Name: delivery of the cas9/sgrna ribonucleoprotein complex in immortalized and primary cells via virus-like particles ("nanoblades")
Uploaded: 2021-03-31T13:00:00
Duration: 9 min 23 s
Description: Watch this Scientific Journal Video about Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades" at JoVE.com

This work was funded by Labex Ecofect (ANR-11-LABX-0048) of the Universit&#233; de Lyon, within the program Investissements d&#39;Avenir (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), Fondation FINOVI, Agence Nationale des Recherches sur le SIDA et les H&#233;patites Virales (ANRS-ECTZ3306) and by the European Research Council (ERC-StG-LS6-805500 to E.P.R.) under the European Union&#39;s Horizon 2020 research and innovation programs.

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Philippe E. Mangeot and Emiliano P. Ricci are named as inventors on a patent relating to the Nanoblades technology (patent applicants: Institut National de la Sant&#233; et de la Recherche M&#233;dicale (INSERM), Centre National de la Recherche Scientifique (CNRS), Ecole Normale Superieure de Lyon, Universit&#233; Claude Bernard Lyon 1, Villeurbanne Cedex; application number: WO 2017/068077 Al; patent status: published, 27th April 2017; all aspects of the manuscript are covered by the patent application. The remaining authors declare no competing interests.

Nanoblades allow for rapid and dose-dependent delivery of the Cas9/sgRNA RNP complex in cell lines and primary cells. In contrast to classical transfection and other viral delivery vectors, but like protein electroporation, Nanoblades have the advantage of transient delivery of the Cas9/sgRNA RNP in a transgene-free manner. Nanoblades offer a highly versatile, simple, and inexpensive platform for protein delivery that can be easily and rapidly adapted to the ever-expanding family of CRISPR variants. Nanoblades can be produced in the HEK293T cell line or its derivatives. HEK293T cell lines used here have been developed to maximize retroviral and lentiviral particle production (see the Table of Materials). However, although other sources of HEK293T cells may be suitable, users must test and compare HEK293T cells from different sources as major differences in particle production have been observed depending on the HEK293T cell-source. Cells have also to be checked for Mycoplasma contamination frequently and passaged every three days (classically &#8539; dilution) to avoid overconfluence, which has a negative impact on particle production.
Cells should not be maintained for over 20 passages. DMEM supplemented with glucose, penicillin/streptomycin, glutamine, and 10% decomplemented fetal bovine serum was used for cell culture. As serum origin may affect the quality of the Nanoblade preparation, different batches of serum should be tested before large-scale production. Nanoblades can be efficiently produced in other media such as RPMI or serum-free modifications of minimum essential medium that can replace DMEM on the day after transfection. As indicated below, although medium replacement after transfection with some DNA-transfection reagents is optional, it may be beneficial to modify the medium into which the VLPs are released, especially to limit serum traces in the particle preparation. However, cultivation of cells in reduced-serum minimum essential medium on the day before transfection has not yet been attempted.
As mentioned, Nanoblades are produced upon overexpression of a mixture of plasmids in producer cells. The overexpression appears to be required for optimal production. Indeed, this laboratory developed a producer cell line where the Gag-Pol-expressing construct was stabilized by antibiotic selection; however, this system failed to produce significant amounts of Nanoblades. A similar observation was made when the sgRNA-coding construct was stably integrated into the genome of producer cells. As described for other particle production systems, a stable cell line expressing at least some constructs involved in Nanoblades production may be useful; however, this would certainly require the processing of large volumes of supernatant and an appropriate technique to purify particles. The above protocol outlines the preferred procedure to produce Nanoblades that exploits specific transfection reagents (see the Table of Materials).
Although transfection reagents from other manufacturers have also been tested with success, the vast majority of this group&#39;s results with Nanoblades follow the procedure described herein. Low-cost transfection can be achieved using calcium phosphate reagents and yield good production efficiency; however, this method absolutely requires the replacement of transfection medium on the day after transfection and may leave calcium phosphate residues in the sedimented particle preparation. Consistent with the necessity of high expression levels for Nanoblade components within producer cells is the observation that the amount of sgRNAs associated with the Cas9 protein can be a limiting factor for efficient genome-editing. To improve sgRNA loading, two technical approaches have been recently developed by independent groups using protein delivery vectors similar to Nanoblades. These rely on the use of T7 polymerase-dependent cytoplasmic expression of sgRNA6 or through the addition of a retroviral encapsidation signal to the sgRNA sequence to mediate binding to the Gag polyprotein6. These approaches could indeed improve sgRNA loading within Nanoblades although they have not been tested yet.
Transduction of target cells is a critical step in the procedure. In most immortalized cell lines, transduction with Nanoblades has little or no cytopathic effect. However, in primary cells, toxicity can be an issue. Transduction has therefore to be optimized for each cell type. Specifically, the exposure time to Nanoblades is an important factor to modify when optimizing the transduction protocol. For sensitive cells such as primary neurons or bone marrow cells, 4-6 h of incubation with Nanoblades before replacing the medium allows for efficient delivery of the Cas9 protein while minimizing cell toxicity. Furthermore, adjuvants such as cationic polymers, among others, can significantly improve the efficiency of transduction in some cells (see the Table of Materials). It is important to note that Nanoblades are VLPs and can induce an immunogenic response. This can be a limitation if working with certain types of primary cells, such as macrophages or dendritic cells, in which incubation with Nanoblades could induce important changes in gene expression and the phenotype of the cells. If macrophages and dendritic cells are derived from hematopoietic stem cell precursors (such as mouse bone marrow cells), it is preferable to transduce cells with the Nanoblades before they are fully differentiated to avoid inducing a cellular response against the Nanoblades. Otherwise, Cas9 protein electroporation could represent a viable alternative when working with differentiated immune cells.
Nanoblades can be used in vivo to transduce mouse zygotes or embryos to generate transgenic animals. Similar to classical retroviral or lentiviral vectors, they can also be injected directly into tissues from adult animals. However, Nanoblades (similar to retroviral and lentiviral vectors) can be inactivated by the immune response of the host animal; hence, the dose to be injected has to be optimized for each application. This immune response can also limit the distribution of functional VLPs to tissues close to the injection site. Finally, unlike lentiviral vectors, Nanoblades are transgene-free and deliver the Cas9 in a restricted timeframe. Therefore, they cannot be used to perform genome-wide functional screenings that require high-throughput sequencing of sgRNAs upon selection of cells. Nanoblades are useful when rapid, dose-dependent, and transgene-free genome-editing is required20. Furthermore, similar to protein electroporation, Nanoblades lead to fewer off-target effects than prolonged expression of Cas9/sgRNA through DNA transfection or classical viral vectors3. Future development of Nanoblades is focused on incorporating Cas9 variants for different technological applications such as base-editing and RNA targeting.

