Ethics statement
The work carried out in this project involved the manipulation of human cells obtained from the MRC Centre for Neuromuscular Diseases BioBank. Primary myoblasts used for this work were obtained from a DMD patient carrying an in-frame duplication of DMD exons 3–16. Clinically, this patient performs within the better end of the DMD spectrum. The disease manifested when the patient was 3 years old with global developmental delay; later on he was diagnosed with autistic spectrum disorder and aggressive behaviour. Since the age of 5, the patient has been taking steroids. He is currently 13 and still ambulant. The patient has a positive Gowers’ sign (+ 4 s), with only partial antigravity power in hip and shoulder proximal muscles.
Ethical approval and consent for research have been obtained to facilitate pharmacological, gene and cell therapy trials in neuromuscular disorders (NRES Committee London- West London & GTAC, REC reference number 06/Q0406/33) and to allow the use of cells as a model system to study pathogenesis and therapeutic strategies for neuromuscular disorders (NRES Committee London—Stanmore, REC reference 13/LO/1826), in compliance with national guidelines regarding the use of human-derived cell lines for research. Written informed consent was obtained from the patient’s guardians.
sgRNA design and cloning in integrating lentiviral CRISPR/Cas9 expression vectors
The full intron 9 sequence of the human DMD gene was retrieved through the Ensemble genome browser (www.ensembl.org/). We selected genomic regions spanning from 176 to 225 bp throughout DMD intron 9, and run them through the algorithm developed by Feng Zhang laboratory at MIT (http://crispr.mit.edu/). For each genomic target, we chose the returned sgRNAs with the highest quality score (inversely correlated to the number of their potential off-target effects). Details about each genomic sequence and its genomic location are provided in Supplementary Table 1 together with the sequence of sgRNAs 1–4. sgRNAs were cloned into the integrating LentiCRISPRv1 plasmid (Addgene #49535) from Feng Zhang’s laboratory. The best sgRNA (sgRNA2) was also cloned into the integrating pL-CRISPR.EFS.GFP plasmid from Benjamin Ebert’s laboratory (Addgene #57818), following the specified protocol.
Lipofectamine transfection of HEK 293T cells
HEK293T cells were plated in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco) supplemented with 10% foetal bovine serum (FBS) (Life Technology). Plasmid transfection occurred using Lipofectamine 2000 (LF2000), at a ratio of 1 μg DNA to 2 μl LF2000. After 24 h, transfection medium was replaced with fresh culture medium and cells were kept in culture at 37 °C and 5% CO2 for further 24 h.
Culture and differentiation of patient-derived myoblasts
Experiments were performed in muscle-derived primary DUPmyo myoblasts and DUPmyo-i cell line. DUPmyo-i is the result of DUPmyo myoblast immortalization, carried out by Dr. Vincent Mouly24. Primary and immortalized myoblasts were cultured in complete Skeletal Muscle Growth medium (PromoCell) containing 3 mM GlutaMax (Invitrogen), 10% (v/v) FBS and 40 μg/ml Gentamicin (Sigma). Cells were maintained at 37 °C and 5% CO2. Terminal differentiation of human myoblasts was achieved by culturing cells for 5–7 days with a confluence of 70% with DMEM (MegaCell), 2% (v/v) FBS, 1X non-essential amino-acids, 2 mM Glutamine, 0.5 mM, β-mercaptoethanol and 5 ng/ml basic fibroblast growth factor (bFGF).
Desmin immunostaining and myogenicity assay
Terminally differentiated cells were fixed with 4% paraformaldehyde and permeabilized with Triton-X. After two phosphate-buffered saline (PBS) washes, 250 μl 10% goat serum was added and incubated for 30 min at room temperature. Desmin primary antibody (Dako, mouse anti-human) was diluted 1:100 in 10% goat serum and PBS. This was added to the cells and incubated for 60 min at room temperature. DAPI (Thermo Fisher Scientific) was also added (1:10,000) and incubated together with desmin antibody to stain the nuclei in each cell. Following three PBS washes, cells were incubated with the secondary antibody (Invitrogen, goat anti-mouse conjugated with Alexa488) prepared in PBS (1:100 dilution). Myogenicity was assessed by microscope analysis by measuring the ratio between the total number of desmin-positive cells with more than 3 nuclei, and the total number of DAPI stained nuclei.
