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Method Article

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

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DOI:

10.3791/56915

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December 12th, 2017

 , 
1Duke Viral Vector Core, Department of Neurobiology, Duke University School of Medicine

In This Article

Summary

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Abstract

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

Introduction

Precise gene editing forms the cornerstone of major biomedical advances that involve the development of novel strategies to tackle genetic diseases. At the forefront of gene-editing technologies is the method relying on the usage of the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas9 system that was initially identified as a component of bacterial immunity against the invasion of viral genetic material (reviewed in references1,2). A major advantage of the CRISPR/Cas9 system over other gene-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (reviewed in reference3), is the relative simplicity of plasmid design and construction of CRISPR components — a feature that has powered the expansion of gene-editing from a few specialized laboratories to a much wider research community. Additionally, the simplicity of CRISPR/Cas9 programming and its capacity for multiplexed target recognition have further fueled its popularity as a cost-effective and easy-to-use technology. Among the various methods available to researchers to deliver such gene-editing components to cells, viral vectors remain by far the most popular and efficient system.

Lentiviral vectors (LVs) have emerged as the vehicle of choice to deliver the components of CRISPR/Cas9 system in vivo for diverse applications4,5,6,7. Several key features make LVs a popular choice for this process including their ability to infect both dividing and non-dividing cells, low immunogenicity, and minimal cellular toxicity (reviewed in reference8). As a result, LV-mediated gene therapy has been employed in treatments of infectious diseases, such as HIV-1, HBV, and HSV-1, as well as in the correction of defects underlying human hereditary diseases, such as cystic fibrosis and neo-vascular macular degeneration4,5,7,9,10,11. Moreover, LVs have been effectively modified to perform multiplex gene editing at distinct genomic loci using a single vector system12.

However, the inherent property of LVs to integrate into the host genome can be mutagenic and often handicaps their utility as transgene delivery vehicles, especially in clinical settings. Moreover, since stably-integrated LVs express their transgenes at sustainably high levels, this system is ill-suited for the delivery of gene-editing components such as CRISPR/Cas9; overexpression of Cas9-guide RNA (gRNA), and similar proteins such as ZFNs, are associated with elevated levels of off-target effects, which include undesirable mutations13,14,15,16,17 and can potentially enhance cytotoxicity18. Therefore, to achieve precise gene-editing with minimal off-target effects, it is imperative to design systems that allow for the transient expression of gene editing components.

In recent years, a variety of delivery platforms have been developed to transiently express CRISPR/Cas9 in cells16,19,20,21 (reviewed in reference22). These include methods that rely on directly introducing purified Cas9 along with the appropriate guide RNAs into cells, which was shown to be more effective at targeted gene-editing in comparison to plasmid-mediated transfection16. Studies have demonstrated that ribonucleoprotein (RNP) complexes consisting of guide RNA/Cas9 particles are rapidly turned over after mediating DNA cleavage at their targets, suggesting that short-term expression of these components is sufficient to achieve robust gene editing16. Conceivably, non-integrating viral vector platforms such as adeno-associated viral vectors (AAVs) can provide a viable alternative to deliver gene-editing machinery to cells. Unfortunately, AAV capsids possess significantly lower packaging capability than LVs (<5kb), which severely impedes their ability to package the multi-component CRISPR toolkit within a single vector (reviewed in reference8). It is worth noting that addition of compounds that inhibit histone deacetylases (e.g., sodium butyrate23) or impede the cell cycle (e.g., caffeine24) have been shown to increase lentiviral titers. Despite the recent progress, the transient expression systems developed so far are still impeded by several shortcomings, such as lower production efficiency, which lead to reduced viral titers, and low transduction efficiency of the viruses generated through such approaches25.

Integrase-deficient lentiviral vectors (IDLVs) represent a major advancement in the development of gene-delivery vehicles, as they combine the packaging capability of LVs with the added benefit of AAV-like episomal maintenance in cells. These features help IDLVs largely circumvent the major issues associated with integrating vectors, vis-à-vis continuous overexpression of potentially genotoxic elements and integration-mediated mutagenicity. It was previously demonstrated that IDLVs can be successfully modified to enhance episomal gene expression26,27. With regards to IDLV-mediated CRISPR/Cas9 delivery, low production titers and lower expression of episome-borne genomes relative to integrase-proficient lentiviral systems limits their utility as bona fide tools for delivering genome-editing transgenic constructs. We recently demonstrated that both transgene expression and viral titers associated with IDLV production are significantly enhanced by the inclusion of binding sites for the transcription factor Sp1 within the viral expression cassette28. The modified IDLVs robustly supported CRISPR-mediated gene editing both in vitro (in HEK-293T cells) and in vivo (in post-mitotic brain neurons), while inducing minimal off-target mutations compared to the corresponding ICLV-mediated systems28. Overall, we developed a novel, compact, all-in-one CRISPR toolkit carried on an IDLV platform and outlined the various advantages of using such a delivery vehicle for enhanced gene editing.

