Mar 01, 2024

Public workspace LENTIVIRAL PRODUCTION FOR PCRISPRi DUAL GUIDE mDA NEURON LIBRARY

This protocol is a draft, published without a DOI.
  • 1Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 2Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
  • 4School of Medical Science, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 5Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 6The Centre for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA;
  • 7Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
  • 8The New York Stem Cell Foundation Research Institute, New York, NY, USA
Renuka Ravi Gupta
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Protocol Citation: Renuka Ravi Gupta, Nona Farbehi, Helaine Graziele Santos Vieira, Helen E. King, hendersa, Vikram Khurana, Gist Croft, Robert J Weatheritt, Lorenz Studer, Joseph Powell 2024. LENTIVIRAL PRODUCTION FOR PCRISPRi DUAL GUIDE mDA NEURON LIBRARY. protocols.io https://protocols.io/view/lentiviral-production-for-pcrispri-dual-guide-mda-c9uez6te
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 27, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95846
Keywords: ASAPCRN, Lentiviral Production, CRISPRi machinery, dopaminergic neuron differentiation, Perturb-Seq
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000472
Abstract
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.
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Materials


ABC
MATERIALCOMPANYCATALOG
T25 FlaskCorning430639
15ml polypropylene centrifuge tubesFalcon352096
5ml serological pipettesCorning4487
10ml serological pipettesCorning4488
DNA Low-bind tubes 1.5mlEppendorf022431021
DMEM High glucoseThermoFisher Scientific11965092
FBSBovogen2008A
Lipofectamine 3000 ThermoFisher ScientificL3000015
psPAX2Addgene12260
pCAG-VSV-GAddgene35616
Transfer Plasmid (Plasmid of interest) pCRISPRi dual-guide mDA Neuron LibraryPrepared by Robert Weatherhitt Lab at GarvanNA
Opti-MEM I Reduced Serum MediumThermoFisher Scientific31985062
Lenti-X ConcentratorTakara-Bio631231
  1. Reagent Composition for HEK293T culture media


AB
REAGENTAMOUNT
DMEM (1X)450ml
10% FBS50 ml


  1. Aliquot the media into 50 ml tubes and store it at 4C.
  2. Do not use Pen-Strep in your media for transfection protocol.

Day 0: Seeding HEK 293T
Day 0: Seeding HEK 293T
Seed HEK 293T cells into T25 flasks in 5ml of HEK culture media at a cell seeding density of 1.5x10^6 cells/flask.
Day 1: Transfection OF CRISPRi plasmid pool
Day 1: Transfection OF CRISPRi plasmid pool
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.
In TUBE A, mix the following reagents. Make this volume of reagent per T25 flask.


AB
TUBE AAMOUNT IN ul
Lipofectamine 300015
Opti-MEM235
Total250

In TUBE B, mix the following reagents. Make this volume of reagent per T25 flask.


AB
TUBE BAMOUNT
psPAX24ug
VSV-G2ug
pCRISPRi dual-guide mDA Neuron Library4ug
P300020ul
Opti-MEMX ul
Total250 ul


Transfer tube B contents to tube A, mix gently by pipetting and incubate for 10 mins at room temperature.
Replace the media in the T25 flask with fresh 5 ml of HEK culture media during incubation.
Add the whole mix (~500ul) gently, drop by drop, making sure to cover the entire area of the T25 flask.
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.
Day 2: Media change
Day 2: Media change
After 24 hours, aspirate the media and gently add 5 ml of fresh HEK culture media
Note
Caution: HEK293T cells tend to lift off easily, so be extremely careful while changing the media and add the media on the opposite wall of the flask where the cells are not attached.

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

AB
VOLUME OF CLARIFIED SUPERNATANTVOLUME OF CONCENTRATOR
x = 5 mlx/3 = 1.6 ml

Mix by gentle inversion.
Incubate mixture overnight at 4C.
Day 4: Centrifuge and Resuspension of pellet
Day 4: Centrifuge and Resuspension of pellet
Centrifuge the concentrated virus at 1500g for 45 mins at 4C.
Note
After the centrifugation, an off-white pellet will be visible.

Carefully remove the supernatant without disturbing the pellet. The residual supernatant can be removed with a pipette tip or by brief centrifugation at 1500g.
Gently resuspend the pellet in 1/10th of the original volume (500ul) with DMEM.
Note
The pellet will be sticky at first but will suspend quickly.

Detailed protocol for concentration for the virus can be found in this link: Lenti-X™ Concentrator Protocol-at-a-Glance
Protocol references