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Updated: Jun 26, 2025
Adeno-Associated Virus-Mediated Delivery of CRISPR for Cardiac Gene Editing in Mice
Published on: August 2, 2018
Allele-Specific Suppression of Variant MHC With High-Precision RNA Nuclease CRISPR-Cas13d Prevents Hypertrophic
Ping Yang1, Yingmei Lou1, Zilong Geng1
1Key Laboratory of Systems Biomedicine, Shanghai Center for Systems Biomedicine, Engineering Research Center of Techniques and Instruments for Diagnosis and Treatment of Congenital Heart Disease, Institute for Developmental and Regenerative Medicine, Xin Hua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China (P.Y., Y. Lou, Z. Geng, Z. Guo, S.W., Y. Li, K.S., S.Z., J.X., A.F.C., L.D., K.S., B.Z.).
Insights
A novel CRISPR-Cas13 variant precisely targets and corrects MYH7 gene mutations responsible for hypertrophic cardiomyopathy. This gene therapy approach shows promise for treating inherited cardiac diseases by preventing cardiac hypertrophy in mouse models.
Area of Science:
- Cardiovascular Genetics
- Molecular Biology
- Gene Therapy
Background:
- Familial hypertrophic cardiomyopathy (HCM) is a severe genetic disorder with serious clinical outcomes.
- Heterozygous single nucleotide variants (SNVs) in sarcomere genes, particularly MYH7, are the primary cause of HCM.
- CRISPR-Cas13, a gene editing tool, has potential for genetic disorders but its application in cardiomyopathy is unexplored.
Purpose of the Study:
- To develop a highly precise CRISPR-Cas13 variant for targeting MYH7 SNVs in hypertrophic cardiomyopathy.
- To evaluate the efficacy and specificity of the engineered CRISPR-Cas13 variant in vitro and in vivo.
- To explore the therapeutic potential of CRISPR-Cas13-based gene therapy for inherited cardiac diseases.
Main Methods:
- Developed a sensitive reporter system to screen Cas13d variants for mutagenic activity.
- Engineered a high-precision Cas13d variant (hpCas13d) with specific cleavage of MYH7 variant RNAs.
- Validated hpCas13d in vitro for precision and low collateral cleavage; tested in HCM mouse models using AAV9 delivery to cardiomyocytes.
- Conducted large-scale library screening to assess hpCas13d's ability to resolve 45 human MYH7 pathogenic SNVs.
Main Results:
- Wild-type Cas13d failed to distinguish heterozygous MYH7 alleles with SNVs.
- The engineered hpCas13d variant demonstrated minimized collateral RNase activity and resolved various MYH7 pathological sequence variations.
- In vivo application in two distinct HCM mouse models suppressed the altered MYH7 allele and prevented cardiac hypertrophy.
Conclusions:
- CRISPR-Cas nucleases offer significant potential for precise treatment of inheritable cardiomyopathy.
- This study establishes a new therapeutic strategy for managing inherited cardiac diseases.
- High-precision gene editing presents a promising avenue for genetic cardiomyopathy treatment.
Background:
Familial hypertrophic cardiomyopathy has severe clinical complications of heart failure, arrhythmia, and sudden cardiac death. Heterozygous single nucleotide variants (SNVs) of sarcomere genes such as MYH7 are the leading cause of this type of disease. CRISPR-Cas13 (clustered regularly interspaced short palindromic repeats and their associated protein 13) is an emerging gene therapy approach for treating genetic disorders, but its therapeutic potential in genetic cardiomyopathy remains unexplored.
Methods:
We developed a sensitive allelic point mutation reporter system to screen the mutagenic variants of Cas13d. On the basis of Cas13d homology structure, we rationally designed a series of Cas13d variants and obtained a high-precision Cas13d variant (hpCas13d) that specifically cleaves the MYH7 variant RNAs containing 1 allelic SNV. We validated the high precision and low collateral cleavage activity of hpCas13d through various in vitro assays. We generated 2 HCM mouse models bearing distinct MYH7 SNVs and used adenovirus-associated virus serotype 9 to deliver hpCas13d specifically to the cardiomyocytes. We performed a large-scale library screening to assess the potency of hpCas13d in resolving 45 human MYH7 allelic pathogenic SNVs.
Results:
Wild-type Cas13d cannot distinguish and specifically cleave the heterozygous MYH7 allele with SNV. hpCas13d, with 3 amino acid substitutions, had minimized collateral RNase activity and was able to resolve various human MYH7 pathological sequence variations that cause hypertrophic cardiomyopathy. In vivo application of hpCas13d to 2 hypertrophic cardiomyopathy models caused by distinct human MYH7 analogous sequence variations specifically suppressed the altered allele and prevented cardiac hypertrophy.
Conclusions:
Our study unveils the great potential of CRISPR-Cas nucleases with high precision in treating inheritable cardiomyopathy and opens a new avenue for therapeutic management of inherited cardiac diseases.
