We would like to thank the Department of Neurobiology, Duke University School of Medicine and Dean&#39;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).

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&#160;antibiotic-antimycotic solution (100x&#160;solution contains 10,000 units penicillin, 10 mg streptomycin and 25 &#181;g amphotericin B per mL). The media is also supplemented with 1x&#160;sodium pyruvate, 1x&#160;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 cm&#178;). 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.
<ol>
	<li>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 &#176;C with 5% CO2 in a standard tissue-culture incubator. Use a standard hemocytometer to count cells for all subsequent steps.</li>
	<li>Once the cells reach 90 - 95% confluent growth, reseed into 15 cm tissue culture plates (below, Steps 1.3 - 1.5).</li>
	<li>To reseed, aspirate media from the confluent plate and gently rinse with sterile 1x&#160;PBS. Incubate cells with 2 mL of a dissociation reagent (e.g., Trypsin-EDTA) at 37 &#176;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.</li>
	<li>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.</li>
	<li>Bring the total volume of each plate to 25 mL with warm (37 &#176;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 &#176;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).</li>
</ol>
2. Transfecting HEK-293T Cells Using a Calcium Phosphate-based Protocol
<ol>
	<li>Transfection reagents
	<ol>
		<li>To prepare 2x&#160;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 &#181;M filter unit. Store at -20 &#176;C.</li>
		<li>Prepare 1M CaCl2. Filter solution via a 0.22 &#181;M filter. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Observe the plates that were seeded a day prior. Cells are ready for transfection once they reach 70 - 80% confluency.</li>
	<li>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.</li>
	<li>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 &#181;g of the CRISPR/Cas9-transfer vector (pBK198 or pBK189), 25 &#181;g of pBK43 (psPAX2-D64E), 12.5 &#181;g pMD2.G, and 6.25 &#181;g of pREV (Figure 1c).</li>
	<li>Add 312.5 &#181;L 1M CaCl2&#160;to the plasmid mix. To this, add up to 1.25 mL of sterile dd-H20.</li>
	<li>Slowly (drop-wise) add 1.25 mL of 2x&#160;BBS solution while vortexing the mix. Incubate for 30 min at room temperature.</li>
	<li>Add the transfection mixture dropwise to each 15-cm plate (2.5 mL per plate). Swirl the plates gently and incubate at 37 &#176;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&#160;BBS (pH = 6.95).</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56915/56915fig2v2.jpg" /> 
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&#160;PBS for further usage (see text for further details). <a href="//cloudfront.jove.com/files/ftp_upload/56915/56915fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Day After Transfection
<ol>
	<li>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 &#176;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).</li>
</ol>
4. Harvesting Virus
<ol>
	<li>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.</li>
	<li>Clear the suspension by centrifugation at 400 - 450 x g for 10 min using a tabletop centrifuge. Filter the supernatant through a 0.45 &#181;m vacuum filter unit. 
	NOTE: The filtered supernatant can be stored at 4 &#176;C for up to 4 days before proceeding with concentration, or aliquoted and stored at -80 &#176;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.</li>
</ol>
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).
<ol>
	<li>Load the conical ultracentrifugation tubes in the following order to create a sucrose gradient: 0.5 mL 70% sucrose (dissolved 1x&#160;PBS), 0.5 mL 60% sucrose (dissolved in DMEM), 1 mL 30% sucrose (dissolved in DMEM), and 2 mL 20% sucrose (dissolved in 1x&#160;PBS).</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Balance the tubes with 1x&#160;PBS and centrifuge samples at 70,000 x g for 2 h at 17 &#176;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.</li>
