Name: a protocol for the production of integrase-deficient lentiviral vectors for crispr/cas9-mediated gene knockout in dividing cells
Uploaded: 2017-12-12T14:00:00
Description: Watch this Scientific Journal Video about A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells at JoVE.com

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...

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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>Equipment</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-&#956;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 &#956;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Cell culture reagents</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>p24 ELISA reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal anti-p24 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-p24 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Restriction enzymes</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...

Watch this Scientific Journal Video about A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells at JoVE.com

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 ...

The overall goal of this protocol is to describe the production and potential applications of optimized integrase-deficient lentiviral vectors capable of delivering CRISPR/Cas9 transgenes to cells in vivo and in vitro for rapid and efficient gene editing. CRISPR/Cas was a highly-efficient lab for gene editing manipulation. Here is a protocol outline in the production of a highly-concentrated optimized integrase-deficient lentiviral vectors for efficient delivery of CRISPR/Cas gene editing tools.The technique has proven to be very efficient in the causal cell and post-mitotic neurons. Seed the HEK-293T cells a day before the transfection so that the confluency reaches between 70 to 80%the next day. Before transfection, aspirate the old media from the culture plates and add freshly-prepared media lacking serum.In a 15-milliliter conical tube, add all the four plasmids to prepare a plasmid mix for transfecting a 15-centimeter dish. Then, add 312.5 microliters of one molar calcium chloride to the plasmid mix and adjust the volume to 1.25 milliliters with sterile double-distilled water. Keep adding drops of 2X BES buffered solution gradually while vortexing.And then, incubate the mixture at room temperature for 30 minutes. Add 2.5 milliliters of plasmid mixture in drops to each of the 15-centimeter plates. Then, gently swirl the plates and incubate at 37 degrees Celsius with 5%carbon dioxide for two to three hours.After three hours, add 2.5 milliliters of 10%serum to each plate and continue for overnight incubation. Use a 10-milliliter tissue culture pipette to carefully aspirate the supernatant from the transfected cells seeded on each culture dish. Then, pool the entire supernatant in 50-milliliter centrifuge tubes.Place the tube inside a tabletop centrifuge and start the centrifugation. Then, filter the supernatant through a 0.45-micrometer vacuum filter unit. Load the conical ultracentrifugation tubes with different sucrose concentrations to form layers with sucrose gradient.Carefully add the virus containing supernatant to the sucrose gradient. To process the entire 100-milliliter volume of viral supernatant, use six ultracentrifugation tubes in each spin. Then, balance the ultracentrifuge tubes with 1X PBS buffer and spin the samples at 70, 000 G for two hours at 17 degrees Celsius.In clean tubes, collect the 30%and 60%sucrose fractions and add cold 1X PBS buffer to adjust the volume to 100 milliliters. Mix the samples by pipetting it up and down several times. Then, add 20 to 25 milliliters of the viral solution on top of four milliliters of 20%sucrose in 1X PBS solution to prepare the sucrose cushion.Adjust the volume with sterile 1X PBS buffer if the tubes are less than 3/4 full and balance it. Then, spin the samples for two hours at 17 degrees Celsius. Then, pour the supernatant off and drain the remaining liquid by inverting the tubes on paper towels.After aspirating the last remaining droplets, a pellet containing the virus is barely visible as a translucent spot. Next, resuspend the pellet in 70 microliters of 1X PBS by thoroughly pipetting the suspension. Again, transfer the suspension to another tube and thoroughly mix to completely dissolve the pellet.Then, rinse the tube with an additional 50 microliters of cold 1X PBS buffer and mix. Keep the samples on a tabletop microcentrifuge and centrifuge at 10, 000 G for 30 seconds. After centrifugation, add 10 microliter aliquots of the supernatant to each fresh microfuge tube and store them at negative 80 degrees Celsius.Coat a high-binding 96-well plate with 100 microliters of monoclonal anti-p24 antibody at a dilution of one to 1, 500 according to NIH AIDS vaccine program for HIV-1 p24 antigen capture assay kit. Incubate the plate overnight at four degrees Celsius. The next day, wash the plate with 200 microliters of 0.05%TWEEN-20 in cold PBS three times.To avoid non-specific binding, block the plate with 200 microliters of 1%bovine serum albumin for one hour at room temperature. Then, wash the plate with 200 microliters of 0.05%TWEEN-20 in cold PBS three times. Then, prepare the concentrated and non-concentrated vector samples to a final concentration of 10%by diluting the vector with double-distilled water and Triton X-100.Next, prepare the HIV-1 standards using two-fold serial dilution starting at five nanograms per milliliter. After diluting the concentrated and non-concentrated vector samples, pipette it on the plate in triplicates and incubate at four degrees Celsius overnight. The next day, wash the plate, then add 100 microliters of polyclonal rabbit anti-p24 antibody on each plate and incubate at 37 degrees Celsius for four hours.After six washes, incubate the plate with goat anti-rabbit horseradish peroxidase at 37 degrees Celsius for one hour. After multiple washing, incubate the plate with TMB peroxidase substrate at room temperature for 15 minutes. Add one normal hydrochloric acid to stop the reaction.And read the absorbance at 450 nanometers with an absorbance plate reader. Seed the cells in a six-well plate. Next day, when the cells reach 90%confluency, transduce with purified virus at a predetermined multiplicity of infection ranging from one to 10.Then, incubate the plate at 37 degrees Celsius with 5%carbon dioxide. Monitor the plates to record difference in the GFP signals at regular intervals for one to seven days. Look for GFP positive cells under fluorescent microscope fitted with GFP filter set with excitation and emission wavelengths at 470 and 525 nanometers respectively.To differentiate between the population of GFP positive and negative cells, use untransduced cells. A representative agarose gel showing the positive mutated integrase clone. The double-digested clone with restriction enzymes EcoRV and SPH1 in red-dashed box represents the mutated integrase clone.The clone was further sequenced to check the mutation of aspartic acid at position 64 to glutamic acid. A representative bar graph for p24 ELISA assay has been shown depicting the viral titers for SP1-integrase-deficient lentiviral vector CRISPR/Cas9 plotted in the black bar and SP1-integration-competent lentiviral vectors CRISPR/Cas9 plotted in the white bar. The bar graph shows integrase-deficient and competent viral titers in the range of one times 10 to the 10.The bar plots represent the copy number of viral particles per milliliter. Representative fluorescent images showing the mean fluorescent intensity of GFP positive cells under viral treatment. The cells were transduced with integration-competent lentiviral vectors CRISPR or integrase-deficient lentiviral vector CRISPR viral components.A sharp reduction in GFP signal was observed at multiplicity of infection one and five respectively after transducing the integrase-competent or deficient lentiviral components with respect to the untransduced control cells. The main advantage of this technique stem from the ability of integrase-deficient lentiviral vectors to a deliver genome editing tool transiently which significantly enhance its specificity and the safety. If properly conducted, the protocols should take one week to complete.Conducting this protocol, one should expect to generate highly-concentrated lentiviral vectors in the range of 10 to the nine, 10 to the 10 viral genome parameter.

a-protocol-for-production-integrase-deficient-lentiviral-vectors-for

Research

JoVE Journal

Genetics

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...