TITLE:  LENTIVIRAL PRODUCTION FOR PCRISPRi DUAL GUIDE  mDA NEURON LIBRARY
AUTHORS: Renuka Ravi Gupta, Nona Farbehi, Helaine Graziele Santos Vieira, Helen E. King, hendersa, Vikram Khurana, Gist Croft, Robert J Weatheritt, Lorenz Studer, Joseph Powell

[DESCRIPTION]
This protocol outlines the production of lentiviral supernatant from a CRISPRi Library plasmid. The plasmid is transfected into HEK293T cells and used for CRISPRi perturbation in both human pluripotent stem cells (hPSCs) and hPSC-derived dopaminergic neural cells. To enhance the viral titer, the virus is concentrated using the Lenti-X concentrator from Takara-Bio following collection of the viral supernatant from HEK 293T cells.

[STEPS]
SECTION: Day 0: Seeding HEK 293T
1. Seed HEK 293T cells into T25 flasks in 5ml of HEK culture media at a cell seeding density of 1.5x10^6 cells/flask. 
SECTION: Day 1: Transfection OF CRISPRi plasmid pool
2. After 24 hours, check whether the HEK293T cells are at least 60% confluent and proceed with transfection and bring the Opti-MEM to room temperature.
SECTION: Day 1: Transfection OF CRISPRi plasmid pool
3. In TUBE A, mix the following reagents. Make this volume of reagent per T25 flask. 


 

SECTION: Day 1: Transfection OF CRISPRi plasmid pool
4. In TUBE B, mix the following reagents. Make this volume of reagent per T25 flask. 


 


SECTION: Day 1: Transfection OF CRISPRi plasmid pool
5. Transfer tube B contents to tube A, mix gently by pipetting and incubate for 10 mins at room temperature.
SECTION: Day 1: Transfection OF CRISPRi plasmid pool
6. Replace the media in the T25 flask with fresh 5 ml of HEK culture media during incubation.
SECTION: Day 1: Transfection OF CRISPRi plasmid pool
7. Add the whole mix (~500ul) gently, drop by drop, making sure to cover the entire area of the T25 flask.
SECTION: Day 1: Transfection OF CRISPRi plasmid pool
8. Gently swirl the media in the flask to ensure the proper distribution of the packaging mixture, and incubate the flask in the incubator for 24 hours.
SECTION: Day 2: Media change
9. After 24 hours, aspirate the media and gently add 5 ml of fresh HEK culture media
 

SECTION: Day 2: Media change
10. Return the flask to the incubator.
SECTION: Day 3: Harvesting the supernatant and concentrating the virus
11. Harvest the lentivirus-containing supernatant into 15 ml tubes.
SECTION: Day 3: Harvesting the supernatant and concentrating the virus
12. Centrifuge the supernatant at 500 g for 10 mins. 
SECTION: Day 3: Harvesting the supernatant and concentrating the virus
13. Transfer the clarified supernatant (5 ml) into a sterile 15 ml falcon tube.
SECTION: Day 3: Harvesting the supernatant and concentrating the virus
14. Depending on the volume of the supernatant, combine 1 volume of Lenti-X Concentrator with 3 volumes of clarified supernatant.



 

SECTION: Day 3: Harvesting the supernatant and concentrating the virus
15. Mix by gentle inversion.
SECTION: Day 3: Harvesting the supernatant and concentrating the virus
16. Incubate mixture overnight at 4C.
SECTION: Day 4: Centrifuge and Resuspension of pellet
17. Centrifuge the concentrated virus at 1500g for 45 mins at 4C.
 

SECTION: Day 4: Centrifuge and Resuspension of pellet
18. Carefully remove the supernatant without disturbing the pellet. The residual supernatant can be removed with a pipette tip or by brief centrifugation at 1500g.
SECTION: Day 4: Centrifuge and Resuspension of pellet
19. Gently resuspend the pellet in 1/10th of the original volume (500ul) with DMEM.
 

SECTION: Day 4: Centrifuge and Resuspension of pellet
20. Detailed protocol for concentration for the virus can be found in this link: Lenti-X™ Concentrator Protocol-at-a-Glance