Production of lentiviral particles expressing functional CRISPR/Cas9
Lentiviral particles expressing pL-CRISPR.EFS.GFP plasmid were generated by transfecting HEK293T cells by means of FuGENE6 transfection reagent (Promega), using a DNA:FuGENE6 ratio of 1:3. Transfected plasmids included pL-CRISPR.EFS.GFP (16 μg/plate), psPax2 plasmid (Addgene #12260, 12 μg/plate) and pCMV-VSVg plasmid (Addgene #8454, 4 μg/plate). Transfection complexes were prepared according to the manufacturer’s instructions and added dropwise to the HEK293T cells. Transfected cells were incubated at 37 °C and 5% CO2 to allow the assembly and release of lentiviral particles into the culture medium. At days 3 or 4 post-transfection, cell culture medium was collected and filtered through a 40 μm filter (Corning) to remove cells and debris. Filtered media, containing lentiviral particles, was placed in 25 × 83 mm polyallomer centrifuge tubes (Beckman Coulter) and centrifuged for 2 h at 60,000×g at 4 °C in a Sorvall Discovery 90SE centrifuge. Following ultracentrifugation, viral particles were resuspended in Opti-MEM (Thermo Fisher Scientific), aliquoted and stored at -80 °C.
Primary myoblasts transduction
Lentiviral particles (MOIs 0.4, 1.6, 6.4, 25.6) were added to the cells culture medium, rocking the plate to ensure homogeneous distribution. Cells were then incubated overnight at 37 °C and 5% CO2. The following day, cells were washed using PBS and cultured for 48 h in complete Skeletal Muscle Growth medium (PromoCell) containing 3 mM GlutaMax (Invitrogen), 10% (v/v) FBS (Life Technology) and 40 μg/ml Gentamicin (Sigma).
Primary myoblasts transfection
Transfection of 2 × 105 DUPmyo cells/well of a 6-well plate (10 × 103 cells/cm2) was performed in serum-free Opti-MEM (Thermo Fisher Scientific) as indicated in manufactures’ instructions. pCMV-GFP plasmid (Addgene #11153) from Connie Cepko’s laboratory was used to test transfection methods. 2.5 μg of plasmid DNA was transfected in combination with different amounts of Lipofectamine 2000 (ThermoFisher Scientific) (6 μl, 9 μl, 12 μl and 15 μl). TurboFect transfection (ThermoFisher Scientific) was done with 2 μg of plasmid DNA mixed with different volumes of transfection reagent (4 μl, 6 μl and 8 μl). 2 μg DNA were also used to transfect cells with 6 μl of GeneJuice reagent (Sigma-Aldrich). After transfection, cells were incubated at 37 °C and 5% CO2 for the amount of time specified by each protocol. Finally, the transfection medium was removed and replaced by the complete Skeletal Muscle Growth medium (PromoCell) and transgene expression evaluated after 48 h.
Nuclear electroporation of human myoblasts—NEON
5 × 105 DUPmyo (or DUPmyo-i) myoblasts were electroporated with 1 μg of the highly pure CRISPR/Cas9-GFP plasmids by using the NEON device (Thermo Fisher Scientific) provided by Julie Dumonceaux and Virginie Mariot, as specified by the manufacturer instructions. Number of pulses, duration and voltage intensity were suggested by Dumonceaux and Mariot, as follows: 1 pulse, 20 ms, 1400 V. Electroporated plasmids were U6gRNA-CMVCas9-GFP (Sigma-Aldrich) expressing sgRNA0 and sgRNA2 (named CR0 and CR2).
