Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes

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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were amplified by PCR (KOD Fx Neo, TOYOBO) as follows: 94 °C for 2 min, followed by 30 cycles of 98 °C for 2 10 s, annealing temperature (depending on primer sequences) for 30 s and 68 °C for 30 s. Primer sequences are listed 3 in Supplementary Table. After purification of PCR products using QIAquick PCR purification kit (QIAGEN), PCR 4 fragments both from untreated and treated allele were hybridized to form hetero DNA duplex. Then, hybridized PCR 5 hetero duplexes were enzymatically digested by mismatch-specific endonuclease, Cel-I 42 °C for 60 min 6 (SURVEYOR Mutation Detection Kit). Genomic DNA were extracted from mouse tail using a ready-made extraction solution (Express Extract, KAPA 10 Biosystems). PCR reactions were performed using 5 l ReadyMix (HiFi HotStart ReadyMix, KAPA Biosystems), 11 0.5 l of 10 M forward and reverse primers, 0.4 l of DNA extract and PCR-grade water to a total amount of 10 l.

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For Cas9 KI mice, 10 M forward and two reverse primers were mixed. PCR conditions are as follows: 95 °C for 3 13 min, followed by 33 cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C for 10s. Amplified DNA was separated by 14 electrophoresis in a 2 % TBE agarose gel.

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Proteins were separated by SDS-PAGE and transferred to PVDF membrane. Antibodies were diluted by 3% nonfat 21 milk. After blocking with 3% nonfat milk for 1 h, the transferred membrane was incubated with primary antibody 22 at 4°C overnight and with secondary antibody at room temperature for 30 min. The membrane signals were 23 detected by chemiluminescence using ECL or ECL prime reagent (GE). Gapdh was used as control for equal 24 loading and transfer. The protein expression level was quantified using ImageQuant TL (GE). Theoretical 25 4 molecular weight of the protein was calculated using website tool (ExPASy: http://web.expasy.org/compute_pi/).

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The number of moles of each protein band were estimated by normalizing protein expression levels by molecular 2 weight and relative molar ratio of tdTomato-tagged protein was calculated.

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The whole system was maintained at 39 °C. Following perfusion, hearts were placed in a 60-mm dish containing 20 50 μM Ca 2+ buffer (50 μM CaCl 2 in 0-Ca 2+ buffer) and minced with micro-scissors into small pieces. Non-21 cardiomyocytes were quickly removed according to their size (smaller than cardiomyocytes) using cell strainer.

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Isolated cardiomyocytes were incubated for 7 days after AAV6 transduction.

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Data are expressed as means ± S.D. of at least three independent experiments, unless otherwise indicated. The two-1 tailed Student's t-test or one-way ANOVA with repeated measures followed by Post-hoc Tukey test were used to 2 analyze differences between two groups. p-value < 0.05 was considered statistically significant.  2 A) Cleavage activity of each sgRNA targeting exon 6 of mouse Actb was evaluated by single-strand annealing 3 assay (see Methods) (n=3, means ± SD). 4 B) C2C12 cells were transfected with pX459 vector encoding sgRNA against exon 6 of mouse Actb. Two days 5 after transfection, cells were exposed to 3 g/mL puromycin for two days. Genomic DNA was extracted, and 6 cleavage activities were evaluated by Cel-I assay. White arrowhead indicates the cleaved PCR products.     n=3, means ± SD). C2C12 cells were transfected with pX459 vector encoding sgRNA against Myl2 (#2). Two 6 days after transfection, cells were exposed to 3 g/mL puromycin for 2 days. Genomic DNA was extracted, and 7 cleavage activities were evaluated by Cel-I assay (right).

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IV. Create a linked One to One target set to link'Nucleus'(primary target set) with'Cardiomyocyte' 12 (secondary target set), to count the number of cardiomyocytes. Set Overlap conditions so that more than 13 80% of the primary target is within the secondary target. 14 B) Cardiomyocytes seeded in 96-well plate were transduced with AAV6 encoding HDR components 6 h after 15 addition of EdU, followed by continuous labeling. After 4 days of culture, cells were fixed and immunostained.

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Representative images of cardiomyocytes positive for tdTomato with EdU staining. Scale bar: 100 m.

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18 Figure S4 19 A) C2C12 cells were transduced with pX459 vector encoding 3xFLAG-Cas9, with or without sgRNA #2 against 20 Tnnt2 combined with the repair template plasmid. Two days after transfection, cells were exposed to 3 g/mL 21 puromycin for two days. Genomic DNA was extracted, and the targeted region was amplified by PCR. Purified 22 PCR products were digested by XhoI. Arrowheads indicate the cleaved PCR products.

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B) Genotyping results of K210 knock-in allele in neonatal mice obtained after crossing mice carrying 24 heterozygous K210 knock-in allele and homozygous Cas9 knock-in allele. The higher PCR band indicates the K210 knock-in allele 5 . 1 C) Neonatal cardiomyocytes isolated from Cas9 knock-in mice were transduced with AAV6 encoding LacZ or 2 editing components shown in Fig. 4F (1.88 x 10 5 viral genomes/cell). Forty-eight hours after transduction, 3 whole-cell lysates were extracted. Troponin T and Gapdh were detected by western blot.