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Genome engineering has been transformed by CRISPR technology, which makes it possible to precisely alter DNA sequences. This approach targets the DMD gene in mdx/utrophin+/- mice, a model for DMD, using CRISPR-mediated genome editing. Because of its effectiveness in modifying the dystrophin gene and regaining its function, the SaCas9/gRNA system given by rAAVrh.74 was selected. Heart failure significantly contributes to the morbidity and death of those with DMD, a severe genetic condition marked by progressive muscle weakness.
| Name | Comments | Related Products and Services |
| C2C12 cell line | Used to evaluate the gRNA and SaCas9-containing plasmids' effectiveness in gene editing. | Cas9 Stable Cell Line - C2C12 Gene Knockdown Stable Cell Lines Gene Knockout Stable Cell Lines Gene Overexpression Stable Cell Lines |
| AD293 cell line | Used in the creation of rAAV (recombinant adeno-associated virus) vectors. | |
| rAAVrh.74 | Recombinant AAV serotype rh.74 is utilized as a vector to introduce genetic material into target cells for gene editings, such as gRNA and SaCas9. | AAV Rh74-CAG-GFP CMV-GFP AAV (Serotype BR1) Adeno-associated Virus (AAV) Guide Recombinase AAV Particles |
| pHelper | The adenoviral helper activities required for AAV packing and replication during rAAV generation in AD293 cells are encoded by this plasmid. | |
| pXR (rh.74 or other serotypes) | Contains the genetic code necessary to package the rAAV genome during the creation of the viral vector. This code is found in the AAV capsid protein of the appropriate serotype (such as rh.74 or others). | |
| pX601-CMV-SaCas9-mPA-U6-gRNA | The primary vector that the CMV promoter controls for the cloning and production of the SaCas9 nuclease and guide RNA (gRNA). This plasmid acts as the foundation for building the gene editing system. | pX601-mCherry pX601 |
| pX601 plasmid | Used to help determine the viral titer precisely by creating a standard curve for the quantification of rAAV particles during titering investigations. | |
| pX601-i23 pX601-i20 | These plasmids have particular gRNA sequences that target dystrophin introns 23 (i23) and 20 (i20), respectively. For expression, they are ligated into the pX601 vector. |
a. gRNA selection: For SaCas9, select two gRNAs that target dystrophin introns 20 and 23 (i20 and i23). i20: 5'-GGGCGTTGAAATTCTCATTACCAGAGT and i23: 5'-CACCGATGAGAGGGAAAGGTCCCTGAAT are the sequences in question. (Note: PAM sequences have italics and underlining.)
b. gRNA Oligo PCR Annealing: The gRNA oligos should be annealed in 1x annealing buffer using the following PCR parameters: The temperature was 95 °C for 10 minutes, 90 cycles from 95 to 59 °C with a 20-second 0.4 °C drop each cycle, 90 cycles from 59 to 32 °C with a 20-second 0.3 °C drop per cycle, and 20 cycles from 32 to 26 °C with a 20-second 0.3 °C drop per cycle.
c. Ligation of gRNA Oligos into Vector: Using BsaI and T4 DNA ligase in a single procedure, ligate the annealed gRNA oligos into the pX601-CMV-SaCas9-mPA-U6-gRNA. Reaction conditions: 5 cycles of 37 °C for 5 minutes and 16 °C for 10 minutes; 20 °C for 37 minutes; and 80 °C for 5 minutes.
d. Modification and Inspection: Transform the ligation product into competent cells, then cultivate the cells by plating them on an agar plate containing ampicillin. PCR is used to screen colonies.
e. Positive clone culture: select colonies and cultivate in LB medium supplemented with ampicillin.
f. PCR Screening: Use particular primers to screen positive clones by PCR.
g. Culture Expansion: Set up a positive clone culture and let it grow overnight.
h. Plasmid Purification and Verification: Use Sanger sequencing to confirm the purity of the plasmid DNA.
i. Cell Culture: Grow C2C12 cells to a confluency of about 70%.
j. Electroporation: In C2C12 cells, electroporate a mixture of the SaCas9/gRNA plasmid.
k. DNA Extraction: After 48 hours, harvest the cells and separate the genomic DNA.
l. PCR Analysis: Use mouse dystrophin locus genomic DNA for PCR analysis.
a. Cell Culture: Seed AD293 cells and maintained until ~90% confluency.
b. Transfection: PEI transfect AD293 cells using diluted plasmids.
c. Virus Collection: Gather cell pellets and medium after 72 hours.
d. Lysis and Storage: After lysing and storing lysates, resuspend the cell pellets.
e. Precipitation and Purification: Filter culture media, add PEG8000, incubate, and centrifuge. Resuspend pellets.
a. Gradient Centrifugation: Centrifuge the crude rAAV preparation onto an iodixanol gradient, then gather the fractions.
b. Dilution and Concentration: Concentrate after diluting the rAAV particles and centrifuging them with a filter device.
a. Extract genomic DNA from concentrated rAAV by means of DNA extraction.
b. Standard Curve: To quantify rAAV, create a standard curve.
c. Quantitative real-time PCR analysis: Use this method to determine the rAAV titer.
a. Injection: Give newborn mice systemic delivery of virus particles.
a. Allow the mice to heal themselves.
b. Tissue Collection: After ten weeks, euthanize the mice in order to harvest tissue.
a. Genomic DNA PCR: Extract and examine genomic DNA from biological materials.
b. RT-PCR Analysis: This method involves RNA extraction, reverse transcription, and dystrophin expression analysis.
c. qPCR Analysis: Use quantitative RT-PCR to estimate the effectiveness of gene editing.
d. Dystrophin expression staining using immunofluorescence staining: fix tissues, stain, and image.
a. Forecast: Estimate probable off-target locations.
b. Amplify genomic areas spanning putative off-target sites using PCR amplification.
c. Digestion and Analysis: Use the T7E1 enzyme to digest the products, then analyze them using electrophoresis.
Figure 1. Immunofluorescence staining of heart sections from mdx/utrophin+/- mice 10 weeks post-AAV-SaCas9/gRNAs treatment. Representative images depict dystrophin (red) and caveolin-3 (green) expression in WT, mdx/utrophin+/-, and treated mdx/utrophin+/- mice. Nuclei are labeled with DAPI (blue). Scale bar: 100 µm. (Xu, L., et al., 2018)
Using rAAVrh.74-SaCas9/gRNA vectors, this approach offers a thorough manual for postnatal cardiac gene editing in mdx/utrophin+/-mice. It covers the procedures for designing gRNA, creating vectors, producing rAAV, injecting mice, and analyzing the results of gene editing. This strategy may be used to create cutting-edge treatments for DMD and other hereditary conditions that impact heart function.
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