy for targeting one of the 4 conserved intron 1 CArG boxes (vertical green and black lines) in mouse Cnn1 gene. A synthetic crRNA (underlined sequence) was designed to include the first seven nucleotides of the CArG box (green) immediately 5’ of a protospacer adjacent motif (PAM, blue box) comprising the remaining three nucleotides of the CArG box. The blue arrow indicates the predicted double strand break three nucleotides upstream of the PAM sequence. The crRNA was cloned upstream of the invariant tracrRNA (curved line) and this gRNA was then combined with Cas9 mRNA and the HDR template harboring three nucleotide substitutions of the CArG box following two nucleotide changes (red) that together create a novel SalI restriction site (pink underlined sequence), for cytoplasmic injection of fertilized mouse eggs. Genotyping of F1 intercrossed mice was done using a SalI RFLP digest (B), a novel multiplex PCR assay (C) and Sanger sequencing (D). Thick arrow in panel B represents the wildtype PCR product and thin arrows represent the expected size products of one or both alleles with correct SalI site. Arrows in panel C represent the PCR products with multiplex PCR (top gel) and PCR using wildtype (middle) or mutant (bottom) forward primers (see Figure I in the online-only Data Supplement). The red box in panel D shows the confirmed sequence edit creating novel SalI site (pink underlined sequence) that changes the normal CArG box (green underlined sequence).

Figure 2

Figure 2. Expression of Cnn1 mRNA and CNN1 protein in F1 mice

(A) Mouse aorta or bladder analyzed for expression of Cnn1 mRNA by quantitative RT-PCR with normalized Cnn1+/+ set to 1.0; other genotypes expressed relative to Cnn1+/+. (asterisks indicate p<0.01 compared to wildtype). See also Supplemental Figures II and III. (B) Expression of CNN1 protein in aorta by Western blotting. (C) In vivo chromatin immunoprecipitation assay of SRF binding to wildtype versus mutant intronic CArG box in bladder tissue. (D) Confocal immunofluorescence microscopy of CNN1 in wildtype versus mutant carotid arteries of two different founder lines. (E) SMC DNA synthesis (arrows) revealed with an antibody to Ki-67. Similar findings were seen in an independent founder line. Scale bars are 30 µm and 10 µm for panels D and E, respectively.

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