Mar 22, 2024

Public workspaceCRISPR/Cas9-Mediated Knockdown in LUHMES Cells: Nucleofection and Validation Protocol V.1

DOI
dx.doi.org/10.17504/protocols.io.36wgq32d3lk5/v3
  • 1Washington University, Saint Louis. McDonnell Genome Institute (MGI);
  • 2Washington University in St. Louis;
  • 3Washington University School of Medicine;
  • 4Washington University School of Medicine @FIVE;
  • 5McDonnell Genome Institute;
  • 6Washington University: MGI
  • WashU FIVE @ MGI
Mallory Wright
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DOI: dx.doi.org/10.17504/protocols.io.36wgq32d3lk5/v3
Protocol Citation: Mallory Wright, William J Buchser, Colin Kremitzki, Serena Elia, Graham Bachman, Jason Waligorski, Nicholas Tu, Lina Mohammed Ali, Emanuel Gerbi 2024. CRISPR/Cas9-Mediated Knockdown in LUHMES Cells: Nucleofection and Validation Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq32d3lk5/v3Version created by Mallory Wright
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: Working
We use this protocol and it's working
Created: March 21, 2024
Last Modified: March 22, 2024
Protocol Integer ID: 97114
Keywords: LUHMES, Ribonucleoprotein Complex (RNP), Neurodegenerative disease models, Gene knockout, Neuroscience, CRISPR /Cas9, Gel electrophoresis, PCR
Abstract
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
Materials

ReagentAlt-R CRISPR-Cas9 sgRNA,10Integrated DNA Technologies, Inc. (IDT) , Length 100
ReagentP3 Primary Cell 4D-Nucleofector X Kit S LonzaCatalog #V4XP-3032
ReagentAlt-R S.p. Cas9 Nuclease/NickaseIntegrated DNA Technologies, Inc. (IDT)
ReagentCas9 (Nuclear Localized Signaling) purified proteinMacro labs (Berkeley)Catalog #1111
Reagent1XPBS Fisher ScientificCatalog #10-010-023

Lonza Single Nucleocurvette (100uL) Lonza Nucleocurvette strip (25 uL)
Nucleofection solution (P3)82 uL 16.4 uL
Nucleofection supplement (P3)18 uL3.6 uL
Amount of cells~ 1.3 million~500,000 cells
pmaxGFP Vector (0.5ug/uL) (optional positive control)Add 4 uLAdd 1.2 uL

Ribonucleoprotein components(RNP): gRNA + Cas9 + 1XPBS
ComponentsStock ConcentrationLonza Single Nucleocuvette (100uL)Lonza Nucleocuvette strip (20uL)Final Concentration
gRNA (IDT)100 uM6 uL1.2 uL (120 pmol)4.8 μM
Cas9 (IDT)62 uMAdd 8.5 μLAdd 1.7 uL 4.216 μM
1X PBS1XAdd 10.5 uL Add 2.1 uL
Total volume of RNP Complex:25 uL5 uL
For a second Cas9 option, use Cas9 NLS (Berkeley) at 40 μM:
  • Add 10.54 μL to a single nucleocuvette.
  • Add 2.635 μL to a cuvette strip to achieve 4.216 μM.


*By adding 1.7μL of Alt-R Cas9 enzyme from a 62 μM stock, 105.4 pmol is introduced into the RNP mixture. The inclusion of the RNP Complex into the nucleofection solution along with the supplement yields a final concentration of 4.216 uM.

*Adding 8.5 μL from the same stock introduces 527 pmol into the RNP complex. The final Alt-R Cas9 concentration in a 125 μL volume is approximately 4.216 μM.


Protocol materials
Reagenttrypsin-EDTA (TE)Fisher ScientificCatalog #R001100
ReagentLysis Buffer 1X Working solutionTakara Bio Inc.Catalog #635013
Nucleofection Protocol
Nucleofection Protocol
Maintain cell confluency between 70–85% to optimize Nucleofection efficiencies; optimal results typically occur with cells in the logarithmic growth phase.
Coat a new 6-well plate freshly with poly-L-ornithine (50ug/mL) and fibronectin (2ug/mL) to facilitate LUHMES attachment.
Add Amount2 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.

