This work was funded by grants from Association Fran&#231;aise contre les myopathies (AFM-T&#233;l&#233;thon) and from Auvergne-Rh&#244;ne Alpes R&#233;gion (AURA).

Muscle biopsies were obtained from the Bank of Tissues for Research (Myobank, a partner in the EU network EuroBioBank, Paris, France) in accordance with European recommendations and French legislation. Written informed consent was obtained from all individuals. Immortalized myoblasts were kindly produced by Dr. V. Mouly (Myology Institute, Paris, France), and the protocols were approved by Myology Institute ethic committee (MESRI, n AC-2019-3502).
1. CRISPR guide design
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A major step on the way to the characterization of genes of unknown function involved in pathologies is the development of relevant cellular models to study the function of these genes. The use of gene editing using CRISPR/Cas9 is an exponentially growing field of research, and the development of knock-out models as presented here is among its most widely used applications. In this context, we propose here a versatile protocol to develop a human cell line knock-out in any gene of interest, allowing the characterization o...

With the progress of gene sequencing and the identification of mutations in genes of unknown functions in a specific tissue, the development of relevant cellular models to understand the function of a new target gene and confirm its involvement in the related pathophysiological mechanisms constitutes an essential tool. In addition, these models are of major importance for future therapeutic developments1,2, and constitute an interesting alternative to the development of knock-out animal models in straight line with the international recommendations for reduction in the use of animals in experimentation. Gene editing using CRISPR/Cas9 is among the most powerful tools currently available, which has allowed the development of many knock-out/knock-in models, and targeted gene validation using CRISPR/Cas9 is among the most widely used application of CRISPR/Cas93. The success of gene editing relies on the ability to introduce the CRISPR tools (the guide RNAs and the nuclease Cas9) in the target cell model, which can be a challenge in many difficult-to-transfect cells, such as muscle cells4. This challenge can be overcome with the use of virus, usually lentivirus, which has the great advantage to transduce efficiently many cell types and deliver its transgene. But its major drawback is the integration of the transgene in the host cell genome, leading to potential alteration of genes localized at the integration site and to the permanent expression of the transgene, which in the case of the nuclease Cas9 would result in damaging consequences5. A smart solution has been proposed by Merienne and colleagues6, which consists of introduction into the cells of a guide-RNA targeting the Cas9 gene itself, leading to Cas9 inactivation. An adaptation of this strategy is presented here as a user friendly and versatile protocol allowing to knock-out virtually any gene in difficult-to-transfect cells.
The goal of the protocol presented here is to induce the inactivation of a gene of interest in immortalized muscle cells. It can be used to knock-out any gene of interest, in different types of immortalized cells. The protocol described here contains steps to design the guide RNAs and their cloning into lentiviral plasmids, to produce the CRISPR tools in lentiviral vectors, to transduce the cells with the different lentiviruses, and to clone the cells to produce a homogenous edited cell line.
Using this protocol, immortalized human skeletal muscle cells have been developed with deletion of the type 1 ryanodine receptor (RyR1), an essential calcium channel involved in intracellular calcium release and muscle contraction7. The knock-out (KO) of the gene has been confirmed at the protein level using Western blot, and at the functional level using calcium imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-CACNA1S antibody</td>
			<td>Sigma-Aldrich</td>
