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Protocol

Dharmacon Edit-R Lentiviral sgRNA glycerol stocks

  • Hide Machado 1,2
  • 1 - Horizon Team
  • 2 - Horizon/Perkin Elmer

Aug 10, 2021

Abstract

The Dharmacon™ Edit-R™ Lentiviral sgRNA vector is part of the Edit-R CRISPR-Cas9 system for genome engineering. The purpose is to provide the researcher with the most effective tools to deliver a gene-specific sgRNA and, together with Cas9 expression, allow gene editing in cells. In addition to the Edit-R Lentiviral sgRNA, the Edit-R Lentiviral Gene Engineering platform also requires the Cas9 nuclease, which can be expressed in a Cas9-integrated cell line or from a Cas9 expression vector, such as the Edit-R Lentiviral Cas9 Nuclease or the Edit-R Cas9 Nuclease Expression plasmids.
 


Introduction

In the Edit-R Lentiviral sgRNA vector, the gene-specific CRISPR RNA (crRNA) and the trans-activating CRISPR RNA (tracrRNA) are expressed as a chimeric single guide RNA (sgRNA) under the control of a human U6 promoter. Additionally, expression of the puromycin resistance marker (PuroR) is driven from the mouse CMV promoter and allows for rapid selection of cells with integrated sgRNA or transiently transfected plasmid DNA.
 


Reagents and Equipment

Edit-R Lentiviral sgRNA is available in glycerol stock or lentiviral particles format. If using lentiviral particles, please refer to the Edit-R CRISPR-Cas9 Gene Engineering with Lentiviral Cas9 and sgRNA technical manual for constitutive Cas9 expression or the Edit-R Inducible Lentiviral Cas9 Nuclease Technical Manual for inducible Cas9 expression.

Trans-Lentiviral shRNA packaging kit (Cat #TLP5912 or TLP5917). 

 


Procedure

Antibiotic resistance
Edit-R Lentiviral sgRNA plasmids contain two antibiotic resistance markers (Table 2). 

Plasmid preparation
Culture conditions for individual plasmid preparations For plasmid preparation, grow all Edit-R Lentiviral sgRNA clones at 37 °C in LB broth medium plus 100 µg/mL carbenicillin.

  1.  Upon receiving your glycerol stock(s) containing the sgRNA(s), store at -80 °C until ready to begin.
  2. To prepare plasmid DNA, first thaw your glycerol stock culture and pulse vortex to resuspend any E. coli that may have settled to the bottom of the tube.
  3. Take a 10 µL inoculum from the glycerol stock into 3-5 mL of LB broth medium with 100 µg/mL carbenicillin. Return the glycerol stock(s) to -80 °C.
    If a large culture volume is desired, incubate the 3-5 mL culture for 8 hours at 37 °C with shaking and use as a starter inoculum. Dilute the starter culture 1:500-1:1000 into the larger volume
  4. Incubate at 37 °C for 18-19 hours with vigorous shaking.
  5. Pellet the culture and begin preparation of plasmid DNA. Plasmid DNA can be isolated using a plasmid miniprep kit of your choice.
  6. Edit-R Lentiviral sgRNA plasmid is 7.5 kb. See the vector map (Figure 1)

Due to the tendency of lentiviral vectors to recombine, we recommend keeping the incubation times as short as possible and avoid subculturing. Return to your original glycerol stock for each plasmid preparation. 

Replication of plates
Prepare target plates by dispensing ~ 160 μL of LB broth medium supplemented with 8% glycerol and 100 μg/mL carbenicillin.

Prepare Source Plates

  1. Remove foil seals from the source plates while they are still frozen. This minimizes cross-contamination.
  2. Thaw the source plates with the lid on. Wipe any condensation underneath the lid with a paper wipe soaked in ethanol.

Replicate

  1. Gently place a disposable replicator in the thawed source plate and lightly move the replicator around inside the well to mix the culture. Make sure to scrape the bottom of the plate of the well.
  2. Gently remove the replicator from the source plate and gently place in the target plate and mix in the same manner to transfer cells.
  3. Dispose of the replicator.
  4. Place the lids back on the source plates and target plates.
  5. Repeat steps 1-4 until all plates have been replicated.
  6. Return the source plates to the -80 °C freezer.
  7. Place the inoculated target plates in a 37 °C incubator for 18-19 hours.
  8. Freeze at -80 °C for long-term storage.