Here, the production protocol of the IDLV-CRISPR/Cas9 system is described, including the various steps involved in the assembly, purification, concentration, and titration of IDLVs, as well as strategies to validate the gene-editing efficacy of these vectors. This protocol is easily scalable to meet the needs of different investigators and is designed to successfully generate LV vectors with titers in the range of 1 x 1010 transducing units (TU)/mL. The vectors generated through this protocol can be utilized to efficiently infect several different cell types, including difficult-to-transduce embryonic stem cells, hematopoietic cells (T-cells and macrophages), and cultured and in vivo-injected neurons. Furthermore, the protocol is equally well-suited for the production of integrase-competent lentiviral vectors in similar quantities.

Gene editing; zinc-binding, catalytic, DNA-binding domains; gel electrophoresis, plasmid diagram.
Figure 1: IDLV packaging. (a) Schematic of the wild type integrase protein (b) The modified plasmid was derived from psPAX2 (see Methods, plasmid construction for details). Representative agarose gel image of clones screened for mutated integrase clones. DNA samples prepared using a standard plasmid DNA isolation mini-kit were analyzed by digestion with EcoRV and SphI. The correctly-digested clone (number 5, dashed red box) was further verified by direct (Sanger) sequencing for the D64E substitution in INT. The integrase-deficient packaging cassette was named pBK43. (c) Schematic of the transient transfection protocol employed to generate IDLV-CRISPR/Cas9 vectors, showing 293T cells transfected with VSV-G, packaging, and transgene cassettes (Sp1-CRISPR/Cas9 all-in-one plasmid). Viral particles that bud out from the cell membrane contain the full-length RNA of the vector (expressed from the transgene cassette). The second generation of the IDLV-packaging system was used, which includes the regulatory proteins Tat and Rev. Rev expression is further supplemented from a separate cassette (RSV-REV-plasmid). Abbrev: LTR-Long-terminal repeat, VSV-G, vesicular stomatitis virus G-protein, pCMV-cytomegalovirus promoter; Rous sarcoma virus (RSV) promoter; RRE- (Rev Response Element). Other regulatory elements on the expression cassette include Sp1-binding sites, Rev Response element (RRE), Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a core-elongation factor 1α promoter (EFS-NC), the vector packaging element ψ (psi), human Cytomegalovirus (hCMV) promoter, and human U6 promoter. Please click here to view a larger version of this figure.

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Protocol

1. Culturing HEK-293T Cells and Seeding Cells for Transfection

NOTE: Human Embryonic Kidney 293T (HEK-293T) cells are grown in DMEM, high glucose media supplemented with 10% bovine calf serum supplemented with iron and growth promoters, and 1x antibiotic-antimycotic solution (100x solution contains 10,000 units penicillin, 10 mg streptomycin and 25 µg amphotericin B per mL). The media is also supplemented with 1x sodium pyruvate, 1x non-essential amino acid mix, and 2 mM L-Glutamine (stock 200 mM L-alanyl-L-glutamine dipeptide in 0.85% NaCl). Cells are cultured in 100 mm tissue culture plates (approximate growth surface area is 55 cm²). A sub-cultivation ratio of 1:10 is used with sub-culturing every 2 - 3 days. Trypsin-EDTA 0.05% is used for dissociation of cells between passages. To maintain consistency between experiments, we recommend testing calf sera when switching to a different lot/batch and monitor any changes in cell growth, transfection efficiency, and vector production.