			<td>HPA048892</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Blp I</td>
			<td>NE BioLabs</td>
			<td>R0585S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>CalPhos Mammalian Transfection Kit</td>
			<td>Takara</td>
			<td>631312&#160;</td>
			<td>Transfection kit</td>
		</tr>
		<tr>
			<td>Easy blot anti Mouse IgG</td>
			<td>GeneTex</td>
			<td>GTX221667-01</td>
			<td>HRP secondary antibody</td>
		</tr>
		<tr>
			<td>Easy blot anti Rabbit IgG</td>
			<td>GeneTex</td>
			<td>GTX221666</td>
			<td>HRP secondary antibody</td>
		</tr>
		<tr>
			<td>Fluo-4 direct</td>
			<td>Molecular Probes</td>
			<td>F10472</td>
			<td>Calcium imaging</td>
		</tr>
		<tr>
			<td>GAPDH(14C10) Rabbit mAb&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>#2118</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>Fermentas</td>
			<td>ER0501</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>InFusion HD Precision Plus</td>
			<td>Takara</td>
			<td>638920</td>
			<td>Ligation kit</td>
		</tr>
		<tr>
			<td>MasterMix Phusion High Fidelity with GC</td>
			<td>ThermoFisher Scientific</td>
			<td>F532L</td>
			<td>Mix for PCR reaction with High fidelity Taq polymerase and dNTPs</td>
		</tr>
		<tr>
			<td>Myosin Heavy Chain antibody</td>
			<td>DHSB</td>
			<td>MF20</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi EF</td>
			<td>Macherey-Nagel</td>
			<td>REF&#160;740424</td>
			<td>Maxipreparation kit for purification of plasmids</td>
		</tr>
		<tr>
			<td>NucleoSpin Gel and PCR Clean-up</td>
			<td>Macherey-Nagel</td>
			<td>740609</td>
			<td>DNA purification</td>
		</tr>
		<tr>
			<td>NucleoSpin Tissue</td>
			<td>Macherey-Nagel</td>
			<td>740952</td>
			<td>Kit for DNA extraction from cell</td>
		</tr>
		<tr>
			<td>One Shot Stbl3 Chemically Competent&#160;E. coli</td>
			<td>ThermoFisher Scientific</td>
			<td>C737303</td>
			<td>Chemically competent cells</td>
		</tr>
		<tr>
			<td>Plasmid #87904</td>
			<td>Addgene</td>
			<td>87904</td>
			<td>Lentiviral plasmid encoding the SpCas9 (for LV-Cas9)</td>
		</tr>
		<tr>
			<td>Plasmid #87919</td>
			<td>Addgene</td>
			<td>87919</td>
			<td>Lentiviral backbone for insertion of cassette with guides (for LV-guide-target)</td>
		</tr>
		<tr>
			<td>Plasmid #12260</td>
			<td>Addgene</td>
			<td>12260</td>
			<td>Lentiviral plasmid encoding lentiviral packaging GAG POL</td>
		</tr>
		<tr>
			<td>Plasmid #8454</td>
			<td>Addgene</td>
			<td>8454</td>
			<td>Lentiviral plasmid encoding envelope protein for producing lentiviral and MuLV retroviral particles</td>
		</tr>
		<tr>
			<td>V5 Tag Monoclonal Antibody</td>
			<td>Invitrogene</td>
			<td>R96025&#160;</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>XL10-Gold Ultracompetent Cells</td>
			<td>Agilent</td>
			<td>200317</td>
			<td>Chemically competent cells</td>
		</tr>
		<tr>
			<td>Xma I</td>
			<td>NE BioLabs</td>
			<td>R0180S</td>
			<td>Restriction enzyme</td>
		</tr>
	</tbody>
</table>

development of knock-out muscle cell lines using lentivirus-mediated crispr/cas9 gene editing

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

This protocol was applied to immortalized myoblasts from a healthy subject15 (so-called HM cells, for human myoblasts), in which the RyR1 has been previously characterized16, in order to knock out the RYR1 gene encoding the RyR1 protein. The design of the guides RNA was made to delete the sequence encompassing part of exon 101 and intron 101 of the gene. Deletion of part of exon 101 is foreseen to result in disruption of the reading frame. In addition, exon 101 enc...

Watch this Scientific Journal Video about Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing at JoVE.com

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, ...

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.

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3.9K 조회수.  University Grenoble Alpes, INSERM, U1216, CHU Grenoble Alpes, Grenoble Institut Neurosciences. 이 프로토콜은 근육에서이 유전자의 기능을 더 연구하기 위해 모든 유전자에서 근육 세포 녹아웃의 개발을 허용합니다. 장점은 어떤 유전자를 연구하기 위해 녹아웃 동물의 개발보다 저렴하고 빠르다는 것입니다. 이 기술은 예를 들어, 근병증에서 새로 확인된 유전자의 관여를 연구하는데 사용될 수 있다.여기에 기술된 기술이 iPS 세포에 적용된다면 임의의 유형의 세?...