通过病毒样颗粒（“纳米刀”）递送Cas9/sgRNA核糖核蛋白复合物至永生化细胞和原代细胞

摘要

我们开发了一种简单且低成本的方案，用于将Cas9/单导向RNA（sgRNA）核糖核蛋白复合物装载到病毒样颗粒中。这些被称为“Nanoblades”的颗粒能够高效地将Cas9/sgRNA复合物递送至永生化细胞、原代细胞以及体内环境。

摘要

成簇规律间隔短回文重复序列（CRISPR）-Cas系统已使真核细胞的基因组编辑实现普及化，并推动了众多创新应用的发展。然而，将Cas9蛋白和单导向RNA（sgRNA）递送至靶细胞在技术上仍具挑战性。经典的病毒载体，例如源自慢病毒（LVs）或腺相关病毒（AAVs）的载体，能够在多种原代细胞及体内实现编码Cas9蛋白及其相关sgRNA的转基因的高效递送。尽管如此，这些载体仍存在一些缺陷，例如转基因在靶细胞基因组中的整合、装载容量有限，以及Cas9蛋白和导向RNA在靶细胞中长期表达等问题。

为克服上述部分问题，研究人员开发了一种基于小鼠白血病病毒（MLV）的递送载体，可在不包含任何编码转基因的情况下，将Cas9蛋白及其相关的向导RNA包装递送。通过将Cas9蛋白与MLV结构蛋白Gag的C末端融合，形成了装载Cas9蛋白和sgRNA的病毒样颗粒（VLPs），被称为"Nanoblades"。Nanoblades可从生产细胞的培养基中收集，经纯化、定量后用于转导靶细胞，递送具有活性的Cas9/sgRNA复合物。Nanoblades能够在多种原代细胞和永生化细胞中瞬时且快速地递送其核糖核蛋白（RNP）货物，并可通过使用修饰的Cas9蛋白，编程实现其他应用，例如靶向基因的瞬时转录激活。Nanoblades能够在注射后的成年小鼠肝脏以及卵母细胞中实现体内基因组编辑，从而生成转基因动物。最后，它们还可与供体DNA形成复合物，实现"无需转染"的同源定向修复。Nanoblades的制备过程简单、成本相对较低，可在任何细胞生物学实验室中轻松完成。

引言

与其他可编程核酸酶相比，CRISPR-Cas 系统极大地简化并普及了真核细胞中序列特异性的基因组靶向与切割操作。通过简单表达一条单导向RNA（sgRNA），用户即可将Cas9蛋白（或其优化变体）编程至几乎任意的细胞基因位点¹。在此情况下，Cas9蛋白与sgRNA的递送成为实现定点诱变的主要限制因素。在永生化细胞中，sgRNA和Cas蛋白可通过转染质粒实现高效表达，从而在大多数细胞中完成有效的基因组靶向。然而，Cas9/sgRNA复合物的持续表达可能增加Cas9蛋白的脱靶活性，导致非特异性位点发生非预期的遗传改变²。在原代细胞中，DNA转染在技术上往往难以实现，可能导致表达水平低下或转染细胞比例过低。除经典DNA转染外，替代方法包括使用病毒载体递送编码Cas9和sgRNA的转基因，或通过电穿孔将重组Cas9蛋白与合成sgRNA共同导入细胞。然而，这些方法可能导致转基因整合至宿主细胞基因组（如经典的逆转录病毒和慢病毒表达载体），受到细胞内因子的限制，并引发Cas9蛋白和sgRNA的持续表达。

通过电穿孔将Cas9/sgRNA核糖核蛋白复合物导入细胞，可克服上述大多数问题，实现原代细胞及体内高效且瞬时的递送，并允许产生剂量依赖性反应。然而，该方法通常依赖昂贵的设备和试剂，且当需要处理大量细胞时，难以实现规模化扩增。作为上述技术的替代方案，这些研究人员开发了"Nanoblades"——一种用于递送Cas9蛋白和sgRNA的逆转录病毒载体³，其概念与其他基于病毒的衣壳蛋白递送系统相似^4,5,6,7,8。Nanoblades利用逆转录病毒Gag多蛋白在培养细胞中单独表达时，能够自发形成并释放至细胞外培养基中的病毒样颗粒（VLPs）这一天然特性⁹。通过将Cas9蛋白与小鼠白血病病毒（MLV）Gag多蛋白的C末端融合，并共表达sgRNA和病毒包膜糖蛋白，可将Cas9蛋白包裹于释放的VLPs（即Nanoblades）中。经纯化后，Nanoblades可与靶细胞共孵育，或直接注射至体内，从而实现Cas9/sgRNA核糖核蛋白复合物的快速、瞬时且具有剂量依赖性的递送³。

