The discovery of gene mutations linked to diseases as a result of advances in gene sequencing has made it necessary to create pertinent cellular models in order to investigate the pathophysiological mechanisms and gene function. CRISPR/Cas9 technology has been extensively employed for targeted gene validation and provides a potent tool for gene editing. But there are obstacles when trying to transfect tough cells, such as muscle cells. In order to overcome these difficulties, this technique uses Cas9 regulation to guarantee accurate gene knockout and lentiviral vectors for effective transduction.

Experimental Materials and Recommended Services

Procedure

Figure 1. Protocol Overview: Steps from in silico design to in vitro cloning and cell selection. (Beaufils, M., et al., 2022)

1. CRISPR Guide Design

1. Using genome browser resources such as ensembl.org or genome.ucsc.edu, identify the target gene area and acquire its genomic sequence coordinates.

2. Get the ensemble's FASTA sequence for exon 101 and the gene. Choose the particular exon after selecting the gene and retrieving its genomic sequence.

3. Using online resources, create two gRNAs that border the deletion area.

- Make that the chosen gRNAs, which each have a 20-nucleotide sequence that does not contain the PAM motif, are spaced a few hundred base pairs apart.

- Arrange the guides so that they eliminate an important exon or sequence while avoiding areas that are only present in one transcript.

- Find each gRNA's reverse complement sequences.

- Order the primers listed in Table 1 for plasmid cloning, maintaining a primer concentration of 10 nM in sterile H2O throughout the procedure.

Figure 2. Guide RNA Localization and Design. (A) Guides for the production of RYR1-KO cell lines were located. While Guide 2 induces a 326 bp loss and frameshift in intron 101, Guide 1 targets the end of exon 101. While Guide mCherry targets the end of mCherry, Guide Killer targets the initiation codon of SpCas9. (B) Localization of the guides' genomic sequences; each 20 bp guide sequence is highlighted and bolded with its corresponding PAM. (Beaufils, M., et al., 2022)

Figure 3. Cloning Procedure for Cassette Production. Diagram illustrating the stages in PCR (A, B, C, and D) that produce guide-containing cassettes for lentivirus backbone plasmid insertion. For every step, PCR programs 1 and 2 are displayed. (Beaufils, M., et al., 2022)

2. Plasmid cloning

1. Obtain plasmids:

- Obtain plasmid A' and plasmid #87904.

2. Cassette construction:

- Run PCR (A) with plasmid A', primer_XmaIF, primer_Guide1R, polymerase mix, and H2O.

- Purify the 300 bp fragment.

- Run PCR (B) with plasmid A', primer_Guide1F, primer_Guide2R, polymerase mix, and H2O.

- Purify the 400 bp fragment.

- Run PCR (C) with plasmid A', primer_Guide2F, primer_BlpIR, polymerase mix, and H2O.

- Purify the 600 bp fragment.

- Run PCR (D) with elution PCR A and B, primer_XmaIF, primer_Guide2R, polymerase mix, and H2O.

- Purify the 700 bp fragment.

- Run final PCR with elution PCR C and D, primer_XmaIF, primer_BlpIR, polymerase mix, and H2O.

- Purify the final cassette (~1300 bp).

1. Acquire plasmids: - Acquire plasmids A' and #87904.

2. Construction of cassettes:

- Use plasmid A', primers_XmaIF and Guide1R, polymerase mix, and H2O to do PCR (A).

- Clean up the 300 bp piece.

- Use plasmid A', primers Guide1F and Guide2R, polymerase mix, and water to run PCR (B).

- Clean up the 400 bp piece.

- Use plasmid A', primers Guide2F and BlpIR, polymerase mix, and H2O to do PCR (C).

- Clean up the 600 bp piece.

- Proceed with PCR (D) using primer_XmaIF, primer_Guide2R, polymerase mix, H2O, and elution PCR A and B.

- Clean up the 700 bp piece.

- Conduct the last PCR using primers_XmaIF, primer_BlpIR, polymerase mix, H2O, and elution PCR C and D.

- Clean the last cassette (around 1300 bps).

3. Putting the lentiviral plasmid into insert:

- Make plasmid A' linear with XmaI and BlpI.

- To create p_guides, linearize a plasmid and ligate gRNA cassette.

- Change E. Coli using p_guides.

- Conduct PCR amplification and mini prep on chosen colonies.

- To verify cassette insertion, run DNA sequencing.

3. Lentivirus Production

1. Prepare and purify all required plasmids using an endotoxin-free maxi-prep kit. Prepare aliquots at 2 µg/µL concentration and store at -20 °C.

2. Preparation of Cells (Day 1)

- Seed 1 x 10⁶ HEK293 cells per 145 cm² plate in 16 mL of medium.