	<li>Carefully collect 30 - 60% sucrose fractions into clean tubes (Figure 2). Add cold 1x&#160;PBS to the pooled fractions and bring up the volume to 100 mL; mix by pipetting up and down several times.</li>
	<li>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&#160;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&#160;PBS.</li>
	<li>Carefully balance and centrifuge the samples at 70,000 x g for 2 h at 17 &#176;C, as before. Pour off the supernatant and allow the remaining liquid to drain by inverting tubes on paper towels.</li>
	<li>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.</li>
	<li>Resuspend pellets by adding 70 &#181;L of 1x&#160;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.</li>
	<li>Rinse the tubes with an additional 50 &#181;L of cold 1x&#160;PBS and mix as before. Ensure that the combined volume of the final suspension is ~120 &#181;L, and appears slightly milky; clear it by centrifugation at 10,000 x g for 30 s on a tabletop microcentrifuge.</li>
	<li>Transfer the supernatant to a fresh microfuge tube, make 10 &#181;L aliquots, and store them at -80 &#176;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).</li>
</ol>
6. Estimation of Viral Titers
<ol>
	<li>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.
	<ol>
		<li>The next day, wash the wells three times with 200 &#181;L 0.05% Tween 20 in cold PBS (PBS-T solution). Coat the plate with 100 &#181;L of monoclonal anti-p24 antibody at a dilution of 1:1500 in 1x&#160;PBS and incubate overnight at 4 &#176;C.</li>
		<li>To eliminate non-specific binding, block the plate with 200 &#181;L 1% BSA in PBS; wash three times with 200 &#181;L 0.05% Tween 20 in cold PBS (PBS-T solution) for at least 1 h at room temperature.</li>
		<li>Prepare samples: For concentrated vector preparations, dilute 1 &#181;L of the sample 100-fold by adding 89 &#181;L of dd-H20 and 10 &#181;L of Triton X-100 (final concentration of 10%). For non-concentrated preparations, prepare ten-fold diluted samples (add 80 &#181;L of dd-H20 and 10 &#181;L of Triton X-100 (final concentration of 10%) to 10 &#181;L of the sample). 
		NOTE: Samples can be stored at -20 &#176;C at this step for an extended period of time for later usage.</li>
		<li>Prepare HIV-1 standards by applying a 2-fold serial dilution (with a starting concentration 5 ng/mL).</li>
		<li>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.</li>
		<li>Apply samples on the plate in triplicates and incubate overnight at 4 &#176;C.</li>
		<li>The next day, wash the wells six times and incubate at 37 &#176;C for 4 h with 100 &#181;L polyclonal rabbit anti-p24 antibody, diluted 1:1000 in RPMI 1640, 10% FBS, 0.25% BSA, and 2% normal mouse serum (NMS).</li>
		<li>Wash six times as above and incubate at 37 &#176;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.</li>
		<li>Wash the plate, as above, and incubate with TMB peroxidase substrate at room temperature for 15 min.</li>
		<li>Stop the reaction by adding 100 &#181;L of 1 N HCL. Measure sample absorbance at 450 nm using the absorbance plate reader.</li>
	</ol>
	</li>
	<li>Measurement of fluorescent reporter intensity
	<ol>
		<li>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.
		<ol>
			<li>Make a ten-fold serial dilution of the preparation (from 10-1 to 10-5) of the viral preparation in 1x&#160;PBS.</li>
			<li>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 &#181;L of each viral dilution to the cells and incubate cells at 37 &#176;C for 48 h.</li>
			<li>Harvest cells for FACS analysis as follows: add 200 &#181;L of 0.05% Trypsin-EDTA solution and incubate cells at 37 &#176;C for 5 min. Add 2 mL of a complete DMEM media and collect samples into 15 mL conical tubes.</li>
			<li>Pellet cells by centrifugation at 400 x g at 4 &#176;C, and resuspend the pellet in 500 &#181;L of cold 1x&#160;PBS.</li>