Transfection 
If the plasmid is transfected directly into cells for gene knockout, a Cas9 nuclease expression plasmid is also required for co-transfection unless a cell line stably expressing Cas9 nuclease is used. Consult our website for a list of available Edit-R Cas9 Nuclease expression plasmids.

Quantities and volumes should be scaled up according to the number of cells/ well to be transfected (Table 3). This example is for co-transfection of equal amounts of Edit-R Lentiviral sgRNA and an Edit-R Cas9 Nuclease plasmid DNA in 24-well plate format.

  1. In each well, seed ~ 5 × 104 adherent cells or ~ 5 × 105 suspension cells in 0.5 mL of growth medium 24 hours prior to transfection. 
    The recommended confluency for adherent cells on the day of transfection is 70-90%. Suspension cells should be in logarithmic growth phase at the time of transfection. 
  2. Dilute 0.5 µg each of Edit-R Lentiviral sgRNA and Cas9 Nuclease plasmid DNA (1 µg total DNA) in 50 µL of DMEM or other serum-free growth medium.
  3. Gently mix DharmaFECT kb transfection reagent and add 3 µL to the diluted DNA. Mix immediately by pipetting.
    Prepare immediately prior to transfection. We recommend starting with 1 µg of DNA and 3 µL of DharmaFECT kb reagent per well in a 24-well plate (see scale-up Table 3). Subsequent optimization may further increase transfection efficiency depending on the cell line and transgene used.
  4. Incubate 10 minutes at room temperature. Remove medium from wells and replace with 0.45 mL of fresh growth medium.
    The transfection efficiency with DharmaFECT kb™ transfection reagent (Cat #T-2006-01) is equally high in the presence of serum. This is not the case with other transfection reagents. 
  5. Add 50 µL of the DharmaFECT kb reagent/DNA mixture gently to each well.
  6. Gently rock the plate to achieve even distribution of the complexes.
  7. Incubate at 37 °C in a CO2 incubator.
  8. Analyze the cells for gene editing or expected gene knockout phenotype 48-72 hours later, or longer if necessary depending on the experimental design and cell type.

Edit-R Lentiviral sgRNA vectors are not compatible with third generation packaging systems due to the requirement of the expression of tat, which third generation systems do not contain. We recommend the Trans-Lentiviral shRNA packaging system (Cat #TLP5912, #TLP5917) for use with Dharmacon lentiviral vectors.

Packaging lentiviral particles
The Edit-R Lentiviral sgRNA constructs are Tat-dependent; therefore, you must use a packaging system that expresses the tat gene. We recommend the Trans-Lentiviral shRNA packaging kit (Cat #TLP5912 or TLP5917). The Trans-Lentiviral packaging system allows creation of replication-incompetent, HIV-1-based lentiviral particles that can be used to deliver and express your sgRNA of interest in either dividing or non-dividing mammalian cells, and it is based on the trans-lentiviral system developed by Kappes (Kappes and Wu 2001). For protocols and information on packaging Edit-R Lentiviral sgRNA constructs with the Trans-Lentiviral packaging system, please see the product manual available on our website.

Edit-R Lentiviral sgRNA constructs do not express a fluorescent protein reporter. Therefore, after packaging plasmid DNA, we recommend titering the lentiviral particles using a functional titration protocol such as limiting dilution with cell viability assay by crystal violet staining or genomic qPCR assay (Kutner et al. 2009).

Generation of a stable cell line
For generation of a stable cell line expressing Cas9 nuclease and a sgRNA construct, we recommend using the Edit-R Lentiviral Cas9 Nuclease and sgRNA packaged into lentiviral particles format. See the Edit-R CRISPR-Cas9 Gene Engineering with Lentiviral Cas9 and sgRNA technical manual for protocols.


Time Taken

2 hours

Notes and Comments

Important safety
note Please follow the safety guidelines for use and production of vector-based lentiviral particles as set by your institution’s biosafety committee.

  • For glycerol stocks of E. coli containing lentiviral plasmids, BSL1 guidelines should be followed.
  • For handling and use of lentiviral products to produce lentiviral particles, BSL2 or BSL2+ guidelines should be followed. 
  • For handling and use of lentiviral particle products, BSL2 or BSL2+ guidelines should be followed. 