  1. Start a new culture by using low passage cells (it is recommended to not use cells after passage 15 or if growth slows down) by seeding into a 10-cm tissue culture plate. Use DMEM supplemented with 10% serum for cell growth. Grow cells at 37 °C with 5% CO2 in a standard tissue-culture incubator. Use a standard hemocytometer to count cells for all subsequent steps.
  2. Once the cells reach 90 - 95% confluent growth, reseed into 15 cm tissue culture plates (below, Steps 1.3 - 1.5).
  3. To reseed, aspirate media from the confluent plate and gently rinse with sterile 1x PBS. Incubate cells with 2 mL of a dissociation reagent (e.g., Trypsin-EDTA) at 37 °C for 3 - 5 min. Add 8 mL of media containing serum to inactivate the dissociation reagent, and triturate 10 - 15 times with a 10 mL serological pipette to create a single cell suspension. Resuspend cells in the culture media to obtain a cell density of approximately 4 x 106 cells/mL.
  4. To enhance adherence to the substrate, pre-coat the 15 cm plates with 0.2% gelatin, adding 8 mL of gelatin per plate. Spread evenly across the surface of the plate, incubate at room temperature for 10 min, and siphon off the liquid.
  5. Bring the total volume of each plate to 25 mL with warm (37 °C) HEK-293 T media (see Note under Step 1) and seed the plates by adding 2.5 mL of cells (total ~1 x 107 cells/plate). Incubate plates at 37 °C with 5% CO2 overnight or until 70 - 80% confluency is reached.
    NOTE: Up to six 15 cm plates can be used for production. Adjust volume of media per plate to ~20 - 22 mL when using more than four plates (refer to Step 5 of the protocol for rationale).

2. Transfecting HEK-293T Cells Using a Calcium Phosphate-based Protocol

  1. Transfection reagents
    1. To prepare 2x BES-buffered solution BBS (50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4), combine 16.36 g of NaCl, 10.65 g of BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid), and 0.21 g of Na2HPO4. Add double-distilled H2O (dd-H2O) up to 900 mL. Dissolve, titrate to pH 6.95 with 1M NaOH, and bring volume to 1 L. Filter via 0.22 µM filter unit. Store at -20 °C.
    2. Prepare 1M CaCl2. Filter solution via a 0.22 µM filter. Store at 4 °C.
  2. Observe the plates that were seeded a day prior. Cells are ready for transfection once they reach 70 - 80% confluency.
  3. Aspirate old media from the plates and gently add freshly-prepared media without serum.
    NOTE: See Supplementary File 1-Plasmids for details about the chosen plasmids and their preparation.
  4. Prepare the plasmid mix by aliquoting the four plasmids into a 15-mL conical tube. For a single 15-cm dish preparation, use 37.5 µg of the CRISPR/Cas9-transfer vector (pBK198 or pBK189), 25 µg of pBK43 (psPAX2-D64E), 12.5 µg pMD2.G, and 6.25 µg of pREV (Figure 1c).
  5. Add 312.5 µL 1M CaCl2 to the plasmid mix. To this, add up to 1.25 mL of sterile dd-H20.
  6. Slowly (drop-wise) add 1.25 mL of 2x BBS solution while vortexing the mix. Incubate for 30 min at room temperature.
  7. Add the transfection mixture dropwise to each 15-cm plate (2.5 mL per plate). Swirl the plates gently and incubate at 37 °C with 5% CO2 for 2 - 3 h. Thereafter, add 2.5 mL (10%) serum per plate and continue incubating overnight (12 - 18 h).
    NOTE: Some labs use 3% CO2 incubators to stabilize media pH. However, we do not observe any difference in transfection efficiency between 3% and 5% CO2. Also, the size of CaPO4 precipitates is crucial for transfection efficiency; the transfection mix has to be clear prior to its addition onto the cells. If the mix becomes cloudy during incubation, prepare fresh 2x BBS (pH = 6.95).

Viral purification diagram showing centrifugation, sucrose gradient, and PBS resuspension steps.
Figure 2: Concentration of the viral particles using double-sucrose gradient protocol. Viral particles collected from the supernatant (SN) are loaded onto gradient sucrose gradient. 70%, 60%, 30%, and 20% sucrose solutions are used to create the gradient. Following centrifugation, the particles collected from the 30 - 60% sucrose fractions are further loaded onto a 20% sucrose cushion and precipitated. The final pellet containing purified viral particles is resuspended in 1x PBS for further usage (see text for further details). Please click here to view a larger version of this figure.

3. Day After Transfection

  1. Observe the cells to ensure that they are nearing 100% confluency at this point with little to no cell death. Replace media by adding 25 mL of fresh DMEM + 10% serum to each plate. Continue incubating at 37 °C with 5% CO2 for an additional 48 h.
    NOTE: Adjust volume of media in step 3.1 if using more than six 15 cm plates (see Step 5.2, Note).

