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.

<ol>
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E., Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+analysis+of+CRISPR-Cas+experiments.">Design and analysis of CRISPR-Cas experiments.</a> Nature Biotechnology. 38 (7), 813-823 (2020).</li><li>Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning a laboratory manual. Third edition. , (2001).</li><li>Jiang, W., 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=An+optimized+method+for+high-titer+lentivirus+preparations+without+ultracentrifugation.">An optimized method for high-titer lentivirus preparations without ultracentrifugation.</a> Scientific Reports. 5, 13875 (2015).</li><li>Rueden, C. 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K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Research. 42 (22), 168 (2014).</li><li>Bollen, Y., Post, J., Koo, B. -. K., Snippert, H. J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+create+state-of-the-art+genetic+model+systems:+strategies+for+optimal+CRISPR-mediated+genome+editing.">How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing.</a> Nucleic Acids Research. 46 (13), 6435-6454 (2018).</li><li>Brinkman, E. K., 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=Easy+quantification+of+template-directed+CRISPR/Cas9+editing.">Easy quantification of template-directed CRISPR/Cas9 editing.</a> Nucleic Acids Research. 46 (10), 58 (2018).</li><li>Dussaud, S., Pardanaud-Glavieux, C., Sauty-Colace, C., Ravassard, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+mediated+production+of+transgenic+mice:+a+simple+and+highly+efficient+method+for+direct+study+of+founders.">Lentiviral mediated production of transgenic mice: a simple and highly efficient method for direct study of founders.</a> Journal of Visualized Experiments. (140), e57609 (2018).</li><li>Montagna, 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=VSV-G-enveloped+vesicles+for+traceless+delivery+of+CRISPR-Cas9.">VSV-G-enveloped vesicles for traceless delivery of CRISPR-Cas9.</a> Molecular Therapy. Nucleic Acids. 12, 453-462 (2018).</li><li>Marnef, 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=A+cohesin/HUSH-+and+LINC-dependent+pathway+controls+ribosomal+DNA+double-strand+break+repair.">A cohesin/HUSH- and LINC-dependent pathway controls ribosomal DNA double-strand break repair.</a> Genes &amp; Development. 33 (17-18), 1175-1190 (2019).</li></ol>

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 ...

delivery-cas9sgrna-ribonucleoprotein-complex-immortalized-primary

Research

JoVE Journal

Biology

4.7K Views.  LBMC, Laboratoire de Biologie et Modélisation de la Cellule Univ Lyon, ENS de Lyon, Université Claude Bernard Lyon 1, CNRS, UMR 5239, INSERM, U1210, Lyon, 69007, France. 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.

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

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

High Yield Expression of Recombinant Human Proteins with the Transient Transfection of HEK293 Cells in Suspension

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and function. The rapid establishment and high recovery from transiently-transfected mammalian cell lines addresses this barrier and is an effective means of expressing proteins that are naturally channeled through the ER and Golgi-mediated secretory pathway. Here is one protocol for protein expression using the human HEK293F and HEK293S cell lines transfected with a mammalian expression vector designed for high protein yields. The applicability of this system is demonstrated using three representative glycoproteins that expressed with yields between 95-120 mg of purified protein recovered per liter of culture. These proteins are the human Fc&#947;RIIIa and the rat &#945;2-6 sialyltransferase, ST6GalI, both expressed with an N-terminal GFP fusion, as well as the unmodified human immunoglobulin G1 Fc. This robust system utilizes a serum-free medium that is adaptable for expression of isotopically enriched proteins and carbohydrates for structural studies using mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, the composition of the N-glycan can be tuned by adding a small molecule to prevent certain glycan modifications in a manner that does not reduce yield.

Laboratory-scale production of eukaryotic proteins with appropriate post-translational modification represents a significant barrier. Here is a robust protocol with rapid establishment and turnaround for protein expression using a mammalian expression system. This system supports selective amino acid, selective labeling of proteins and small molecule modulators of glycan composition.

The art of producing recombinant proteins with complex post-translational modifications represents a major challenge for studies of structure and ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ ...

Isolation of Human Primary Valve Cells for In vitro Disease Modeling

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve causes aortic stenosis and contributes to heart failure and stroke. Disease pathogenesis is multifactorial, and stresses such as inflammation, extracellular matrix remodeling, turbulent flow, and mechanical stress and strain contribute to the osteogenic differentiation of valve endothelial and valve interstitial cells. However, the precise initiating factors that drive the osteogenic transition of a healthy cell into a calcifying cell are not fully defined. Further, the only current therapy for CAVD-induced aortic stenosis is aortic valve replacement, whereby the native valve is removed (surgical aortic valve replacement, SAVR) or a fully collapsible replacement valve is inserted via a catheter (transcatheter aortic valve replacement, TAVR). These surgical procedures come at a high cost and with serious risks; thus, identifying novel therapeutic targets for drug discovery is imperative. To that end, the present study develops a workflow where surgically removed tissues from patients and donor cadaver tissues are used to create patient-specific primary lines of valvular cells for in vitro disease modeling. This protocol introduces the utilization of a cold storage solution, commonly utilized in organ transplant, to reduce the damage caused by the often-lengthy procurement time between tissue excision and laboratory processing with the benefit of greatly stabilizing cells of the excised tissue. The results of the present study demonstrate that isolated valve cells retain their proliferative capacity and endothelial and interstitial phenotypes in culture upwards of several days after valve removal from the donor. Using these materials allows for the collection of control and CAVD cells, from which both control and disease cell lines are established.

This protocol describes the collection of human aortic valves extracted during surgical aortic valve replacement procedures or from cadaveric tissue, and the subsequent isolation, expansion, and characterization of patient specific primary valve endothelial and interstitial cells. Included are important details regarding the processes needed to ensure cell viability and phenotype specificity.

Calcific aortic valve disease (CAVD) is present in nearly a third of the elderly population. Thickening, stiffening, and calcification of the aortic valve ...