Additional information on the safety features incorporated in the Edit-R Lentiviral sgRNA vector and the Dharmacon™ Trans-Lentiviral™ packaging system can be found on page 3.

Additional safety information
Historically, the greatest safety risk associated with a lentiviral delivery platform stems from the potential generation of recombinant viruses that are capable of autonomous replication. The Edit-R Lentiviral sgRNA minimizes these hazards to the greatest degree by combining a disabled lentiviral genome with the proprietary Trans-Lentiviral packaging process. Starting with the HXB2 clone of HIV-1 (GenBank, Accession #K03455), the lentiviral backbone has been modified to eliminate all but the most essential genetic elements necessary for packaging and integration (such as 5’ LTR, Psi sequences, polypurine tracts, Rev responsive elements and 3’ LTR). The resultant self-inactivating (SIN) vector greatly reduces the probability of producing recombinant particles and limits cellular toxicity often associated with expression of HIV genes. 

Troubleshooting
For help with transfection or transduction of your lentiviral constructs, please email technical support at ts.dharmacon@horizondiscovery.com with the answers to the questions below, your sales order or purchase order number and the catalog number or clone ID of the construct with which you are having trouble.

  1. Are you using direct transfection or transduction into your cell line?
  2. What was the 260/280 ratio of DNA? Over 1.8?
  3. What was the transfection efficiency if you used direct transfection? What transfection reagent was used?
  4. Were positive and negative controls used (such as our PPIB or DNMT3B validated positive control and the validated non-targeting negative control)?
  5. What were the results of the controlled experiments?
  6. How was knockout measured (for example, DNA mismatch detection assay or western blot analysis)?
  7. What is the abundance and the half-life of the protein? Does the protein have many isoforms?
  8. What packaging cell line was used if you are using transduction rather than transfection?
  9. What was your lentiviral titer in your cells?
  10. What was your MOI?
  11. Did you maintain the cells in puromycin selection medium after transfection or transduction?
  12. How much time elapsed from transfection/transduction to puromycin selection?

If transfection into your cell line is unsuccessful, you may need to consider the following list of factors influencing successful transfection.

  1. Concentration and purity of plasmid DNA and nucleic acids—determine the concentration of your DNA using 260 nm absorbance. Avoid cytotoxic effects by using pure preparations of nucleic acids.
  2. Insufficient mixing of transfection reagent or transfection complexes.
  3. Transfection in serum-containing or serum-free medium—our studies indicate that the transfection efficiency with DharmaFECT transfection reagent is equally high in the presence of serum. This is not the case with other transfection reagents.
  4. We do not recommend antibiotics (e.g., pen-strep) in the transfection complexing medium.
  5. Cell history, density, and passage number—it is very important to use healthy cells that are regularly passaged and in growth phase. The highest transfection efficiencies are achieved if cells are plated the day before; however, adequate time should be given to allow the cells to recover from the passaging (generally > 12 hours). Plate cells at a consistent density to minimize experimental variation. If transfection efficiencies are low or reduction occurs over time, thawing a new batch of cells or using cells with a lower passage number may improve the results. 

References

  1. Cong, L., Ran, F.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.
  2. Jinek, M., Chylinski, K., et al. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821.
  3. Kappes, J.C., and Wu, X. (2001). Safety considerations in vector development. Somat. Cell Mol. Genet. 26, 147-58. 
  4. Kappes, J.C., Wu, X., and Wakefield, J.K. (2003). Production of translentiviral vector with predictable safety. Methods Mol. Med. 76, 449-65.
  5. Kutner, R.H., Zhang, X-Y., and Reiser, J. (2009). Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors. Nat. Protoc. 4, 495-505.
  6. Pauwels, K., Gijsbers, R., et al. (2009). State-of-the-art lentiviral vectors for research use: risk assessment and biosafety recommendations. Curr. Gene Ther. 9, 459-474.
  7. Wu, X., Wakefield, J.K., et al. (2000). Development of a novel trans-lentivral vector that affords predictable safety. Mol. Ther. 2, 47-55.
  8. Zufferey, R., Dull, T., et al. (1998). Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J. Virol. 72, 9873-9880.

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