TITLE: CRISPR/Cas9-Mediated Knockdown in LUHMES Cells: Nucleofection and Validation Protocol
AUTHORS: Mallory Wright, William J Buchser, Colin Kremitzki, Serena Elia, Graham Bachman, emanuel gerbi, Jason Waligorski, Nicholas Tu, Lina Mohammed Ali

[DESCRIPTION]
Utilizing a CRISPR RNP complex and nucleofection, this protocol enables precise gene knockout. LUHMES cells, sourced from human fetal mesencephalic tissue, provide a valuable model for investigating dopaminergic dysfunction. They exhibit stimulus-induced dopamine release, pertinent electrophysiological traits, and distinctive dopaminergic markers, validating their phenotypic relevance.

LUHMES Cell Line: (ATCC Catalog Number: CRL-2927)
Nucleofection kit:  P3 Primary Cell 4D-Nucleofector X Kit S Catalog #V4XP-3032

[STEPS]
SECTION: Nucleofection Protocol
1. Maintain cell confluency between 70–85% to optimize Nucleofection efficiencies; optimal results typically occur with cells in the logarithmic growth phase.
SECTION: Nucleofection Protocol
2. Coat a new 6-well plate freshly with poly-L-ornithine (50ug/mL) and fibronectin (2ug/mL) to facilitate LUHMES attachment.
SECTION: Nucleofection Protocol
4. Rinse LUHMES with 5 mL  1XPBS, then add   to dissociate the cells, using4 mL for a T-75 flask, and incubate for 3 min

SECTION: Nucleofection Protocol
5. Centrifuge1200 rpm for 5 min, then carefully discard the supernatant

SECTION: Nucleofection Protocol
6. Re-suspend cells with5 mL of 1X PBS and count cells.

SECTION: Nucleofection Protocol
11. Add cells to the cuvette, ensuring the sample covers the bottom of the cuvette. Gently tap to distribute evenly and avoid bubbles.
SECTION: Nucleofection Protocol
14. Following nucleofection, immediately add an additional500 µL of pre-warmed media to each individual single cuvette or 50 uL to a curvette strip.

Maintain cell stability by avoiding any disturbance for at least15 min at room temperature 2 °Cor within an incubator


SECTION: Nucleofection Protocol
16. A typical analysis time is 24-Hours post-nucleofection.

SECTION: Nucleofection Protocol
9. While centrifuging, prepare the RNP complex as outlined in the table below and configure the Nucleofector 4D accordingly. Select the plate layout (e.g., single cuvette or strip), program CA-137, and specify the cell type.
 


SECTION: Nucleofection Protocol
13. Insert the nucleocuvettes into the nucleofector cuvette slot, ensuring they are properly aligned, then press start.
SECTION: Nucleofection Protocol
3. Add 2 mL of the LUHMES growth media to the 6-well plate and pre-incubate/equilibrate the plates in a humidified incubator set at 37°C with 5% CO2.

SECTION: Nucleofection Protocol
7. After cell counting, distribute the cells into individual 15mL tubes according to the number of samples. 

Add approximately 1 million cells per single cuvette and 500,000 cells per well in a nucleocuvette strip.

SECTION: Nucleofection Protocol
8. Centrifuge the samples a second time at a low speed90 x g for10 min, ensuring minimal cell agitation before nucleofection.

SECTION: Nucleofection Validation
22. Add the Snap cap tube to fridge3 min  in  4 °C

SECTION: Nucleofection Validation
17. 24 hours after nucleofection, rinse the cells using 5 mL 1X PBS, apply TE dissociation solution, allow a3 minincubation period, transfer to a 15 mL tube, and then centrifuge1200 rpm, 5 min 

SECTION: Nucleofection Validation
18.  

Re-suspend the cells in 100 µL 1X lysis buffer, gently triturating approximately 5 times to ensure efficient cell membrane disruption and DNA extraction.
                  
 

SECTION: Nucleofection Validation
20. Add the 1.5 mL snap cap tube to a65 °C heat block for 15 min to disrupt cellular structures, denature proteins, and aid DNA extraction.

SECTION: Nucleofection Validation
21. Follow with a second heat treatment at 95 °C for3 min

SECTION: Nucleofection Validation
19. Add cell suspension to a 1.5 mL snap cap tube
SECTION: PCR Protocol
24. 
Prepare the PCR reaction mix in a sterile microcentrifuge tube by combining the following components:

Template DNA: The DNA you wish to amplify, need ~100 ng of DNA per 25 µL reaction
Forward and Reverse Primers: Short DNA sequences that bind to the start and end of the target DNA region you want to copy.
MyTaq Red Mix (Meridian Life Science Catalog #BIO-25047): A ready-to-use PCR master mix containing DNA polymerase, dNTPs, buffer, and a red dye.
DMSO (Dimethyl Sulfoxide):  Added to enhance PCR specificity and amplification of GC-rich templates

 



SECTION: PCR Protocol
23. Utilize a nanodrop device to determine the nucleic acid concentration of your bulk sample.
SECTION: Nucleofection Protocol
10.  Carefully re-suspend each cell pellet in room temperature 4D-Nucleofection Solution + supplement.
 
 
SECTION: Nucleofection Protocol
12. For each single nucleocuvette, add25 µL of the RNP Complex, or for each well of a strip, add 5 µL of the RNP complex.

SECTION: Nucleofection Protocol
15. Transfer the cells to the previously coated flask or plate containing pre-warmed LUHMES growth media, and incubate.
SECTION: Running an Agarose Gel for DNA Electrophoresis
25. Prepare Agarose Gel: 

- Measure 1 gof agarose. 
- Dissolve in70 mL of 0.5X TBE buffer in a beaker
- Microwave for 75 sor until completely dissolved.
- Add SybrSafe dye at 1 µL for ever10 mLof 0.5X TBE Buffer to the agarose solution for staining the DNA bands.
- Cool for about5 min

SECTION: Running an Agarose Gel for DNA Electrophoresis
26. Pour Gel and Insert Combs:

- Pour the cooled agarose into a gel tray. 
- Insert combs to create wells for loading samples. 
- Remove any bubbles in gel
- Let the gel sit undisturbed for about 30 minto solidify.

SECTION: Running an Agarose Gel for DNA Electrophoresis
27. Prepare Samples: 

-Remove combs 
- Add 5 µL of DNA ladder 100bp (Gold bio)
-Add 5 µL of each DNA sample to the wells. Ensure proper labeling to keep track of the samples.

SECTION: Running an Agarose Gel for DNA Electrophoresis
28. Run the Gel: 

-Connect wires to matching color ports on the power supply (red to red, black to black)
-Set voltage to 170V, 400mA and run gel for 25 min

Monitor gel progress as DNA fragments move through agarose: smaller ones faster, longer ones slower.

SECTION: Running an Agarose Gel for DNA Electrophoresis
29. Once complete:

- Carefully remove the gel from the casting tray and place it in a gel imaging system. 
- Visualize the DNA bands under UV light. 
- The Sybr Safe dye will fluoresce upon binding to DNA, allowing for the visualization of DNA fragments. 

Analyze the gel to identify the presence of contaminants, estimate the size of amplified DNA fragments, and confirm whether they match the expected sizes based on primer design.
SECTION: Running an Agarose Gel for DNA Electrophoresis
30. Last, perform PCR2 to incorporate barcodes and then submit the samples for next-generation sequencing.