Compared to other programmable nucleases, the CRISPR-Cas system dramatically simplified and democratized the procedure of sequence-specific genome targeting and cleavage in eukaryotic cells. Through the simple expression of a sgRNA, users can program the Cas9 protein (or optimized variants) for almost any cellular locus1. In this scenario, delivery of the Cas9 protein and sgRNA becomes the main limitation when performing site-directed mutagenesis. In immortalized cells, the sgRNA and the Cas protein can be easily expressed from transfected plasmids to achieve efficient genome targeting in most cells. However, constitutive expression of the Cas9/sgRNA complex can increase off-target activity of the Cas9 protein and introduce undesired changes in non-specific loci2. In primary cells, DNA transfection can be technically difficult to achieve and lead to poor expression or a small percentage of transfected cells. Alternatives to classic DNA transfection comprise the use of viral vectors that deliver a transgene coding for the Cas9 and sgRNA or the electroporation of recombinant Cas9 protein coupled to a synthetic sgRNA. However, these approaches can lead to transgene integration within the cell host genome (as is the case for classical retroviral and lentiviral expression vectors), restriction by cellular factors, and lead to constitutive expression of the Cas9 protein and sgRNA.
Electroporation of the Cas9/sgRNA RNP complex can overcome most of these problems and lead to efficient and transient delivery in primary cells and in vivo as well as allow a dose-dependent response. Nevertheless, it usually relies on expensive equipment and reagents and is also difficult to upscale if a large number of cells have to be treated. As an alternative to the above-mentioned techniques, these authors have developed &#34;Nanoblades&#34;-a retroviral delivery vector for the Cas9 protein and sgRNA3 that is conceptually similar to other viral-derived capsid protein delivery systems4,5,6,7,8. Nanoblades exploit the natural capacity of the Gag polyprotein from retroviruses to produce, when expressed alone in cultured cells, VLPs that are released in the extracellular medium9. By fusing the Cas9 protein to the C-terminal end of the murine leukemia virus (MLV) Gag polyprotein and co-expressing the sgRNA and viral envelope glycoproteins, the Cas9 protein can be encapsidated within released VLPs or Nanoblades. Upon purification, the Nanoblades can be incubated with target cells or injected in vivo to mediate rapid, transient, and dose-dependent delivery of the Cas9/sgRNA RNP complex3.
Nanoblades can be programmed with multiple sgRNAs for simultaneous editing at different loci or with Cas9 variants to perform other applications such as target-specific transcriptional activation or repression3. In contrast to protein electroporation, which relies on recombinant expression, newly described Cas variants from the literature can be easily cloned into the Gag fusion expression vector, making it a versatile platform. Nanoblades can be further complexed or loaded with single-stranded and double-stranded oligodeoxynucleotides (ssODNs) to perform homology-directed repair3. Nanoblade production is relatively simple and cheap. Moreover, Nanoblades can be stored at 4 &#176;C for many days or at -80 &#176;C for long-term storage. Typically, Nanoblades mediate efficient, transgene-free genome-editing in most immortalized and primary cultured cells. However, some primary cells might be sensitive to the presence of viral particles, resulting in increased mortality. Cells of the innate immune system can also react to the presence of Nanoblades (because of their viral origin) and become activated. In these cases, the transduction protocol has to be optimized to limit the exposure time to Nanoblades and minimize nonspecific effects. Nanoblades represent a viable and easy-to-implement alternative to other available CRISPR delivery methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13.2 mL, Thinwall Polypropylene Tubes, 14 x 89 mm - 50Pk</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>331372</td>
