title: A mouse model for adult cardiac-specific gene deletion with CRISPR/Cas9.
abstract: Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)9 genomic editing has revolutionized the generation of mutant animals by simplifying the creation of null alleles in virtually any organism. However, most current approaches with this method require zygote injection, making it difficult to assess the adult, tissue-specific functions of genes that are widely expressed or which cause embryonic lethality when mutated. Here, we describe the generation of cardiac-specific Cas9 transgenic mice, which express high levels of Cas9 in the heart, but display no overt defects. In proof-of-concept experiments, we used Adeno-Associated Virus 9 (AAV9) to deliver single-guide RNA (sgRNA) that targets the Myh6 locus exclusively in cardiomyocytes. Intraperitoneal injection of postnatal cardiac-Cas9 transgenic mice with AAV9 encoding sgRNA against Myh6 resulted in robust editing of the Myh6 locus. These mice displayed severe cardiomyopathy and loss of cardiac function, with elevation of several markers of heart failure, confirming the effectiveness of this method of adult cardiac gene deletion. Mice with cardiac-specific expression of Cas9 provide a tool that will allow rapid and accurate deletion of genes following a single injection of AAV9-sgRNAs, thereby circumventing embryonic lethality. This method will be useful for disease modeling and provides a means of rapidly editing genes of interest in the heart.
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148;
authors.forename: Kelli J
authors.initials: KJ
authors.lastname: Carroll
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148;
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Makarewich
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148;
authors.forename: John
authors.initials: J
authors.lastname: McAnally
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148;
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Anderson
authors.affiliation: Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, I-34149 Trieste, Italy.
authors.forename: Lorena
authors.initials: L
authors.lastname: Zentilin
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148;
authors.forename: Ning
authors.initials: N
authors.lastname: Liu
authors.affiliation: Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology, I-34149 Trieste, Italy.
authors.forename: Mauro
authors.initials: M
authors.lastname: Giacca
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148;
authors.forename: Rhonda
authors.initials: R
authors.lastname: Bassel-Duby
authors.affiliation: Department of Molecular Biology and the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148; Eric.Olson@utsouthwestern.edu.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Olson
mesh_terms: D000375:Aging / Q000235:genetics*; D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D006332:Cardiomegaly / Q000150:complications / Q000473:pathology; D002469:Cell Separation; D000229:Dependovirus / Q000378:metabolism; D017353:Gene Deletion; D055785:Gene Knockdown Techniques; D006333:Heart Failure / Q000150:complications / Q000473:pathology; D008822:Mice, Transgenic; D023421:Models, Animal; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics; D009928:Organ Specificity / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism
chemical_list: C584019:Myh6 protein, mouse; D000094704:RNA, Guide, CRISPR-Cas Systems; D018995:Myosin Heavy Chains
keywords: CRISPR-associated endonuclease; cardioediting; cardiovascular pathology; gene knockdown; transgenic mouse