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.

Genome Editing of Monogenic Neuromuscular Diseases

“…With regard to Cas9 immunogenicity, high-level expression of Cas9 in various tissues of transgenic mice evoked no overt abnormalities. 57,58 However, in humans, the potential immunogenicity of Cas9 remains to be determined.…”

Die hypertrophe Kardiomyopathie

“…[19]. Die großen Fortschritte auf dem Gebiet der In-vivo-Editierung des Genoms durch die CRISPR-Technik lassen inzwischen auch gentherapeutische Ansätze denkbar erscheinen, nach entsprechender ethischer Abwägung auch bei Patienten, bei denen ansonsten die Herztransplantation im Säuglingsalter die einzige Therapiealternative ist, zumal deren Verfügbarkeit immer weiter eingeschränkt ist [5]. …”

Considerations for Cardiac CRISPR

“…As the heart is responsible for a large disease burden, it represents a particularly attractive tissue to target for therapeutic purposes 4 . While several studies have shown that cardiomyocytes can be edited in the postnatal murine heart using viral delivery of one or more of the CRISPR/Cas9 system components 5–7 , the efficiency of this phenomenon has been less well characterized.…”