			<td>Ultracentrifugation tubes for Nanoblades purification</td>
		</tr>
		<tr>
			<td>Amersham Protran Premium Western blotting membranes, nitrocellulose</td>
			<td>Merck</td>
			<td>GE10600004</td>
			<td>Nitrocellulose membrane for quantifying Cas9 levels within purified Nanoblades</td>
		</tr>
		<tr>
			<td>BIC-Gag-CAS9</td>
			<td>Addgene</td>
			<td>119942</td>
			<td>Encodes a GAG (F-MLV)-CAS9(sp) fusion. Allows the production of GAG-CAS9 Virus like particles from producer cells in association with over expressed gRNA(s) and appropriate envelopes</td>
		</tr>
		<tr>
			<td>BICstim-Gag-dCAS9-VPR</td>
			<td>Addgene</td>
			<td>120922</td>
			<td>Encodes a GAG-dCAS9-VPR fusion for targeted transcriptional activation</td>
		</tr>
		<tr>
			<td>BLADE</td>
			<td>Addgene</td>
			<td>134912</td>
			<td>Empty backbone for cloning sgRNA sequence to be used in Nanoblades system</td>
		</tr>
		<tr>
			<td>BsmBI-v2</td>
			<td>New England Biolabs</td>
			<td>R0739S</td>
			<td>Restriction enzyme to digest the BLADE and SUPERBLADES vectors for sgRNA cloning</td>
		</tr>
		<tr>
			<td>Cas9 (7A9-3A3) Mouse mAb (HRP Conjugate) #97982</td>
			<td>Cell Signaling Technology</td>
			<td>97982S</td>
			<td>Anti-Cas9 antibody for Cas9 quantification by dot-blot</td>
		</tr>
		<tr>
			<td>Cas9 Nuclease, S. pyogenes</td>
			<td>New England Biolabs</td>
			<td>M0386T</td>
			<td>Recombinant Cas9 protein to be used as a reference for absolute quantification of the amount of Cas9 loaded within Nanoblades</td>
		</tr>
		<tr>
			<td>Ethidium bromide solution (10 mg/mL in H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>E1510-10ML</td>
			<td>For staining agarose gels and visualize DNA</td>
		</tr>
		<tr>
			<td>Fisherbrand Wave Motion Shakers</td>
			<td>Fisher Scientific</td>
			<td>88-861-028</td>
			<td>Agitation table to resuspend Nanoblades upon centrifugation</td>
		</tr>
		<tr>
			<td>gelAnalyzer</td>
			<td></td>
			<td></td>
			<td>http://www.gelanalyzer.com; quantifying band intensity after digestion</td>
		</tr>
		<tr>
			<td>Gesicle Producer 293T</td>
			<td>Takara</td>
			<td>632617</td>
			<td>Nanoblades producer cell line</td>
		</tr>
		<tr>
			<td>Gibco DMEM, high glucose, pyruvate</td>
			<td>ThermoFisher Scientific</td>
			<td>41966052</td>
			<td>Cell culture medium for Gesicle Producer 293T cells</td>
		</tr>
		<tr>
			<td>GoTaq G2 DNA Polymerase</td>
			<td>Promega</td>
			<td>M7848</td>
			<td>Taq polymerase for amplification of genomic DNA before T7 endonuclease assays</td>
		</tr>
		<tr>
			<td>jetPRIME Transfection Reagent kit for DNA and DNA/siRNA</td>
			<td>Polyplus</td>
			<td>POL114-15</td>
			<td>Transfection reagent for Nanoblade production in Gesicle Producer 293T cells</td>
		</tr>
		<tr>
			<td>Millex-AA, 0.80 &#181;m, syringe filter</td>
			<td>Millipore</td>
			<td>SLAA033SS</td>
			<td>Syringe filter to remove cellular debris before concentration of Nanoblades</td>
		</tr>
		<tr>
			<td>Millex-GS, 0.22 &#181;m, syringe filter</td>
			<td>Millipore</td>
			<td>SLGS033SS</td>
			<td>Syringe filter to sterilise the sucrose cushion solution</td>
		</tr>
		<tr>
			<td>Millex-HP, 0.45 &#181;m, polyethersulfone, syringe filter</td>
			<td>Millipore</td>
			<td>SLHP033RS</td>
			<td>Syringe filter to remove cellular debris before Nanoblades concentration</td>
		</tr>
		<tr>
			<td>Monarch DNA Gel Extraction Kit</td>
			<td>New England Biolabs</td>
			<td>T1020L</td>
			<td>DNA gel extraction kit for purification of the pBLADES or pSUPERBLADES plasmid fragment upon digestion with BsmBI</td>
		</tr>
		<tr>
