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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(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><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 H&lt;sub&gt;2&lt;/sub&gt;O)</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 &amp;micro;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 &amp;micro;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 &amp;micro;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&amp;ndash;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, &amp;ge;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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Biology

4.8K 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. Nanoblades maakten een snelle, efficiënte en dosisafhankelijke afgifte van het Cas9- en gids-RNA-complex mogelijk, zowel in vereeuwigde als in primaire cellen, en in afwezigheid van anti-transgen. Het grote voordeel van deze methode is dat nanoblades gemakkelijk en relatief goedkoop te bereiden zijn. Ze kunnen het Cas9-en RNA-complex op een dosisafhankelijke en voorbijgaande manier leveren, waardoor off-target effecten worden beperkt, terwijl efficiënte genoombewerking mogelijk is.He...

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

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.

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 ...

Genetics

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. ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

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

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

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

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

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to &#8220;loss-of-function&#8221; approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct &#8220;gain-of-function&#8221; studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.

Enhancer RNAs (eRNAs) are non-coding RNAs produced from active enhancers. An optimal approach to study eRNA functions is to manipulate their levels in the native chromatin regions. Here we introduce a robust system for eRNA studies by using CRISPR-dCas9-fused transcriptional activators to induce the expression of eRNAs of interest.

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence ...

A Guide to Precise Gene Disruption in Primary Bone Marrow-Derived Macrophages

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.

Author Spotlight: Efficient CRISPR/Cas9 Genome Editing in Bone Marrow-Derived Macrophages for Precise Gene Disruption

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the ...

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

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

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

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

Neuroscience

Generation of Knock-out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging in primary NK cells. For example, transgene delivery using lentiviral or retroviral transduction resulted in a limited yield of genetically-engineered NK cells due to substantial procedure-associated NK cell apoptosis. We describe here a DNA-free method for genome editing of human primary and expanded NK cells using Cas9 ribonucleoprotein complexes (Cas9/RNPs). This method allowed efficient knockout of the TGFBR2 and HPRT1 genes in NK cells. RT-PCR data showed a significant decrease in gene expression level, and a cytotoxicity assay of a representative cell product suggested that the RNP-modified NK cells became less sensitive to TGF&#946;. Genetically modified cells could be expanded post-electroporation by stimulation with irradiated mbIL21-expressing feeder cells.

Here, we present a protocol to genetically modify primary or expanded human natural killer (NK) cells using Cas9 Ribonucleoproteins (Cas9/RNPs). By using this protocol, we generated human NK cells deficient for transforming growth factor&#8211;b receptor 2 (TGFBR2) and hypoxanthine phosphoribosyltransferase 1 (HPRT1).

CRISPR/Cas9 technology is accelerating genome engineering in many cell types, but so far, gene delivery and stable gene modification have been challenging ...

Immunology and Infection

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

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

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

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

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...

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

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