			<li>For fixation, add an equal volume of 4% formaldehyde solution to this suspension and incubate for 10 min at room temperature.</li>
			<li>Pellet fixed cells and resuspend in 1 mL of 1x&#160;PBS. Analyze GFP expression using a FACS instrument, as described in Ortinski et al.28 Briefly, use the following formula to determine the virus&#39; functional titer: 
			<img alt="Equation 1" src="/files/ftp_upload/56915/56915eq1.jpg" /> 
			NOTE: Here Tg = number of GFP-positive cells counted; Tn = total number of cells counted; N = total number of cells transduced; V = volume (in &#181;L) used for transduction. For example: If 1 x 106 cells were transduced with 10 &#181;L 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: 
			<img alt="Equation 2" src="/files/ftp_upload/56915/56915eq2.jpg" /></li>
		</ol>
		</li>
		<li>Counting GFP-positive cells
		<ol>
			<li>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.</li>
			<li>Prior to transfection, seed a 6-well plate with approximately 3 - 4 x 105 cells per well. Once the cells reach &#62;90% confluency (usually within 24 h), transduce with the purified virus at pre-determined MOIs.</li>
			<li>Incubate plate at 37 &#176;C with 5% CO2 in a standard tissue culture, and monitor cells at regular intervals for 1 - 7 days for changes in GFP signal.</li>
			<li>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 na&#239;ve (un-transduced) cells to set the population of GFP-negative and positive cells.</li>
			<li>Estimate the final titer by adjusting for the dilution factor and the volume, using the following formula: 
			<img alt="Equation 3" src="/files/ftp_upload/56915/56915eq3.jpg" /> 
			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 &#181;L 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)</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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E., Crosato, M., Brodt, P., Galipeau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+histone+deacetylation+in+293GPG+packaging+cell+line+improves+the+production+of+self-inactivating+MLV-derived+retroviral+vectors.">Inhibition of histone deacetylation in 293GPG packaging cell line improves the production of self-inactivating MLV-derived retroviral vectors.</a> Virol J. 3, 27 (2006).</li><li>Ellis, B. L., Potts, P. R., Porteus, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creating+higher+titer+lentivirus+with+caffeine.">Creating higher titer lentivirus with caffeine.</a> Hum Gene Ther. 22 (1), 93-100 (2011).</li><li>Hoban, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+Genome+Editing+Reagents+to+Hematopoietic+Stem/Progenitor+Cells.">Delivery of Genome Editing Reagents to Hematopoietic Stem/Progenitor Cells.</a> Curr Protoc Stem Cell Biol. 36, 1-10 (2016).</li><li>Bayer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+U3+deletion+causes+increased+in+vivo+expression+from+a+nonintegrating+lentiviral+vector.">A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector.</a> Mol Ther. 16 (12), 1968-1976 (2008).</li><li>Kantor, B., Ma, H., Webster-Cyriaque, J., Monahan, P. E., Kafri, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+activation+of+unintegrated+HIV-1+genomes+by+gut-associated+short+chain+fatty+acids+and+its+implications+for+HIV+infection.">Epigenetic activation of unintegrated HIV-1 genomes by gut-associated short chain fatty acids and its implications for HIV infection.</a> Proc Natl Acad Sci U S A. 106 (44), 18786-18791 (2009).</li><li>Ortinski, P. I., O'Donovan, B., Dong, X., Kantor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrase-Deficient+Lentiviral+Vector+as+an+All-in-One+Platform+for+Highly+Efficient+CRISPR/Cas9-Mediated+Gene+Editing.">Integrase-Deficient Lentiviral Vector as an All-in-One Platform for Highly Efficient CRISPR/Cas9-Mediated Gene Editing.</a> Mol Ther Methods Clin Dev. 5, 153-164 (2017).