4. Harvesting Virus

  1. Carefully collect the supernatant from all the tissue culture plates containing the transfected cells using a sterile 10 mL tissue culture pipette and pool in 50 mL centrifuge tubes.
  2. Clear the suspension by centrifugation at 400 - 450 x g for 10 min using a tabletop centrifuge. Filter the supernatant through a 0.45 µm vacuum filter unit.
    NOTE: The filtered supernatant can be stored at 4 °C for up to 4 days before proceeding with concentration, or aliquoted and stored at -80 °C. The expected titers of IDLV-Sp1-CRISPR/Cas9 (non-concentrated) viral preparations should be ~2 x 107 TU/mL (refer to Step 6 for titer determination). However, avoid subjecting the viral preparations to multiple freeze-thaw cycles, as each round of freezing and thawing results in a 10 - 20% loss in functional titers.

5. Concentration of Viral Particles by Ultracentrifugation

NOTE: We utilize a double-sucrose method of purification that involves two steps: a sucrose gradient step and a sucrose cushion step (Figure 2).

  1. Load the conical ultracentrifugation tubes in the following order to create a sucrose gradient: 0.5 mL 70% sucrose (dissolved 1x PBS), 0.5 mL 60% sucrose (dissolved in DMEM), 1 mL 30% sucrose (dissolved in DMEM), and 2 mL 20% sucrose (dissolved in 1x PBS).
  2. Add the virus-containing supernatant carefully to the gradient. As the total volume of supernatant from four 15 cm plates is 100 mL, use six ultracentrifugation tubes per spin to process the full volume of the virus supernatant.
    1. Each ultracentrifugation tube used for this step has a volume capacity of up to 30 mL (including the volume of sucrose), distribute viral supernatant equally among tubes, leaving at least 10% headspace to prevent spillage.
    2. Adjust the culture volume to ~20 - 22 mL per plate when using more than four 15 cm plates for each experiment, so that the final volume of the pooled viral supernatant can easily be accommodated within six ultracentrifugation tubes.
    3. Fill ultracentrifugation tubes to at least three-fourths their total volume capacity, otherwise breakage of tubes can occur during centrifugation, resulting in possible loss of sample and/or equipment damage.
  3. Balance the tubes with 1x PBS and centrifuge samples at 70,000 x g for 2 h at 17 °C (see Table of Materials for rotor details).
    NOTE: To prevent disruption of the sucrose layer during acceleration, set the ultracentrifuge to slowly accelerate the rotor to 200 rpm during the first 3 min of the spin. Similarly, set the ultracentrifuge to slowly decelerate the rotor from 200 rpm to 0 rpm over 3 min at the end of the spin.
  4. Carefully collect 30 - 60% sucrose fractions into clean tubes (Figure 2). Add cold 1x PBS to the pooled fractions and bring up the volume to 100 mL; mix by pipetting up and down several times.
  5. Proceed to the sucrose cushion step by carefully layering the viral preparation on a sucrose cushion. For this, add 4 mL of 20% sucrose (in 1x PBS) to the tube, followed by ~20 - 25 mL of the viral solution per tube. If tubes are less than three-fourths full, top up with sterile 1x PBS.
  6. Carefully balance and centrifuge the samples at 70,000 x g for 2 h at 17 °C, as before. Pour off the supernatant and allow the remaining liquid to drain by inverting tubes on paper towels.
  7. Aspirate remaining droplets in order to remove all liquid from the pellet. At this step, the virus-containing pellets should be barely visible as small translucent spots.
  8. Resuspend pellets by adding 70 µL of 1x PBS to the first tube and thoroughly pipetting the suspension, subsequently transferring the suspension to the next tube and mixing as before, continuing until all the pellets are resuspended.
  9. Rinse the tubes with an additional 50 µL of cold 1x PBS and mix as before. Ensure that the combined volume of the final suspension is ~120 µL, and appears slightly milky; clear it by centrifugation at 10,000 x g for 30 s on a tabletop microcentrifuge.
  10. Transfer the supernatant to a fresh microfuge tube, make 10 µL aliquots, and store them at -80 °C.
    NOTE: Avoid performing repeated freeze-thaw cycles on the lentiviral samples. Except when centrifugation is required, efforts should be made to carry out the remaining steps in tissue culture hoods, or designated tissue-culture rooms using appropriate biosafety measures (see Discussion).

