Eric Schoger1, Kelli J Carroll2, Lavanya M Iyer1, John McAnally3, Wei Tan3, Ning Liu2, Claudia Noack4, Orr Shomroni5, Gabriela Salinas5, Julia Gross6, Nicole Herzog7, Shirin Doroudgar8, Rhonda Bassel-Duby2, Wolfram Hubertus Zimmermann9, Laura C Zelarayan1. 1. Institute of Pharmacology and Toxicology, University Medical Center Goettingen, GERMANY. 2. Molecular Biology, UT Southwestern Medical Center, UNITED STATES. 3. Internal Medicine, UT Southwestern Medical Center, UNITED STATES. 4. Target Discovery Technologies, R and D Pharmaceuticals, GERMANY. 5. NGS-Integrative Genomics (NIG) Institute Human Genetics, University Medical Center Goettingen. 6. Department of Internal Medicine III (Cardiology, Angiology, and Pneumology), Heidelberg University Hospital. 7. Internal Medicine III, Center for Molecular and Translational Cardiology, GERMANY. 8. Internal Medicine III, University Hospital Heidelberg, GERMANY. 9. Pharmacology and Toxicology, Georg August University, Goettingen, GERMANY.
Abstract
Rationale: Genome editing by CRISPR/Cas9 is evolving rapidly. Recently, second generation CRISPR/Cas9 activation systems based on nuclease inactive "dead" (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (two well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription.Methods and Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the myosin heavy chain (Myh) 6 promotor was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR), up to three different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype (AAV) 9. Upon gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects.Conclusions: The developed mouse model permits enhancement of gene expression by utilizing endogenous regulatory genomic elements. Proof-of-concept in two independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.
Rationale: Genome editing by CRISPR/Cas9 is evolving rapidly. Recently, second generation CRISPR/Cas9 activation systems based on nuclease inactive "dead" (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. Objective: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (two well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription.Methods and Results: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the myosin heavy chain (Myh) 6 promotor was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR), up to three different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype (AAV) 9. Upon gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects.Conclusions: The developed mouse model permits enhancement of gene expression by utilizing endogenous regulatory genomic elements. Proof-of-concept in two independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.
Entities:
Keywords:
endogenous gene activation; genetic mouse model; in vivo CRIPSR/Cas9 system
Authors: Franziska S Rathjens; Alica Blenkle; Lavanya M Iyer; Anke Renger; Fahima Syeda; Claudia Noack; Andreas Jungmann; Matthias Dewenter; Karl Toischer; Ali El-Armouche; Oliver J Müller; Larissa Fabritz; Wolfram-Hubertus Zimmermann; Laura C Zelarayan; Maria-Patapia Zafeiriou Journal: Cardiovasc Res Date: 2021-07-07 Impact factor: 10.787