</li><li>Kantor, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notable+reduction+in+illegitimate+integration+mediated+by+a+PPT-deleted,+nonintegrating+lentiviral+vector.">Notable reduction in illegitimate integration mediated by a PPT-deleted, nonintegrating lentiviral vector.</a> Mol Ther. 19 (3), 547-556 (2011).</li><li>Berkhout, B., Verhoef, K., van Wamel, J. L., Back, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+instability+of+live,+attenuated+human+immunodeficiency+virus+type+1+vaccine+strains.">Genetic instability of live, attenuated human immunodeficiency virus type 1 vaccine strains.</a> J Virol. 73 (2), 1138-1145 (1999).</li><li>Gomez-Gonzalo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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.</a> J Biol Chem. 276 (38), 35435-35443 (2001).</li><li>Kim, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+zinc+finger+fusions+targeting+Sp1-binding+sites+and+the+trans-activator-responsive+element+potently+repress+transcription+and+replication+of+HIV-1.">Artificial zinc finger fusions targeting Sp1-binding sites and the trans-activator-responsive element potently repress transcription and replication of HIV-1.</a> J Biol Chem. 280 (22), 21545-21552 (2005).</li><li>Ortinski, P. I., Lu, C., Takagaki, K., Fu, Z., Vicini, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+distinct+alpha+subunits+of+GABAA+receptor+regulates+inhibitory+synaptic+strength.">Expression of distinct alpha subunits of GABAA receptor regulates inhibitory synaptic strength.</a> J Neurophysiol. 92 (3), 1718-1727 (2004).</li><li>Van Lint, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+sites+downstream+of+the+human+immunodeficiency+virus+type+1+transcription+start+site+are+important+for+virus+infectivity.">Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for virus infectivity.</a> J Virol. 71 (8), 6113-6127 (1997).</li><li>Van Lint, C., Ghysdael, J., Paras, P., Burny, A., Verdin, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transcriptional+regulatory+element+is+associated+with+a+nuclease-hypersensitive+site+in+the+pol+gene+of+human+immunodeficiency+virus+type+1.">A transcriptional regulatory element is associated with a nuclease-hypersensitive site in the pol gene of human immunodeficiency virus type 1.</a> J Virol. 68 (4), 2632-2648 (1994).</li><li>Xu, W., Russ, J. L., Eiden, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+residual+promoter+activity+in+gamma-retroviral+self-inactivating+(SIN)+vectors.">Evaluation of residual promoter activity in gamma-retroviral self-inactivating (SIN) vectors.</a> Mol Ther. 20 (1), 84-90 (2012).</li><li>. Testing for Replication Competent Retrovirus (RCR)/Lentivirus (RCL) in Retroviral and Lentiviral Vector Based Gene Therapy Products - Revisiting Current FDA Recommendations Available from: <a target="_blank" href="https://sites.duke.edu/dvvc/files/2016/05/FDA-recommendation-for-RCR-testing.pdf">https://sites.duke.edu/dvvc/files/2016/05/FDA-recommendation-for-RCR-testing.pdf</a> (2017)</li><li>. Biosafety in Microbiological and Biomedical Laboratories Available from: <a target="_blank" href="https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf">https://www.cdc.gov/biosafety/publications/bmbl5/bmbl.pdf</a> (2017)</li><li>Dull, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J Virol. 72 (11), 8463-8471 (1998).</li><li>Liu, X. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editing+DNA+Methylation+in+the+Mammalian+Genome.">Editing DNA Methylation in the Mammalian Genome.</a> Cell. 167 (1), 233-247 (2016).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome+editing+using+Staphylococcus+aureus+Cas9.">In vivo genome editing using Staphylococcus aureus Cas9.</a> Nature. 520 (7546), 186-191 (2015).</li><li>Zetsche, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cpf1+is+a+single+RNA-guided+endonuclease+of+a+class+2+CRISPR-Cas+system.">Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.</a> Cell. 163 (3), 759-771 (2015).</li></ol>