6. Estimation of Viral Titers

  1. p24 -enzyme-linked immunosorbent assay (ELISA) method
    NOTE: The assay is carried out using high-binding 96-well plates as per the instructions of the NIH AIDS Vaccine Program for HIV-1 p24 Antigen Capture Assay Kit (see Table of Materials) with modifications29.
    1. The next day, wash the wells three times with 200 µL 0.05% Tween 20 in cold PBS (PBS-T solution). Coat the plate with 100 µL of monoclonal anti-p24 antibody at a dilution of 1:1500 in 1x PBS and incubate overnight at 4 °C.
    2. To eliminate non-specific binding, block the plate with 200 µL 1% BSA in PBS; wash three times with 200 µL 0.05% Tween 20 in cold PBS (PBS-T solution) for at least 1 h at room temperature.
    3. Prepare samples: For concentrated vector preparations, dilute 1 µL of the sample 100-fold by adding 89 µL of dd-H20 and 10 µL of Triton X-100 (final concentration of 10%). For non-concentrated preparations, prepare ten-fold diluted samples (add 80 µL of dd-H20 and 10 µL of Triton X-100 (final concentration of 10%) to 10 µL of the sample).
      NOTE: Samples can be stored at -20 °C at this step for an extended period of time for later usage.
    4. Prepare HIV-1 standards by applying a 2-fold serial dilution (with a starting concentration 5 ng/mL).
    5. Dilute concentrated samples (from 1:100 pre-diluted stocks) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to establish 1:10,000, 1:50,000, and 1:250,000 dilutions. Dilute non-concentrated samples (from 1:10 pre-diluted stocks) in RPMI 1640 supplemented with 0.2% Tween 20 and 1% BSA to establish 1:500, 1:2500, and 1:12,500 dilutions.
    6. Apply samples on the plate in triplicates and incubate overnight at 4 °C.
    7. The next day, wash the wells six times and incubate at 37 °C for 4 h with 100 µL polyclonal rabbit anti-p24 antibody, diluted 1:1000 in RPMI 1640, 10% FBS, 0.25% BSA, and 2% normal mouse serum (NMS).
    8. Wash six times as above and incubate at 37 °C for 1 h with goat anti-rabbit horseradish peroxidase IgG diluted 1:10,000 in RPMI 1640 supplemented with 5% normal goat serum, 2% NMS, 0.25% BSA, and 0.01% Tween 20.
    9. Wash the plate, as above, and incubate with TMB peroxidase substrate at room temperature for 15 min.
    10. Stop the reaction by adding 100 µL of 1 N HCL. Measure sample absorbance at 450 nm using the absorbance plate reader.
  2. Measurement of fluorescent reporter intensity
    1. FACS Method
      NOTE: The extent of GFP signal depletion in cells can be accurately estimated by measuring mean fluorescence intensity of the transduced cells via flow cytometry. Please refer to the recent paper 28 for FACS data analysis, presentation, and interpretation. The protocol is described as follows.
      1. Make a ten-fold serial dilution of the preparation (from 10-1 to 10-5) of the viral preparation in 1x PBS.
      2. Seed approximately 5 x 105 293T cells in each well of a 6-well plate in a final volume of 2 mL per well. Add 10 µL of each viral dilution to the cells and incubate cells at 37 °C for 48 h.
      3. Harvest cells for FACS analysis as follows: add 200 µL of 0.05% Trypsin-EDTA solution and incubate cells at 37 °C for 5 min. Add 2 mL of a complete DMEM media and collect samples into 15 mL conical tubes.
      4. Pellet cells by centrifugation at 400 x g at 4 °C, and resuspend the pellet in 500 µL of cold 1x PBS.
      5. For fixation, add an equal volume of 4% formaldehyde solution to this suspension and incubate for 10 min at room temperature.
      6. Pellet fixed cells and resuspend in 1 mL of 1x PBS. Analyze GFP expression using a FACS instrument, as described in Ortinski et al.28 Briefly, use the following formula to determine the virus' functional titer:
        Transducing units calculation formula, showing equation for determining TU per mL with variables.
        NOTE: Here Tg = number of GFP-positive cells counted; Tn = total number of cells counted; N = total number of cells transduced; V = volume (in µL) used for transduction. For example: If 1 x 106 cells were transduced with 10 µL of virus, 2 x 104 cells were counted and 5 x 103 were GFP-positive, based on the above equation the functional titer would be:
        Calculation formula for lentiviral titer, includes titration, gene therapy, scientific analysis.
    2. Counting GFP-positive cells
      1. Calculate the multiplicity of infection (MOI) used for transduction. Test a broad range of MOIs (1 - 10), with increasing MOIs resulting in a higher transduction efficiency.
      2. Prior to transfection, seed a 6-well plate with approximately 3 - 4 x 105 cells per well. Once the cells reach >90% confluency (usually within 24 h), transduce with the purified virus at pre-determined MOIs.
      3. Incubate plate at 37 °C with 5% CO2 in a standard tissue culture, and monitor cells at regular intervals for 1 - 7 days for changes in GFP signal.
      4. Count the number of GFP-positive cells with a fluorescent microscope (PLAN 4X objective, 0.1 N.A, 40X magnification) fitted with a GFP filter set (excitation wavelength-470 nm, emission wavelength-525 nm), using naïve (un-transduced) cells to set the population of GFP-negative and positive cells.
      5. Estimate the final titer by adjusting for the dilution factor and the volume, using the following formula:
        Transducing units formula equation: TU/mL = (N)×(D)×(M)/V, relevant for gene transfer analysis.
        NOTE: Here, N = number of GFP-positive cells, D = dilution factor, M = magnification factor (usually 20X), V = volume of virus used for transduction. For example, for 20 GFP-positive cells (N) counted at a dilution of 10-4 (1:10,000) in a 10 µL sample (V) at 20X magnification (M) (D) would result in a functional titer of (20 x 104) x (20) x (10) x (100*) = 4 x 108 TU/mL.
        (*to adjust to per mL)