			<td>NEB Stable Competent E. coli (High Efficiency)</td>
			<td>New England Biolabs</td>
			<td>C3040I</td>
			<td>Competent bacteria for plasmid transformation and amplification</td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Midi kit for transfection-grade plasmid DNA</td>
			<td>Macherey-Nagel</td>
			<td>740410.50</td>
			<td>Maxipreparation kit for purification of plasmid DNA from cultured bacteria</td>
		</tr>
		<tr>
			<td>Nucleospin gDNA extraction kit</td>
			<td>Macherey-Nagel</td>
			<td>740952.50</td>
			<td>Extraction of genomic DNA from transduced cells</td>
		</tr>
		<tr>
			<td>NucleoSpin Plasmid, Mini kit for plasmid DNA</td>
			<td>Macherey-Nagel</td>
			<td>740588.50</td>
			<td>Minipreparation kit for purification of plasmid DNA from cultured bacteria</td>
		</tr>
		<tr>
			<td>NucleoSpin Tissue, Mini kit for DNA from cells and tissue</td>
			<td>Macherey-Nagel</td>
			<td>740952.5</td>
			<td>Genomic DNA extraction kit</td>
		</tr>
		<tr>
			<td>Optima XE-90</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>A94471</td>
			<td>Ultracentrifuge</td>
		</tr>
		<tr>
			<td>pBaEVRless</td>
			<td>Els Verhoeyen (Inserm U1111)</td>
			<td>Personnal requests have to be sent to: els.verhoyen@ens-lyon.fr</td>
			<td>Baboon Endogenous retrovirus Rless glycoprotein described in Girard-Gagnepain, A. et al. Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs. Blood 124, 1221&#8211;1231 (2014)</td>
		</tr>
		<tr>
			<td>pBS-CMV-gagpol</td>
			<td>Addgene</td>
			<td>35614</td>
			<td>Enocdes the Murine Leukemia Virus gag and pol genes</td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>8454</td>
			<td>Envelope protein for producing lentiviral and MuLV retroviral particles</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>ThermoFisher Scientific</td>
			<td>14200091</td>
			<td>10X PBS to dilute in millipore water</td>
		</tr>
		<tr>
			<td>Polybrene Transfection Reagent</td>
			<td>Millipore Sigma</td>
			<td>TR-1003-G</td>
			<td>Cationic polymer that enhances the efficiency of retroviral transduction in specific mammalian cells. It can also allow viral-dependent entry of an Oligodeoxynucleotide (ODN) for homology-directed repair</td>
		</tr>
		<tr>
			<td>Sucrose,for molecular biology, &#8805;99.5% (GC)</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-5KG</td>
			<td>Sucrose to prepare a cushion for Nanoblade purification through ultracentrifugation</td>
		</tr>
		<tr>
			<td>SUPERBLADE5</td>
			<td>Addgene</td>
			<td>134913</td>
			<td>Empty backbone for cloning sgRNA sequence to be used in nanoblades system (Optimized for increased genome editing efficiency via Chen B et al., 2013)</td>
		</tr>
		<tr>
			<td>SuperSignal West Dura Extended Duration Substrate</td>
			<td>ThermoFisher Scientific</td>
			<td>34076</td>
			<td>Enhanced chemiluminescence (ECL) HRP substrate for Cas9 dot blots</td>
		</tr>
		<tr>
			<td>SW 41 Ti Swinging-Bucket Rotor</td>
			<td>Beckman Coulter Life Sciences</td>
			<td>331362</td>
			<td>Rotor for ultracentrifugation</td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td>Alternative to ethidium bromide for staining agarose gels and visualize DNA</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td>DNA ligase to ligate the BLADE or SUPERBLADES vectors with the duplexed DNA oligos corresponding to the variable region of the sgRNA</td>
		</tr>
		<tr>
			<td>T7 Endonuclease I</td>
			<td>New England Biolabs</td>
			<td>M0302S</td>
			<td>T7 Endonuclease I recognizes and cleaves non-perfectly matched DNA. Allows to monitor the extent of genome editing at a specific locus</td>
		</tr>
		<tr>
			<td>Triton-containing lysis buffer</td>
			<td>Promega</td>
			<td>E291A</td>
			<td>Lysis buffer to disrupt Nanoblades and allow Cas9 quantification</td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td>For the preparation of TBST</td>
		</tr>
	</tbody>
</table>