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

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...

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 &#8212; 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 (&#60;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-&#224;-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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56915/56915fig1.jpg" /> 
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 1&#945; promoter (EFS-NC), the vector packaging element &#968; (psi), human Cytomegalovirus (hCMV) promoter, and human U6 promoter. <a href="//cloudflare.jove.com/files/ftp_upload/56915/56915fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Optima XPN-80 Ultracentrifuge</td><td>Beckman Coulter</td><td>A99839</td><td></td></tr><tr><td>Allegra 25R tabletop centrifuge</td><td>Beckman Coulter</td><td>369434</td><td></td></tr><tr><td>xMark Microplate Absorbance plate reader</td><td>Bio-Rad</td><td>1681150</td><td></td></tr><tr><td>BD FACS</td><td>Becton Dickinson</td><td>338960</td><td></td></tr><tr><td>Inverted fluorescence microscope</td><td>Leica</td><td>DM IRB2</td><td></td></tr><tr><td>0.45-&amp;mu;m filter unit, 500mL</td><td>Corning</td><td>430773</td><td></td></tr><tr><td>Conical bottom ultracentrifugation tubes</td><td>Seton Scientific</td><td>5067</td><td></td></tr><tr><td>Conical tube adapters</td><td>Seton Scientific</td><td>PN 4230</td><td></td></tr><tr><td>SW32Ti swinging-bucket rotor</td><td>Beckman Coulter</td><td>369650</td><td></td></tr><tr><td>15 mL conical centrifuge tubes</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50mL conical centrifuge tubes</td><td>Corning</td><td>430291</td><td></td></tr><tr><td>High-binding 96-well plates</td><td>Corning</td><td>3366</td><td></td></tr><tr><td>150 mm TC-Treated Cell Culture dishes with 20 mm Grid</td><td>Corning</td><td>353025</td><td></td></tr><tr><td>100mm TC-Treated Culture Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>0.22 &amp;mu;M filter unit, 1L</td><td>Corning</td><td>430513</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (50)</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>Tissue culture pipettes, 5 mL</td><td>Corning</td><td>4487</td><td></td></tr><tr><td>Tissue culture pipettes, 10 mL</td><td>Corning</td><td>4488</td><td></td></tr><tr><td>Tissue culture pipettes, 25 mL</td><td>Corning</td><td>4489</td><td></td></tr><tr><td>Hemacytometer with cover slips</td><td>Cole-Parmer</td><td>UX-79001-00</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Cell culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human embryonic kidney 293T (HEK 293T) cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>293FT cells</td><td>Thermo Fisher Scientific</td><td>R70007</td><td></td></tr><tr><td>DMEM, high glucose media</td><td>Gibco</td><td>11965</td><td></td></tr><tr><td>Cosmic Calf Serum</td><td>Hyclone</td><td>SH30087.04</td><td></td></tr><tr><td>Antibiotic-antimycotic solution, 100X</td><td>Sigma Aldrich</td><td>A5955-100ML</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma Aldrich</td><td>S8636-100ML</td><td></td></tr><tr><td>Non-Essential Amino Acid (NEAA)</td><td>Hyclone</td><td>SH30087.04</td><td></td></tr><tr><td>RPMI 1640 media</td><td>Thermo Fisher Scientific</td><td>11875-085</td><td></td></tr><tr><td>GlutaMAX</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>Trypsin-EDTA 0.05%</td><td>Gibco</td><td>25300054</td><td></td></tr><tr><td>BES (N, N-bis (2-hydroxyethyl)-2-amino-ethanesulfonic acid)</td><td>Sigma Aldrich</td><td>B9879 - BES</td><td></td></tr><tr><td>Gelatin</td><td>Sigma Aldrich</td><td>G1800-100G</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;p&lt;sup&gt;24&lt;/sup&gt; ELISA reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Monoclonal anti-p&lt;sup&gt;24&lt;/sup&gt; antibody</td><td>NIH AIDS Research and Reference Reagent Program</td><td>3537</td><td></td></tr><tr><td>HIV-1 standards</td><td>NIH AIDS Research and Reference Reagent Program</td><td>SP968F</td><td></td></tr><tr><td>Polyclonal rabbit anti-p&lt;sup&gt;24&lt;/sup&gt; antibody</td><td>NIH AIDS Research and Reference Reagent Program</td><td>SP451T</td><td></td></tr><tr><td>Goat anti-rabbit horseradish peroxidase IgG</td><td>Sigma Aldrich</td><td>12-348</td><td>Working concentration 1:1500</td></tr><tr><td>Normal mouse serum, Sterile, 500mL</td><td>Equitech-Bio</td><td>SM30-0500</td><td></td></tr><tr><td>Goat serum, Sterile, 10mL</td><td>Sigma</td><td>G9023</td><td>Working concentration 1:1000</td></tr><tr><td>TMB peroxidase substrate</td><td>KPL</td><td>5120-0076</td><td>Working concentration 1:10,000</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>pRSV-Rev</td><td>Addgene</td><td>12253</td><td></td></tr><tr><td>lentiCRISPR v2</td><td>Addgene</td><td>52961</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Restriction enzymes&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BsrGI</td><td>New England Biolabs</td><td>R0575S</td><td></td></tr><tr><td>BsmBI</td><td>New England Biolabs</td><td>R0580S</td><td></td></tr><tr><td>EcoRV</td><td>New England Biolabs</td><td>R0195S</td><td></td></tr><tr><td>KpnI</td><td>New England Biolabs</td><td>R0142S</td><td></td></tr><tr><td>PacI</td><td>New England Biolabs</td><td>R0547S</td><td></td></tr><tr><td>SphI</td><td>New England Biolabs</td><td>R0182S</td><td></td></tr></tbody></table>

