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Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover
Published on: May 23, 2016
Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes
Takamaru Ishizu1, Shuichiro Higo2, Yuki Masumura1
1Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan.
Insights
Genome editing using CRISPR/Cas9 effectively repairs mutations in non-dividing heart cells (cardiomyocytes). This homology-directed repair (HDR) approach shows promise for treating genetic cardiomyopathies.
Area of Science:
- Cardiovascular Biology
- Molecular Genetics
- Gene Editing Technologies
Background:
- Hereditary cardiomyopathies are linked to genetic mutations, but targeted therapies are lacking.
- The efficacy of homology-directed repair (HDR) in non-dividing cardiomyocytes remains unclear.
Purpose of the Study:
- To investigate the effectiveness of CRISPR/Cas9-mediated HDR in non-dividing cardiomyocytes.
- To assess the potential of HDR as a therapeutic strategy for intractable cardiomyopathies.
Main Methods:
- Utilized adeno-associated virus (AAV) to deliver CRISPR/Cas9 components (sgRNA and repair template) into cardiomyocytes.
- Endogenously tagged Myl2 protein expression was monitored to confirm HDR in non-dividing cells.
- Genome correction of a pathological mutation in Tnnt2 was performed in cardiomyopathy model mice.
Main Results:
- Demonstrated successful HDR in non-dividing cardiomyocytes, independent of DNA synthesis.
- Achieved precise genome correction of the Tnnt2 mutation with ~12.5% efficiency.
- Developed an sgRNA strategy to minimize off-target effects on gene expression.
Conclusions:
- HDR-mediated genome editing is effective in non-dividing cardiomyocytes.
- This technology presents a potential therapeutic avenue for genetic cardiomyopathies.
Abstract:
Although high-throughput sequencing can elucidate the genetic basis of hereditary cardiomyopathy, direct interventions targeting pathological mutations have not been established. Furthermore, it remains uncertain whether homology-directed repair (HDR) is effective in non-dividing cardiomyocytes. Here, we demonstrate that HDR-mediated genome editing using CRISPR/Cas9 is effective in non-dividing cardiomyocytes. Transduction of adeno-associated virus (AAV) containing sgRNA and repair template into cardiomyocytes constitutively expressing Cas9 efficiently introduced a fluorescent protein to the C-terminus of Myl2. Imaging-based sequential evaluation of endogenously tagged protein revealed that HDR occurs in cardiomyocytes, independently of DNA synthesis. We sought to repair a pathological mutation in Tnnt2 in cardiomyocytes of cardiomyopathy model mice. An sgRNA that avoided the mutated exon minimized deleterious effects on Tnnt2 expression, and AAV-mediated HDR achieved precise genome correction at a frequency of ~12.5%. Thus, targeted genome replacement via HDR is effective in non-dividing cardiomyocytes, and represents a potential therapeutic tool for targeting intractable cardiomyopathy.
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