This protocol allows the development of muscle cell knock-out in any gene, in order to further study the function of this gene in muscle. The advantage is that it's cheap and faster than the development of knock-out animals to study any gene. This technique can be used, for example, to study the involvement of a newly identified gene in a myopathy.
If the technique described here is applied to iPS cells it can lead to the development of knock-out cells for any gene in any type of cells. To begin clustered regularly interspaced short palindromic repeats or CRISPR-mediated gene deletion, first use genome browser tools like ensembl. org to identify the target gene along with the DNA sequences on both sides of it.
To get the list of the gene exons, first click on the transcript of the protein coding sequence and then on Exons. Next, click on Download sequence and select only Genomic sequence to download the full consensus sequence of the whole gene. Scroll the list of the exons and introns of the gene to select the targeted ones.
For designing guide RNA, first select nucleotide sequences of the introns immediately upstream and downstream of the target sequence. Then go to a website such as crispr.tefor. net and use the selected sequences to design two guide RNAs, each 20 nucleotides long without the Protospacer Adjacent Motif separated by a few hundred base pairs.
Next determine the reverse complement sequence for each guide RNA and order the primers. To construct guide RNA cassettes for plasmid cloning, first run a PCR with one set of primers using the recipe and program described in the text, then separate the PCR products in 1%agarose gel using Tris-borate-EDTA buffer. Next excise the 300-base pair fragment and purify using a kit.
Similarly, after running another PCR with the other primer set, purify a 400-base pair long DNA fragment into 20 microliters of elution buffer. To insert the guide RNA cassettes into a lentiviral plasmid backbone, first set up a double digestion reaction of the plasmid with the restriction enzymes Xma 1 and Blp 1 as described in the text. After incubating the reaction tube at 37 degrees Celsius for one hour, incubate it at 65 degrees Celsius for 20 minutes, then run the digestion product in a 1%agarose gel and purify the plasmid of about 10 kilobase pairs using an appropriate kit.
Also quantify the purified product by measuring the optical density. To ligate the guide RNA cassette with the plasmid, prepare a reaction mix with the purified guide RNA, the linearized plasmid DNA, enzyme, and water to a final volume of 10 microliters, then incubate the reaction tube at 50 degrees Celsius for 15 minutes to generate the final plasmid, called pGuide. For lentivirus production, first seed a 145-centimeter plate with 1 million HEK 293 cells in DMEM with high glucose and pyruvate supplemented with 10%FBS and 1%penicillin or streptomycin.
Then grow the cells at 37 degrees Celsius for three days in a 5%carbon dioxide incubator. On the fourth day check the cells to ensure 60 to 65%confluency. Next prepare the transfection solution consisting of the plasmid of interest, the plasmid encoding the virus envelope, the lentiviral packaging plasmid psPAX2, and calcium phosphate.
Adjust the final volume to 1000 microliters with water. Then add the transfection solution dropwise to one milliliter of 2X HBS under agitation and incubate at room temperature for at least 10 minutes. After that, add the solution dropwise to the cells.
For a homogenous distribution of the transfection solution, gently tilt the plate in all directions, then incubate the cells at 37 degrees Celsius in a 5%carbon dioxide incubator for at least five hours. Next, remove the media from the plates and wash the cells with PBS to get rid of the transfection reagents, then add 12 milliliters of fresh medium. After 48 hours of incubation at 37 degrees Celsius, pool the media from all the plates into a tube.
Put the tube in a sealed bucket and centrifuge at 800 times G for five minutes at four degrees Celsius, then filter the supernatant using a 0.45-micrometer filter and centrifuge the filtrate at 68, 300 times G for two hours at four degrees Celsius, using a swinging bucket rotor. After removing the supernatant, keep the tubes upside down on a paper towel in a safety cabinet for five to 10 minutes for maximum removal of liquid from the pellets. Add 100 microliters of HEC proliferation medium and keep the tubes at four degrees Celsius for at least two hours.
Then resuspend the pellet by pipetting. After collecting all the resuspended pellets, make aliquots of 10 or 25 microliters, then store the aliquots at minus 80 degrees Celsius after snap freezing in liquid hydrogen. For myoblast transduction, first seed each well of a 96-well plate with 100 microliters of proliferation medium containing 10, 000 myoblast cells.
The next day, add precalculated volumes of LV guides and LV Cas9 to the cells in a safety cabinet. After five days of incubation, release the cells from the well by adding trypsin. After counting the cell numbers, transfer the cells from wells with 40 to 50%confluency to a new plate.
After five hours of incubation, add calculated volumes of LV killer at a multiplicity of infection of 20 and incubate for five to 10 days or at least two passages in between. For functional characterization of the gene-edited clones by calcium imaging, plate 50, 000 cells in the center of a laminin-coated 35-millimeter dish. To induce differentiation, add the differentiation medium as described in the text.
After six days of incubation, take off the culture medium and delineate the cells with a hydrophobic pen. Then add 50 microliters of Fluo-4 Direct, diluted one-to-one in differentiation medium, to the differentiated myotubes. After incubating the dishes at 37 degrees Celsius for 30 minutes, wash the cells twice with Krebs buffer supplemented with one milligram per milliliter of glucose.
Next use an inverted fluorescence microscope or a confocal microscope with a 10X objective to measure the fluorescence variations at a rate of one frame per second for 90 seconds and choose a field with at least 10 myotubes. After removing the additional Krebs buffer, stimulate the cells with two milliliters of potassium chloride for membrane depolarization or two milliliters of florochloral metacresol or 4CmC or RyR1 direct stimulation. Visualize the fluorescence variation after stimulation, then quantify the fluorescence variation in each myotube.
This protocol could be successfully used to knockout the gene RyR1 from human myoblasts, which resulted in shorter genomic DNA in the cells. RyR1 knock-out was also confirmed at the protein level as the RyR1 protein could not be detected in the targeted cells by Western blotting. The absence of RyR1 activity in myotubes differentiated from the RyR1 knock-out cells was further verified by Fluo-4 calcium imaging after direct RyR1 stimulation by 4CmC or indirect stimulation by potassium chloride-induced membrane depolarization.
The sequence that will be deleted should be required to the prediction of a functional protein. This technique is ideal for the development of post-genomic tools to study the involvement of new gene in muscle disease.