delivery of the cas9/sgrna ribonucleoprotein complex in immortalized and primary cells via virus-like particles ("nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the development of numerous innovative applications. However, delivery of the Cas9 protein and single-guide RNA (sgRNA) into target cells can be technically challenge. Classical viral vectors, such as those derived from lentiviruses (LVs) or adeno-associated viruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9 protein and its associated sgRNA in many primary cells and in vivo. Nevertheless, these vectors can suffer from drawbacks such as integration of the transgene in the target cell genome, a limited cargo capacity, and long-term expression of the Cas9 protein and guide RNA in target cells.
To overcome some of these problems, a delivery vector based on the murine Leukemia virus (MLV) was developed to package the Cas9 protein and its associated guide RNA in the absence of any coding transgene. By fusing the Cas9 protein to the C-terminus of the structural protein Gag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA (named &#34;Nanoblades&#34;) were formed. Nanoblades can be collected from the culture medium of producer cells, purified, quantified, and used to transduce target cells and deliver the active Cas9/sgRNA complex. Nanoblades deliver their ribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary and immortalized cells and can be programmed for other applications, such as transient transcriptional activation of targeted genes, using modified Cas9 proteins. Nanoblades are capable of in vivo genome-editing in the liver of injected adult mice and in oocytes to generate transgenic animals. Finally, they can be complexed with donor DNA for &#34;transfection-free&#34; homology-directed repair. Nanoblade preparation is simple, relatively low-cost, and can be easily carried out in any cell biology laboratory.

