The overall goal of this in vivo cardiac gene editing protocol is to restore dystrophin expression in the heart of dystrophic mice using recombinant adeno-associated virus rh. 74 with CRISPR SaCas9 and guide RNA vectors. This method can help answer key questions in the muscular dystrophy field, such as whether gene editing is a reliable therapeutic strategy to treat the root cause of the cardiomyopathy associated with Duchenne's muscular dystrophy.
The main advantage of these techniques are the expression of the corrected dystrophin gene is under its endogenous regulatory control, and this rescue effect is permanent. The implication of this technique extends toward treatment of other genetic disease where correction of mutant gene can be achieved by CRISPR Cas9 gene editing technology. This method not only provides insight into dystrophin restoration in the dystrophic heart, but it can also be used to knock out the gene of interest for reverse genetic studies.
Demonstrating the procedure will be Yandi Gao, a technician from our laboratory. The proper design of the guide RNAs, or gRNAs, is critical to the success of this protocol. Select two gRNAs targeting dystrophin intron 20 and intron 23 for the Staphylococcus aureus CRISPR-associated protein nine.
The protospacer adjacent motif sequence for each gRNA is underlined and italicized. To anneal the gRNA oligonucleotides, set up a reaction containing 100 micromoles per liter of the gRNA oligonucleotides in 1x annealing buffer. Use the following PCR parameters:95 degrees Celsius for 10 minutes, 90 cycles of 95 to 59 degrees Celsius with a 0.4 degree Celsius decrease per cycle for 20 seconds, 90 cycles of 59 to 32 degrees Celsius with a 0.3 degree Celsius decrease per cycle for 20 seconds, and 20 cycles of 32 to 26 degrees Celsius with a 0.3 degree Celsius decrease per cycle for 20 seconds.
Next, prepare to ligate the annealed gRNA oligonucleotides into the CRISP vector in one reaction. Add the following to a tube:one microliter of 50 nanograms per microliter CRISPR vector, one microliter of annealed oligonucleotides, 1.5 microliters of 10x T4 DNA ligase buffer, 0.5 microliters of T4 DNA ligase, one microliter of Bsal, and 10 microliters of double-distilled water. Run the following reaction in a thermocycler:five cycles of 37 degrees Celsius for five minutes and 16 degrees Celsius for 10 minutes, 37 degrees Celsius for 20 minutes, and 80 degrees Celsius for five minutes.
Transform the 15-microliter ligation product into 30 microliters of competent cells, and plate the cells on a agar plate containing 100 microgram per milliliter of ampicillin. Culture at 37 degrees Celsius for 24 hours. On the following day, pick five to 10 colonies from the agar plate and culture in LB medium containing 100 microgram per milliliter of ampicillin at 37 degrees Celsius and 250 rpm for three to four hours.
To screen the colonies by PCR, prepare reactions that each contain 10 microliters of PCR master mix, eight microliters of double-distilled water, one microliter of E.coli culture, one microliter of U6-forward primer, and one microliter of i20 or i23 gRNA reverse primer. Use the following PCR cycling parameters:95 degrees Celsius for five minutes, followed by 35 cycles of 95 degrees Celsius for 30 seconds, 61 degrees Celsius for 30 seconds, and 72 degrees Celsius for 30 seconds. Once the positive clones have been identified, prepare a five-milliliter culture of each positive clone by adding four milliliters of fresh LB medium containing 100 microgram per milliliter ampicillin and culturing at 37 degrees Celsius and 250 rpm overnight.
On the following day, purify the plasmid DNA using the appropriate plasmid extraction kit. The plasmid is subsequently verified by Sanger sequencing with the U6-forward primer. To test the gene editing efficacy of the plasmids, culture C2C12 cells in DMEM supplemented with 10%FBS and 1%Pen-Strep until the cells reach approximately 70%confluency.
Electroporate a total of five micrograms of SaCas9/gRNA plasmid mixture in one-to-one molar ratio into C2C12 cells according to the manufacturer's protocol. 48 hours post-transfection, harvest the C2C12 cells, and extract genomic DNA as described in the text protocol. Use the genomic DNA as template to perform PCR for the mouse dystrophin locus.
Set up reactions containing the forward primer, the reverse primer, the green PCR master mix, double-distilled water, and 100 nanograms of genomic DNA. Use the same PCR conditions as for screening positive clones. Prior to starting this procedure, prepare the recombinant adeno-associated virus, or rAAV, as described in the text protocol.
Administer day three mdx mouse pups with viral particles systemically via an intraperitoneal injection. Allow the mice to recover, and place them back into their home cages. At 10 weeks after rAAV administration, collect tissues from the mice.
Snap-freeze the tissues for genomic DNA and RNA extraction, and use cooled isopentane in liquid nitrogen to freeze tissues with optimal cutting temperature compound for cryosectioning. Begin the immunofluorescence staining protocol by fixing the frozen tissue sections with 4%paraformaldehyde at room temperature for five minutes. After 15 minutes, wash the tissue sections two times with PBS.
Incubate with blocking solution at room temperature for one hour. Next, incubate with the anti-dystrophin primary antibody at four degrees Celsius overnight. On the following morning, wash the slides with PBS, and then incubate with fluorescent secondary antibodies at room temperature for one hour.
Mount the slides using mounting medium with DAPI, and image with an inverted confocal microscope. The efficacy of the gRNAs to induce the deletion of the target genomic DNA region in cell cultures was evaluated by PCR. A PCR product of approximately 500 base pairs indicates successful deletion of the target genomic DNA resulting from gene editing, while the control cells should not yield a band.
Once the efficacy of the gRNAs has been confirmed in cell cultures, the gRNAs are packaged into rAAV. The rAAV viral particles are purified and the purity analyzed by SDS-PAGE. The presence of only three bands corresponding to the three capsid proteins indicates high purity.
These representative immunofluorescence images of heart sections of wild type, mdx, and mdx mice treated with AAV SaCas9 gRNA systemically show that AAV SaCas9 gRNA restores dystrophin expression in dystrophic mice. After watching this video, you should have a good understanding of how to achieve in vivo cardiac gene editings in postnatal mice using rAAV rh. 74-mediated delivery of SaCas9 and gRNAs.
