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]
This protocol uses a CRISPR RNP complex and nucleofection to precisely knock out genes. LUHMES cells, derived from human fetal mesencephalic tissue, offer a valuable model for studying dopaminergic dysfunction, featuring stimulus-induced dopamine release, relevant electrophysiological traits, and unique 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 a cell confluency of 70–85%. Higher cell densities may lead to lower Nucleofection efficiencies.
SECTION: Nucleofection Protocol
2. Coat a new 6-well plate freshly with poly-L-ornithine and fibronectin to facilitate LUHMES attachment.
SECTION: Nucleofection Protocol
4. Rinse LUHMES cells with 1XPBS, then add   to dissociate the cells, using 4 mL in a T-75 flask, and incubate for 3 minutes.

SECTION: Nucleofection Protocol
5. Spin at 1200 revolutions per minute (rpm) in a centrifuge for 5 minutes, then carefully discard the supernatant
SECTION: Nucleofection Protocol
6. Re-suspend cells with 5 mL of 1X PBS.
SECTION: Nucleofection Protocol
10. Carefully re-suspend each cell pellet in room temperature 4D-Nucleofection Solution + supplement.

 

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
13. Replace the existing media with warmed fresh media, followed by an incubation period of 10 to 15 minutes at room temperature or in an appropriate incubator. Avoid any disturbance to the cells post-nucleofection.
SECTION: Nucleofection Protocol
14. A typical analysis time is 24-hours post-nucleofection.
SECTION: Nucleofection Protocol
9. During centrifugation, prepare RNP complex while setting up Nucleofector 4D. Choose plate layout, program, and cell type (e.g., single cuvette or strip). Employ LUHMES program CA-137.

 

SECTION: Nucleofection Protocol
12. Place the nucleocuvettes in the nucleofector cuvette holder, ensuring proper alignment
SECTION: Nucleofection Protocol
3. Following the coating process, add 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. Following cell counting, aliquot cells into separate 15 mL tubes, adding around 1.3 million cells per cuvette and 500,000 cells per well in a nucleocuvette strip
SECTION: Nucleofection Protocol
8. Centrifuge the samples a second time at a low speed (90xg) for 10 minutes, ensuring minimal cell agitation before nucleofection.
SECTION: Nucleofection Validation
20. Add the Snap cap tube to fridge over night in  4 °C

SECTION: Nucleofection Validation
15. 24 hours after nucleofection, rinse the cells using 1X PBS, apply TE dissociation solution, allow a 3-minute incubation period, transfer to a 15 mL tube, and then centrifuge.
SECTION: Nucleofection Validation
16.  

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

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

SECTION: Nucleofection Validation
19. Follow with a second heat treatment at 95 °C for 3 minutes to further denature DNA

SECTION: Nucleofection Validation
17. Add cell suspension to a 1.5 mL snap cap tube
SECTION: PCR Protocol
22. 
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 25uL 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 #CSA-01178): 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
21. Utilize a nanodrop device to determine the nucleic acid concentration of your bulk sample.