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. Approach and Results The CRISPR/Cas9 system was used to produce 3/18 founder mice carrying point mutations in an intronic CArG box of the smooth muscle cell (SMC)-restricted Cnn1 gene. Each founder was bred for germ line transmission of the mutant CArG box and littermate interbreeding to generate homozygous mutant (Cnn1ΔCArG/ΔCArG) mice. Quantitative RT-PCR, 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 SMC-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 SRF to the mutant CArG box. Loss of CNN1 expression was coincident with an increase in Ki-67 positive cells in the normal vessel wall. Conclusion CRISPR/Cas9 genome editing of a single CArG box nearly abolishes Cnn1 expression in vivo and evokes increases in SMC 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.

Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice

“…First, the elimination of SRF-binding CArG boxes upstream of Lmod1 had little to no effect on baseline LMOD1 protein or mRNA expression. This finding should be contrasted with an earlier report showing near abolishment of CNN1, another SMC-restricted gene product (34), upon CRISPR-mediated editing of a single intronic CArG box (17). Similarly, a decrease in expression of smooth muscle myosin light chain kinase with attenuated intestinal smooth muscle contractility was noted after deletion of an upstream CArG box (35).…”

“…It is interesting to contrast the findings reported here with several other smooth muscle-restricted actin-binding proteins that are dispensable for similar functional activity in these organs, including CNN1 (17,36), TAGLN (37), CSRP1 (38), and CALD1 (39), which would suggest that compensatory pathways exist to preserve normal organ function in the absence of each of these actin-binding proteins. On the other hand, genetic inactivation of Myh11, encoding the major thick filament protein in smooth muscle, leads to megacystis, a decrease in intestinal motility, defective contraction of the bladder, and early postnatal death, all of which are reminiscent of the Lmod1 −/− phenotype and human MMIHS (40).…”

“…To provide more formal proof of an association between loss in LMOD1 and MMIHS, we turned to CRISPR-Cas9 genome editing in the mouse. Initial efforts to inactivate Lmod1 were based on transgenic studies showing the critical importance of two conserved SRF-binding CArG boxes for Lmod1 promoter activity in vivo (12) and a more recent report that revealed near abolishment of target gene expression upon CRISPR-Cas9 genome editing of a single CArG box (17). In contrast to the latter finding, there was little change in Lmod1 mRNA or LMOD1 protein in mice homozygous for threecomponent CRISPR genome editing (18) of both CArG boxes (SI Appendix, Fig.…”

“…The guide RNAs were in vitro transcribed using a HiScribe T7 mRNA synthesis kit (New England BioLabs) and purified before assessment on an Agilent Bioanalyzer as described (18). Mouse zygotes were microinjected into the cytoplasm with Cas9 mRNA (100 ng/μL) and each guide RNA (100 ng/μL each) as described previously (17,57). After injection, zygotes were cultured in KSOM+AA medium for 1 h before transfer into the oviductal ampullae (10 to 15 zygotes per oviduct) of either CD1 (twocomponent CRISPR) or C57BL/6J (three-component CRISPR) female mice that had been mated, respectively, with vasectomized CD1 or C57BL/6J males the previous evening.…”

“…PCR conditions were as follows: 3 min incubation at 95°C, followed by 35 cycles of 30 s at 95°C, 30 s at 58°C, and 30 s at 72°C and a 5-min extension at 72°C. PCR genotyping of the CArG mutant mice was done with a duplex PCR assay as previously described (17). All PCR products were resolved in a 2% agarose gel.…”