The protocol for Nanoblade preparation is fairly straightforward and requires simple laboratory equipment besides access to a tissue culture hood, a CO2 incubator, and a swinging bucket centrifuge or an ultracentrifuge. However, some steps require particular attention such as the source and handling of producer cells, as well as transduction conditions. As shown in Figure 1A, it is important to seed cells so that they are homogeneously distributed in the plate and reach ~70-80% confluence on the day of transfection (avoid having clumps of cells). Twenty-four hours after transfection (Figure 1B,C), producer cells will form syncytia leading to cells of larger size with multiple nuclei. Forty hours after transfection (Figure 1D), most cells in the plate will have formed syncytia and start detaching from the plate.
This is perfectly normal and is caused by the expression of the envelope glycoprotein, which induces fusion between neighboring cells. Upon concentration by centrifugation (or even straight from the supernatant of producer cells), the amount of Cas9 loaded within Nanoblades can be quantified in an absolute manner by dot-blot on a nitrocellulose membrane using recombinant Cas9 as reference (Figure 2). This step is important to determine the correct amount of Nanoblades to use for transduction of target cells. When performing the dot-blot assay, it is important to consider only the readings that fall within the linear range of the standard curve. However, independently of the amount of Cas9 present within Nanoblades, it is essential to test the efficiency of genome editing directly on target cells using the T7 endonuclease assay (Figure 3) or Sanger sequencing.
As shown in Figure 3, the efficiency of Nanoblades can differ from batch to batch although it is usually correlated to the amount of Cas9. In the example shown in Figure 3, the batch from lane 1 leads to 20% overall editing efficiency while the batch from lane 3 leads to 60% efficiency. In this case, it is possible to increase the volume of Nanoblades used from batch 1 to achieve an editing efficiency similar to that from batch 3. Figure 4 shows the maximum editing efficiency obtained using Nanoblades in different types of primary cells. It is important to note that the efficiency may vary depending on the sequence of the sgRNA used and the target accessibility.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62245/62245fig01.jpg" /> 
Figure 1: Morphology of producer cells during Nanoblade production. (A) HEK293T cells at 70-80% confluence 24 h after plating. (B and C) HEK293T cell morphology 24 h after transfection. (D) HEK293T cell morphology 40 h after transfection. Scale bars = 400 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62245/62245fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62245/62245fig02.jpg" /> 
Figure 2: Quantification of Cas9 loading within Nanoblades by dot-blot. (A) Recombinant Cas9 or 100x concentrated (by ultracentrifugation) Nanoblade samples (#1, #2, and #3) are diluted 2-fold sequentially and spotted on a nitrocellulose membrane before incubating with anti-Cas9 HRP-coupled antibodies. Signal is revealed by enhanced chemiluminescence. (B) Chemiluminescence signal is acquired and quantified for the recombinant Cas9 dilutions and signal intensity plotted against the known amount of Cas9 spotted on the nitrocellulose membrane. A regression curve is calculated for the dilutions that are within the linear range (see blue crosses), excluding all concentrations that are outside of the linear range (see red crosses). (C) Cas9 concentration (nM) in each Nanoblade preparation was extrapolated using the equation from the linear regression obtained in (B). For this, it is important to only use the quantified signal from the Nanoblade dilutions that fall within the linear range of the regression curve. <a href="https://www.jove.com/files/ftp_upload/62245/62245fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62245/62245fig03.jpg" /> 
Figure 3: Monitoring editing efficiency upon transduction. (A) T7 endonuclease assay measuring cleavage efficiency in Nanoblade-treated cells. Cells transduced with Nanoblades targeting the EMX1 gene were analyzed by T7 endonuclease assay. Lane 1: Nanoblade preparation batch #1 (20% cleavage efficiency); Lane 2: Control cells; Lane 3: Nanoblade preparation batch #2 (60% cleavage efficiency). (B) Knock-in of the Flag-tag sequence within the DDX3 open reading frame. Concentrated Nanoblades programmed with an sgRNA targeting the DDX3 locus were produced from different HEK293T clones (#1, #2) and complexed with increasing doses of a Flag-DDX3 ssODN template and the obtained complexes used for the transduction of HEK293T target cells. Upon transduction, cells were grown for three days before collecting them to extract genomic DNA and total proteins. Flag-DDX3 proteins were immunoprecipitated using anti-Flag agarose beads followed by western-blot analysis of the recovered proteins using an anti-Flag antibody (top panel). Site-directed insertion of the Flag-tag in the Ddx3 locus was also assayed by PCR using either primers flanking the insertion site (middle panel), or using a forward primer that recognizes the Flag-tag sequence and a reverse primer specific to the Ddx3 locus downstream of the Flag insertion site (bottom panel). Abbreviations: EMX1 = Empty Spiracles Homeobox 1; DDX3 = DEAD-box RNA helicase 3; PCR = polymerase chain reaction; ODN = oligodeoxynucleotide; ssODN = sing-stranded ODN; sgRNA = single-guide RNA; IP = immunoprecipitation. <a href="https://www.jove.com/files/ftp_upload/62245/62245fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62245/62245fig04.jpg" /> 
Figure 4: Editing efficiency achieved in different primary cell types using Nanoblades. Abbreviations: PBL = peripheral blood lymphocyte; IL = interleukin; CD = cluster of differentiation; iPSC = induced pluripotent stem cell. <a href="https://www.jove.com/files/ftp_upload/62245/62245fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades" at JoVE.com

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"

We have developed a simple and inexpensive protocol to load Cas9/single-guide RNA (sgRNA) ribonucleoprotein complexes within virus-like particles. These particles, called "Nanoblades", allow efficient delivery of the Cas9/sgRNA complex in immortalized and primary cells as well as in vivo.

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades")

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has democratized genome-editing in eukaryotic cells and led to the ...

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