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Results

Validation of the knockout-efficiency of IDLV-CRISPR/Cas9 vectors
We used GFP-expressing 293T cells as a model to validate the efficiency of CRISPR/Cas9-mediated gene knockout. GFP+ cells were generated by transduction of HEK-293T cells with pLenti-GFP (vBK201a) at an MOI of 0.5 (Figure 3b, "no-virus" panel). The sgRNA-to-GFP/Cas9 all-in-one vector cassette was packaged into IDLV or ICLV particles and the production efficiency was assesse...

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Discussion

IDLVs have begun to emerge as the vehicle of choice for in vivo gene-editing, especially in the context of genetic diseases, owing largely to the low risk of mutagenesis associated with these vectors compared to integrating delivery platforms22,28. In the current manuscript, we sought to detail the protocol associated with production of the improved all-in-one IDLV-CRISPR/Cas9 system that was recently developed in our laboratory28...

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Disclosures

Patent USC-499-P (1175) was filed by the University of South Carolina in relation to the work described in this manuscript.

Acknowledgements

We would like to thank the Department of Neurobiology, Duke University School of Medicine and Dean's Office for Basic Science, Duke University. We also thank members of the Duke Viral Vector Core for comments on the manuscript. Plasmid pLenti CRISPRv2 was gift from Feng Zhang (Broad Institute). The LV-packaging system including the plasmids psPAX2, VSV-G, pMD2.G and pRSV-Rev was a kind gift from Didier Trono (EPFL, Switzerland). Financial support for this work was provided by the University Of South Carolina School Of Medicine, grant RDF18080-E202 (B.K).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Equipment
Optima XPN-80 UltracentrifugeBeckman CoulterA99839
Allegra 25R tabletop centrifugeBeckman Coulter369434
xMark Microplate Absorbance plate readerBio-Rad1681150
BD FACSBecton Dickinson338960
Inverted fluorescence microscopeLeicaDM IRB2
0.45-μm filter unit, 500mLCorning430773
Conical bottom ultracentrifugation tubesSeton Scientific5067
Conical tube adaptersSeton ScientificPN 4230
SW32Ti swinging-bucket rotorBeckman Coulter369650
15 mL conical centrifuge tubesCorning430791
50mL conical centrifuge tubesCorning430291
High-binding 96-well platesCorning3366
150 mm TC-Treated Cell Culture dishes with 20 mm GridCorning353025
100mm TC-Treated Culture DishCorning430167
0.22 μM filter unit, 1LCorning430513
QIAprep Spin Miniprep Kit (50)Qiagen27104
Tissue culture pipettes, 5 mLCorning4487
Tissue culture pipettes, 10 mLCorning4488
Tissue culture pipettes, 25 mLCorning4489
Hemacytometer with cover slipsCole-ParmerUX-79001-00
NameCompanyCatalog NumberComments
Cell culture reagents
Human embryonic kidney 293T (HEK 293T) cellsATCCCRL-3216
293FT cellsThermo Fisher ScientificR70007
DMEM, high glucose mediaGibco11965
Cosmic Calf SerumHycloneSH30087.04
Antibiotic-antimycotic solution, 100XSigma AldrichA5955-100ML
Sodium pyruvateSigma AldrichS8636-100ML
Non-Essential Amino Acid (NEAA)HycloneSH30087.04
RPMI 1640 mediaThermo Fisher Scientific11875-085
GlutaMAXThermo Fisher Scientific35050061
Trypsin-EDTA 0.05%Gibco25300054
BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid)Sigma AldrichB9879 - BES
GelatinSigma AldrichG1800-100G
NameCompanyCatalog NumberComments
p24 ELISA reagents
Monoclonal anti-p24 antibodyNIH AIDS Research and Reference Reagent Program3537
HIV-1 standardsNIH AIDS Research and Reference Reagent ProgramSP968F
Polyclonal rabbit anti-p24 antibodyNIH AIDS Research and Reference Reagent ProgramSP451T
Goat anti-rabbit horseradish peroxidase IgGSigma Aldrich12-348Working concentration 1:1500
Normal mouse serum, Sterile, 500mLEquitech-BioSM30-0500
Goat serum, Sterile, 10mLSigmaG9023Working concentration 1:1000
TMB peroxidase substrateKPL5120-0076Working concentration 1:10,000
NameCompanyCatalog NumberComments
Plasmids
psPAX2Addgene12260
pMD2.GAddgene12259
pRSV-RevAddgene12253
lentiCRISPR v2Addgene52961
NameCompanyCatalog NumberComments
Restriction enzymes
BsrGINew England BiolabsR0575S
BsmBINew England BiolabsR0580S
EcoRVNew England BiolabsR0195S
KpnINew England BiolabsR0142S
PacINew England BiolabsR0547S
SphINew England BiolabsR0182S