- Incubate cells at 37 °C, 5% CO₂ for 3 days.

3. Transfection of Cells (Day 4)

- Ensure cells reach 60%-65% confluence.

- Prepare transfection solution for each plate containing plasmids, calcium phosphate, and H₂O.

- Incubate at room temperature for at least 10 min, then add to cells.

- Incubate at 37 °C, 5% CO₂ for 5 h.

- After 5 h, replace the medium and incubate for 48 h at 37 °C, 5% CO₂.

4. Viral Particle Collection (Day 6)

Gather the medium from each plate and combine it.

- Centrifuge to get rid of cell fragments.

- Use a 0.45 µm filter to filter the supernatant.

- To pellet virus particles, centrifuge.

- Re-suspend the pool and pellets.

- Store lentivirus at -80 °C after aliquoting it.

5. Repeat stages to generate LV-Killer, LV-Cas9, and LV-guides.

4. Lignovirus Titration

Transform seed cells into plates with or without coverslips.

- Transduce lentivirus-diluted plates.

- Label and fix the cells.

- Labeled cells are counted to estimate transduction efficiency.

- Use the obtained transduction efficiency to calculate the titer.

5. Myoblast Transduction

The immortalized myoblasts are transduced successively with three lentiviruses, maintained at a density below 50% in Ham's F10 medium supplemented with FBS, penicillin/streptomycin, and Ultroser G, and cultured at 37°C, 5% CO₂.

1. Lentivirus Volume Calculation:

- Using the above formula, determine the lentivirus volume needed for MOI 10 for LV-Cas9 and LV-guides and MOI 20 for LV-killer. On HEK cells, the MOI is computed.

2. Transduction and Seeding of Cells:

- Plant 10,000 cells in 100 µL of medium per well of 96-well plates. On day 2, transduce cells using LV-guides and LV-Cas9, then put them back in the incubator. Steer clear of large MOIs (>25) when using LV-Cas9 to avoid excessive cell death.

3. Amplification and LV-Killer Transduction:

- Trypsinize, count cells, and seed in a fresh dish at 40–50% confluency on day 7. Transduce with LV-Killer at MOI 20 five hours later. For five to ten days, maintain low confluency while amplifying cells.

6. Cellular Cloning

Count and trypsinize the cells. Seed 96-well plates with 1 cell per well after diluting to 10 cells/mL in the medium. Observe development and progressively increase every well. This phase could take two to six weeks.

7. Clone Selection

To amplify the changed sequence area, create primers. To identify modified clones, gather cells from each clone, extract genomic DNA, and run PCR. Verify editing through sequencing. Expand chosen clones to allow for more characterization, and freeze aliquots for later use.

8. Characterization of Edited Clones

1. Analysis of Protein Expression:

- Differentiated myotubes only express RYR1.

- Use a Western blot to assess RYR1 expression.

- Verify the removal of the Cas9 and RYR1 proteins.

2. Configuration of the Protein Analysis Experiment:

- In a 35 mm plate, place 200,000 cells in a proliferation medium over a surface coated with laminin.

- After adhesion, spend six days in the differentiation medium.

3. Gel electrophoresis and sample preparation:

- Lyse cells to ascertain the protein content.

- Transfer denatured protein onto an acrylamide gel gradient.

4. Blotting in the West:

- Proteins should be transferred to a membrane and saturated with blocking buffer.

- Use primary antibodies to incubate against myosin heavy chain, GAPDH (loading control), RYR1, DHPR, and V5-tag (for Cas9).

5. Signal Recognition:

- Wash the membrane after incubating with secondary antibodies.

- Measure the amount of protein expressed and find the chemiluminescent signal.

9. Examination of Edited Clones Functionally

1. Calcium Imaging Experimental Configuration:

- Place 50,000 cells on plates coated with laminin, then let them to differentiate for six days.

- For every stimulus, get biological triplicates ready.

2. Calcium Imaging Protocol: - Use fluo 4-direct to load myotubes and rinse them with KREBS buffer.

- Use a confocal or fluorescent microscope to measure fluctuations in fluorescence.

3. Stimulation and Data Analysis: - Use KCl or 4 CmC to stimulate cells, then monitor their fluorescence.

- Calculate the peak calcium release amplitude by quantifying the fluctuation in fluorescence in each myotube.

This protocol offers a thorough method for CRISPR/Cas9-mediated gene deletion in challenging-to-transfect cells, making it possible to create knockout cell lines for the investigation of disease causes and gene function. The successful elimination of the RYR1 gene in human muscle cells has demonstrated the protocol's effectiveness and highlighted its applicability for researching diverse genes in different cell types.