a protocol for the production of integrase-deficient lentiviral vectors for crispr/cas9-mediated gene knockout in dividing cells

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.

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, &#34;no-virus&#34; panel). The sgRNA-to-GFP/Cas9 all-in-one vector cassette was packaged into IDLV or ICLV particles and the production efficiency w...

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

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.

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 ...

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Research

JoVE Journal

Genetics

15.1K Views.  Duke University School of Medicine. We beschrijven de strategie van de productie van integrase-deficiënte lentivirale vectoren (IDLVs) als voertuigen voor het leveren van CRISPR/Cas9 naar de cellen. Met een vermogen om te bemiddelen snelle en robuuste gene bewerken in cellen, IDLVs presenteren een veiliger en even effectieve vector platform voor de levering van het gen in vergelijking met integrase-bevoegde vectoren.

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

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Development of Knock-Out Muscle Cell Lines using Lentivirus-Mediated CRISPR/Cas9 Gene Editing

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, specifically to study the function of new genes/proteins associated with a disease, identified during the genetic diagnosis. For the development of such cell lines, two major issues have to be untangled: insertion of the CRISPR tools (the Cas9 and the guide RNA) with high efficiency into the chosen cells, and restriction of the Cas9 activity to the specific deletion of the chosen gene. The protocol described here is dedicated to the insertion of the CRISPR tools in difficult to transfect cells, such as muscle cells. This protocol is based on the use of lentiviruses, produced with plasmids publicly available, for which all the cloning steps are described to target a gene of interest. The control of Cas9 activity has been performed using an adaptation of a previously described system called KamiCas9, in which the transduction of the cells with a lentivirus encoding a guide RNA targeting the Cas9 allows the progressive abolition of Cas9 expression. This protocol has been applied to the development of a RYR1-knock out human muscle cell line, which has been further characterized at the protein and functional level, to confirm the knockout of this important calcium channel involved in muscle intracellular calcium release and in excitation-contraction coupling. The procedure described here can easily be applied to other genes in muscle cells or in other difficult to transfect cells and produce valuable tools to study these genes in human cells.

The protocol describes how to generate knock-out myoblasts using CRISPR/Cas9, starting from the design of guide-RNAs to the cellular cloning and characterization of the knock-out clones.

One important application of clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas 9 is the development of knock-out cell lines, ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Biology

Designing, Packaging, and Delivery of High Titer CRISPR Retro and Lentiviruses via Stereotaxic Injection

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique has been widely used to encode fluorescent proteins, opto- or chemo-genetic controllers of cell activity, or heterologous expression of human genes in model organisms. These viruses have also successfully been used to deliver recombinases to relevant target sites in transgenic animals, or even deliver small hairpin or micro RNAs in order to manipulate gene expression. While these techniques have been fruitful, they rely on transgenic animals (recombinases) or frequently lack high efficacy and specificity (shRNA/miRNA). In contrast, the CRISPR/Cas system uses an exogenous Cas nuclease which targets specific sites in an organism's genome via an exogenous guide RNA in order to induce double stranded breaks in DNA. These breaks are then repaired by non-homologous end joining (NHEJ), producing insertion and deletion (indel) mutations that can result in deleterious missense or nonsense mutations. This manuscript provides detailed methods for the design, production, injection, and validation of single lenti/retro virus particles that can stably transduce neurons to express a fluorescent reporter, Cas9, and sgRNAs to knockout genes in a model organism.