References

  1. Horvath, P., Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 327 (5962), 167-170 (2010).
  2. Marraffini, L. A., Sontheimer, E. J. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet. 11 (3), 181-190 (2010).
  3. Kim, H., Kim, J. S. A guide to genome engineering with programmable nucleases. Nat Rev Genet. 15 (5), 321-334 (2014).
  4. Bellec, J., et al. CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells. Curr Gene Ther. 15 (5), 447-459 (2015).
  5. Kennedy, E. M., et al. Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR/Cas RNA-guided DNA endonuclease. Virology. 476, 196-205 (2015).
  6. Roehm, P. C., et al. Inhibition of HSV-1 Replication by Gene Editing Strategy. Sci Rep. 6, 23146(2016).
  7. Wang, W., et al. CCR5 gene disruption via lentiviral vectors expressing Cas9 and single guided RNA renders cells resistant to HIV-1 infection. PLoS One. 9 (12), e115987(2014).
  8. Kantor, B., Bailey, R. M., Wimberly, K., Kalburgi, S. N., Gray, S. J. Methods for gene transfer to the central nervous system. Adv Genet. 87, 125-197 (2014).
  9. Lebbink, R. J., et al. A combinational CRISPR/Cas9 gene-editing approach can halt HIV replication and prevent viral escape. Sci Rep. 7, 41968(2017).
  10. Xu, L., et al. CRISPR/Cas9-Mediated CCR5 Ablation in Human Hematopoietic Stem/Progenitor Cells Confers HIV-1 Resistance In Vivo. Mol Ther. , (2017).
  11. Yiu, G., Tieu, E., Nguyen, A. T., Wong, B., Smit-McBride, Z. Genomic Disruption of VEGF-A Expression in Human Retinal Pigment Epithelial Cells Using CRISPR-Cas9 Endonuclease. Invest Ophthalmol Vis Sci. 57 (13), 5490-5497 (2016).
  12. Kabadi, A. M., Ousterout, D. G., Hilton, I. B., Gersbach, C. A. Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector. Nucleic Acids Res. 42 (19), e147(2014).
  13. Fu, Y., Sander, J. D., Reyon, D., Cascio, V. M., Joung, J. K. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol. 32 (3), 279-284 (2014).
  14. Hsu, P. D., et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 31 (9), 827-832 (2013).
  15. Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24 (6), 1012-1019 (2014).
  16. Pattanayak, V., et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 31 (9), 839-843 (2013).
  17. Shalem, O., et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 343 (6166), 84-87 (2014).
  18. Schaefer, K. A., et al. Unexpected mutations after CRISPR-Cas9 editing in vivo. Nat Methods. 14 (6), 547-548 (2017).
  19. Choi, J. G., et al. Lentivirus pre-packed with Cas9 protein for safer gene editing. Gene Ther. 23 (7), 627-633 (2016).
  20. Platt, R. J., et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 159 (2), 440-455 (2014).
  21. Truong, D. J., et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43 (13), 6450-6458 (2015).
  22. Nelson, C. E., Gersbach, C. A. Engineering Delivery Vehicles for Genome Editing. Annu Rev Chem Biomol Eng. 7, 637-662 (2016).
  23. Jaalouk, D. E., Crosato, M., Brodt, P., Galipeau, J. Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors. Virol J. 3, 27(2006).
  24. Ellis, B. L., Potts, P. R., Porteus, M. H. Creating higher titer lentivirus with caffeine. Hum Gene Ther. 22 (1), 93-100 (2011).