Rinse LUHMES with Amount5 mL 1XPBS, then add Reagenttrypsin-EDTA (TE)Fisher ScientificCatalog #R001100 to dissociate the cells, usingAmount4 mL for a T-75 flask, and incubate for Duration00:03:00

3m
CentrifugeCentrifigation1200 rpm for Duration00:05:00 , then carefully discard the supernatant

5m
Re-suspend cells withAmount5 mL of 1X PBS and count cells.

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.

Centrifuge the samples a second time at a low speedCentrifigation90 x g forDuration00:10:00 , ensuring minimal cell agitation before nucleofection.

10m
Critical
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.



Carefully re-suspend each cell pellet in room temperature 4D-Nucleofection Solution + supplement.
Lonza Single Nucleocuvette (100uL) Lonza Nucleocuvette strip (20uL)
Nucleofection supplement (P3)18 uL3.6 uL
Nucleofection solution (P3)82 uL16.4 uL
pmaxGFP Vector (0.5ug/uL) (optional positive control)Add 4 uL Add 1.2 uL
Add cells to the cuvette, ensuring the sample covers the bottom of the cuvette. Gently tap to distribute evenly and avoid bubbles.
For each single nucleocuvette, addAmount25 µL of the RNP Complex, or for each well of a strip, add Amount5 µL of the RNP complex.

Insert the nucleocuvettes into the nucleofector cuvette slot, ensuring they are properly aligned, then press start.
Following nucleofection, immediately add an additionalAmount500 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 leastDuration00:15:00 at room temperature Temperature2 °C or within an incubator


15m
Critical
Transfer the cells to the previously coated flask or plate containing pre-warmed LUHMES growth media, and incubate.
A typical analysis time is 24-Hours post-nucleofection.

Nucleofection Validation
Nucleofection Validation
24 hours after nucleofection, rinse the cells using 5 mL 1X PBS, apply TE dissociation solution, allow aDuration00:03:00 incubation period, transfer to a 15 mL tube, and then centrifugeCentrifigation1200 rpm, 00:05:00

8m
ReagentLysis Buffer 1X Working solutionTakara Bio Inc.Catalog #635013

Re-suspend the cells in Amount100 µL 1X lysis buffer, gently triturating approximately 5 times to ensure efficient cell membrane disruption and DNA extraction.
Note
Excessive trituration can result in increased shearing of DNA

Add cell suspension to a 1.5 mL snap cap tube
Add the 1.5 mL snap cap tube to aTemperature65 °C heat block for Duration00:15:00 to disrupt cellular structures, denature proteins, and aid DNA extraction.

15m
Follow with a second heat treatment at Temperature95 °C forDuration00:03:00

3m
Add the Snap cap tube to fridgeDurationOvernight in Temperature4 °C

3m
PCR Protocol
PCR Protocol
Utilize a nanodrop device to determine the nucleic acid concentration of your bulk sample.

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 Amount25 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

ComponentsConcentrations120
Forward Primer (100uM)0.5 μM 0.125 μL2.5 μL
Reverse Primer (100uM)0.5 μM0.125 μL2.5 μL
MyTaq (DNA Polymerase)2X12.5250 uL
DMSO5% 1.25 μL per reaction.25 μL
Water (ddH2O)amount depends on the remaining volume after adding the other components9 uL180 uL
Template DNAneed ~100 ng per 25uL reaction2 uL2 uL
Total amount25 uL
PCR1 components



Running an Agarose Gel for DNA Electrophoresis
Running an Agarose Gel for DNA Electrophoresis
35m
35m
Prepare Agarose Gel:

- Measure Amount1 g of agarose.
- Dissolve inAmount70 mL of 0.5X TBE buffer in a beaker
- Microwave for Duration00:01:15 or until completely dissolved.
- Add SybrSafe dye at Amount1 µL for everAmount10 mL of 0.5X TBE Buffer to the agarose solution for staining the DNA bands.
- Cool for aboutDuration00:05:00

6m 15s
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 Duration00:30:00 to solidify.

30m
Prepare Samples:

-Remove combs
- Add Amount5 µL of DNA ladder 100bp (Gold bio)
-Add Amount5 µL of each DNA sample to the wells. Ensure proper labeling to keep track of the samples.

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 Duration00:25:00

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

25m
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.
Last, perform PCR2 to incorporate barcodes and then submit the samples for next-generation sequencing.