GFP sorting
Transduced/transfected/electroporated cells were washed with PBS and detached from the plate with trypsin–EDTA (Gibco). Cells were centrifuged at 500×g for 5 min at room temperature and washed with PBS. The pellet was resuspended in PBS and transferred into the appropriate Falcon round-bottom polystyrene FACS tubes (Thermo Fisher Scientific). Samples were then placed on ice and brought to the FACS facility (https://www.ucl.ac.uk/child-health/core-scientific-facilities-centres/flow-cytometry- core-facility), where trained personnel performed the FACS by either FACSAria III, FACSCalibur or MoFlow XDP Cell sorter. Sorted cells were then incubated overnight in complete Skeletal Muscle Growth medium (PromoCell), at 37 °C and 5% CO2.
Evaluation of DNA editing
Genomic DNA was extracted by human cell lines using the DNeasy Blood & Tissue Kit (QIAgen). The genomic region surrounding the CRISPR/Cas9 target sites was amplified by touchdown PCR (tdPCR) performed with the high fidelity Q5 DNA polymerase (New England BioLabs). TdPCR parameters were 2 min at 98 °C, followed by 26 amplification cycles of 10 s at 98 °C, 30 s at 68 °C (− 0.5 °C per cycle) and 30 s at 72 °C each. Other 9 amplification cycles, each of 10 s at 98 °C, 30 s at 56 °C and 30 s at 72 °C were then added to complete the protocol.
DNA fragments were purified with the QIAquick PCR purification Kit (QIAgen). T7E1 endonuclease (New England BioLabs) assay was performed on 200 ng of purified tdPCR product according to the manufacturer’s instructions. T7E1 reaction stopped by the addition of 2 μl of 0.25 M EDTA was run on a Novex TBE gel (Life Technologies) as specified by the manufacturer. The TBE gel was then stained for 15 min with SybrGold (Life Technologies) diluted 10000X in 200 ml TBE, at room temperature on an orbital shaker in the dark. The gel was visualized with the Gel Doc XR + system (Bio-Rad). The efficiency of CRISPR/Cas9 genomic cleavage efficiency was quantified by exporting in Excel format the data from the densitometric band analysis, which was done by using Fiji software (https://imagej.net/Fiji). CRISPR/Cas9 cleavage efficiency, expressed in percentage, was calculated as follows: upper cleaved band area value/ (Upper cleaved band + Full-length band area values) × 100.
Quantification of duplicated and wild-type dystrophin transcript
RNA was extracted from myotubes using the RNeasy Mini kit (QIAgen) and retrotranscribed to cDNA with the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). qPCR was run by a StepOne device (Thermo Fisher Scientific) on 10 ng of cDNA as follows: 95 °C for 3 min, and 40 cycles of 10 s at 95 °C and 1 min at 60 °C. qPCR data were exported in Excel format and analysed using the ∆∆Ct method. To avoid the amplification of multiple dystrophin transcript isoforms, DMD exon 20 was chosen as anchor region to normalize and quantify dystrophin transcript correction, as it is located outside the duplication and before any of the dystrophin promoters throughout DMD gene. The following primers were used:
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Dystrophin_Exons8-9_Forward = 5’-TTGCCAAGGCCACCTAAA-3’,
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Dystrophin_Exons8-9_Reverse = 5’-TCTCTCATATCCCTGTGCTAGA-3’.
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Dystrophin_Exon20_Forward = 5’-TGGATCGAATTCTGCCAGTT-3’.
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Dystrophin_Exon20_Reverse = 5’GCTCCAATTGTTGTAGCTGATTAT-3’.