  25. Hoban, M. D., et al. Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells. Curr Protoc Stem Cell Biol. 36, 1-10 (2016).
  26. Bayer, M., et al. A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector. Mol Ther. 16 (12), 1968-1976 (2008).
  27. Kantor, B., Ma, H., Webster-Cyriaque, J., Monahan, P. E., Kafri, T. Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection. Proc Natl Acad Sci U S A. 106 (44), 18786-18791 (2009).
  28. Ortinski, P. I., O'Donovan, B., Dong, X., Kantor, B. Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing. Mol Ther Methods Clin Dev. 5, 153-164 (2017).
  29. Kantor, B., et al. Notable reduction in illegitimate integration mediated by a PPT-deleted, nonintegrating lentiviral vector. Mol Ther. 19 (3), 547-556 (2011).
  30. Berkhout, B., Verhoef, K., van Wamel, J. L., Back, N. K. Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains. J Virol. 73 (2), 1138-1145 (1999).
  31. Gomez-Gonzalo, M., et al. The hepatitis B virus X protein induces HIV-1 replication and transcription in synergy with T-cell activation signals: functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 long terminal repeat promoter. J Biol Chem. 276 (38), 35435-35443 (2001).
  32. Kim, Y. S., et al. Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1. J Biol Chem. 280 (22), 21545-21552 (2005).
  33. Ortinski, P. I., Lu, C., Takagaki, K., Fu, Z., Vicini, S. Expression of distinct alpha subunits of GABAA receptor regulates inhibitory synaptic strength. J Neurophysiol. 92 (3), 1718-1727 (2004).
  34. Van Lint, C., et al. Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity. J Virol. 71 (8), 6113-6127 (1997).
  35. Van Lint, C., Ghysdael, J., Paras, P., Burny, A., Verdin, E. A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1. J Virol. 68 (4), 2632-2648 (1994).
  36. Xu, W., Russ, J. L., Eiden, M. V. Evaluation of residual promoter activity in gamma-retroviral self-inactivating (SIN) vectors. Mol Ther. 20 (1), 84-90 (2012).
  37. Testing for Replication Competent Retrovirus (RCR)/Lentivirus (RCL) in Retroviral and Lentiviral Vector Based Gene Therapy Products - Revisiting Current FDA Recommendations. , Available from: https://sites.duke.edu/dvvc/files/2016/05/FDA-recommendation-for-RCR-testing.pdf (2017).
  38. CDC. Biosafety in Microbiological and Biomedical Laboratories. , Available from: https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf (2017).
  39. Dull, T., et al. A third-generation lentivirus vector with a conditional packaging system. J Virol. 72 (11), 8463-8471 (1998).
  40. Liu, X. S., et al. Editing DNA Methylation in the Mammalian Genome. Cell. 167 (1), 233-247 (2016).
  41. Ran, F. A., et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 520 (7546), 186-191 (2015).
  42. Zetsche, B., et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 163 (3), 759-771 (2015).

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CRISPR Cas9 Gene KnockoutViral Vector ProductionUltracentrifugation PurificationSucrose Gradient Separationp24 ELISA AssayHEK 293T TransductionViral Titer QuantificationCalcium Phosphate TransfectionViral Particle Concentration
A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells
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