The CRISPR/Cas9 system offers the potential to make targeted genome editing accessible and affordable to the scientific community. This protocol is intended to demonstrate how to create viruses that will knockout a gene of interest using the CRISPR/Cas9 system, and then inject them stereotaxically into the adult mouse brain.

Replication defective lentiviruses or retroviruses are capable of stably integrating transgenes into the genome of an infected host cell. This technique ...

Neuroscience

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Immunology and Infection

Lentiviral Vector Preparation for In Vivo Gene Modulation 

Lentiviral Vector Preparation for Efficient Gene and MicroRNA Modulation of Peritoneal Cavity Tissue&#45;Resident Macrophages In Vivo in Mice

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring tissues. Their functions are dictated by microenvironmental cues; thus, it is essential to investigate their behavior in an in vivo physiological niche. Currently, specific peritoneal macrophage-targeting methodologies employ whole-mouse transgenic models. Here, a protocol for effective in vivo modulation of mRNA and small RNA species (e.g., microRNA) expression in peritoneal macrophages using lentivirus particles is described. Lentivirus preparations were made in HEK293T cells and purified on a single sucrose layer. In vivo validation of lentivirus effectivity following intraperitoneal injection revealed predominant infection of macrophages restricted to local tissue. Targeting of peritoneal macrophages was successful during homeostasis and thioglycolate-induced peritonitis. The limitations of the protocol, including low-level inflammation induced by intraperitoneal delivery of lentivirus and time restrictions for potential experiments, are discussed. Overall, this study presents a quick and accessible protocol for the rapid assessment of gene function in peritoneal macrophages in vivo.

Author Spotlight: Exploring Macrophage Immunometabolism Through Lentiviral Vector&#45;Mediated Gene Manipulation

We demonstrate a step-by-step protocol for the investigation of gene function in peritoneal tissue-resident macrophages in vivo, using lentiviral vectors.

Peritoneal tissue-resident macrophages have broad functions in the maintenance of homeostasis and are involved in pathologies within local and neighboring ...

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB transposon machinery, a transcriptionally regulated hyperactive transposase (SB100X) and T2-based transposon are employed. Typically, the transposase and transposon are provided transiently by plasmid transfection and SB100X expression is driven by a constitutive promoter. Here, we describe an efficient method to deliver the SB components to human cells that are resistant to several physical and chemical transfection methods, to control SB100X expression and stably integrate a gene of interest (GOI) through a &#34;cut and paste&#34; SB mechanism. The expression of hyperactive transposase is tightly controlled by the Tet-ON system, widely used to control gene expression since 1992. The gene of interest is flanked by inverted repeats (IR) of the T2 transposon. Both SB components are packaged in integration defective lentiviral vectors transiently produced in HEK293T cells. Human cells, either cell lines or primary cells from human tissue, are in vitro transiently transduced with viral vectors. Upon addition of doxycycline (dox, tetracycline analog) into the culture medium, a fine-tuning of transposase expression is measured and results in a long-lasting integration of the gene of interest in the genome of the treated cells. This method is efficient and applicable to the cell line (e.g., HeLa cells) and primary cells (e.g., human primary keratinocytes), and thus represents a valuable tool for genetic engineering and therapeutic gene transfer.

An Efficient In Vitro Transposition Method by a Transcriptionally Regulated Sleeping Beauty System Packaged into an Integration Defective Lentiviral Vector

This protocol describes a method to achieve stable integration of a gene of interest into the human genome by the transcriptionally regulated Sleeping Beauty system. Preparation of the integration of defective lentiviral vectors, the in vitro transduction of human cells, and the molecular assay on transduced cells are reported.

The Sleeping Beauty (SB) transposon is a non-viral integrating system with proven efficacy for gene transfer and functional genomics. To optimize the SB ...

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery system.

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical ...

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

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