Protein quantification and electrophoresis
Proteins were extracted from myotubes by replacing culture medium with NHC lysis buffer (4 M urea, 125 mM Tris pH 6.8 and 4% SDS) supplemented with 1X Complete Protease Inhibitor Cocktail Tablets (Roche). Samples were and left on ice for 10 min after which they were collected, boiled for 3 min and centrifuged at 14000×g for 10 min at 4 °C. Protein quantification was done with the Pierce BCA protein assay (Thermo Fisher Scientific). 50 μg of protein samples were run on a precast NuPAGE Novex Tris- Acetate 3–8% gradient gel (Invitrogen) according to manufacturer’s instructions. 10 μl of the HiMark pre-stained protein standard (Life Technology) and the Odyssey One-Color protein molecular weight marker (Licor) were included as a reference molecular weight to analyse the protein of interest. The gel was run on ice at 75 V for 45 min to allow dystrophin to slowly enter the gel and then at 150 V for 2 h and 15 min.
Assessment of dystrophin protein restoration
At completion of the run, the gel was placed in contact with a polyvinylidene difluoride (PVDF) low-fluorescence membrane. Protein transfer was performed at 30 V for 3 h with the electrophoresis apparatus XCell Sure Lock Mini-Cell (Thermo Fisher Scientific). 300 ml of diluted Transfer buffer (270 ml distilled water, 15 ml 20X NuPAGE Transfer buffer and 15 ml methanol) were placed in the inner chamber, while the outer chamber was filled with 600 ml of cold water, both replaced after 1.5 h. The transfer apparatus was placed in ice to avoid overheating. Following blotting, the membrane was blocked with 10% non-fat milk powder (, OXOID) diluted in Tris buffered saline (TBS) for 1 h at room temperature. The PVDF membrane was then incubated overnight at 4 °C with 10 ml 5% non-fat milk diluted in TBS-Tween (TBS-T), which contained the primary rabbit anti-human dystrophin (Abcam, 1:200) and mouse anti-human vinculin (Sigma, 1:100.000) antibodies. Vinculin antibody protein was used to control if the samples to be analysed are differentiated to a similar extent. This antibody recognizes both vinculin and its slightly higher molecular weight isoform meta-vinculin, whose expression increases on skeletal muscle differentiation41. The membrane was then washed three times with TBS-T and incubated with biotinylated secondary antibodies (Abcam) (1:1000) for 1 h at room temperature. Following another three TBS-T washes, the membrane was incubated 1 h at room temperature with streptavidin-HRP (1:5000) (Thermo Fisher Scientific) in order to amplify the signal. The membrane was washed with TBS-T and incubated for 1 min with 2 ml of the Luminata Forte Western HRP substrate (Millipore). Signal was detected placing the membrane in a ChemiDoc Imager and densitometric analysis of detected bands was performed by using the ImageLab software (Bio-Rad). The amount of restored protein was calculated as the ratio between the amount of duplicated and wild-type dystrophin protein.
Microscopy and image capture
The Olympus IX Inverted microscope (Olympus Life Science) and Leica DMR microscope (Leica Microsystem) were used to acquire images. Any image processing was done by Fiji software (https://fiji.sc/).
Statistical analysis
Replicate experiments were expressed as the mean ± the standard error of the mean. Statistical analysis aimed to verify the effect of CRISPR/Cas9 treatment versus untreated controls were performed by means of the GraphPad Prism software. Results were analysed by applying the Mann–Whitney test (comparison between two groups) or Kruskal–Wallis (comparison between three groups or more) as, due the small sample size, data were assumed to be not normally distributed. Statistical significance was set at P-values below 0.05.
Primer design
Primers were designed by using the primer3 on-line tool (primer3.ut.ee/) and synthesized by Sigma-Aldrich. All primers were provided as desalted stocks which were diluted in pure water to a concentration of 100 μM and stored at -20 °C. When needed, they were further diluted in sterile water to a concentration of 10 μM. The list of all primers designed to amplify CRISPR/Cas9 targets is provided in Supplementary Table 2.