Nanoblade 可通过多个 sgRNA 进行编程，以实现不同位点的同步编辑，或利用 Cas9 变体执行其他应用，例如靶向特异性的转录激活或抑制³。与依赖重组表达的蛋白质电穿孔技术相比，文献中新描述的 Cas 变体可轻松克隆至 Gag 融合表达载体中，使其成为一个多功能的平台。Nanoblade 还可进一步与单链或双链寡脱氧核苷酸（ssODN）形成复合物或负载此类分子，以实现同源定向修复³。Nanoblade 的制备相对简单且成本较低。此外，Nanoblade 可在 4 °C 下保存多天，或在 -80 °C 下长期保存。通常情况下，Nanoblade 可在大多数永生化细胞和原代培养细胞中实现高效、无转基因的基因组编辑。然而，某些原代细胞可能对病毒颗粒的存在较为敏感，导致细胞死亡率升高。先天免疫系统的细胞也可能因 Nanoblade 具有病毒来源而对其产生反应并被激活。在此类情况下，必须优化转导方案，以缩短细胞与 Nanoblade 的接触时间，并将非特异性效应降至最低。Nanoblade 是现有 CRISPR 递送方法的一种切实可行且易于实施的替代方案。

方案

1. sgRNA Design and cloning

NOTE: Guidelines for the design of sgRNAs can be obtained from multiple sources such as https://blog.addgene.org/how-to-design-your-grna-for-crispr-genome-editing or from Hanna and Doench¹⁰.

1. Once the 20 nucleotide sgRNA sequences have been designed, order the following single-stranded DNA oligonucleotides:
   1. Forward: 5' caccgNNNNNNNNNNNNNNNNNNNN 3' (N corresponds to the targeted locus without the protospacer-adjacent motif (PAM) sequence)
   2. Reverse: 5' aaacNNNNNNNNNNNNNNNNNNNNc 3' (N correspond to the reverse-complement of the targeted locus without the PAM sequence)
      NOTE: No special modifications are required when ordering the oligonucleotides (no requirement for 5' phosphate).
2. Hybridize the two DNA oligonucleotides in a 0.2 mL polymerase chain reaction (PCR) tube by mixing 5 µL of annealing buffer (500 mM NaCl; 100 mM Tris-HCl; 100 mM MgCl2; 10 mM DTT; pH 7.9 at 25 °C), 1 µL of each DNA oligonucleotide (100 µM stock solution in water), and 42 µL of water.
3. On a PCR block, incubate samples at 95 °C for 15 s and then decrease the temperature to 20 °C with a ramp of 0.5 °C/s. Keep at room temperature or store at -20 °C.
   NOTE: The protocol can be paused here.
4. Digest 10 µg of the BLADE or SUPERBLADE sgRNA expression plasmids with 10 units of BsmBI-v2 restriction enzyme for 3 h at 55 °C in a total reaction volume of 50 µL.
   NOTE: The digested vector should release a DNA insert of ~1.9 kb and a second DNA fragment of ~3.3 kb.
5. Load the restriction reaction on a 1% agarose gel stained with 5 µg/mL of ethidium bromide (or a safer alternative DNA gel stain).
   NOTE: Wear appropriate protection gear when manipulating ethidium bromide, which is suspected of causing genetic defects.
   1. On an ultraviolet (UV) table set at a wavelength of 312 nm (to avoid damaging the DNA), cut the 3.3 kb DNA fragment from the gel, and place it in a 1.5 mL microcentrifuge tube.
      NOTE: Wear appropriate protection gear (gloves and UV protection goggles) when manipulating ethidium bromide and working on the UV table.
   2. Extract DNA from the sliced gel containing the 3.3 kb DNA fragment using a dedicated DNA gel extraction kit (see the Table of Materials). Quantify the amount of purified DNA using a spectrophotometer.
      ​NOTE: The protocol can be paused here.
6. Ligate the hybridized forward and reverse DNA oligonucleotides from step 1.2 to the BsmB1-digested, gel-purified BLADES or SUPERBLADE vector from step 1.5.2. For this, add 2 µL of T4 DNA ligase buffer, 50 ng of the gel-purified vector (from step 1.5.2), 1 µL of the hybridized DNA oligonucleotides (from step 1.2), water to make up the volume to 19 µL, and 1 µL of T4 DNA ligase. Incubate the reaction at 25 °C for 10 min.
   1. Transform the ligation product into competent bacteria (see the Table of Materials) as described in¹¹. Plate the transformed bacteria on an ampicillin Luria Bertani agar plate and incubate overnight at 37 °C.
   2. Select several isolated colonies on the agar plate to perform DNA minipreparation¹¹ (see the Table of Materials), and perform Sanger sequencing using a U6 forward primer (5' GACTATCATATGCTTACCGT 3') to check for correct ligation of the sgRNA variable sequence.
      ​NOTE: Other sgRNA expression plasmids can be used if they do not code for the Cas9 protein, which could interfere with Nanoblade production.

2. Plasmid preparation

1. Perform maxipreparation (see the Table of Materials) of all required plasmids, and prepare 10 µg aliquots at 1 µg/mL to store at -20 °C. Avoid repeated freeze/thawing cycles of the plasmids; use aliquots twice before discarding them.

3. Nanoblade preparation

1. On Day 1, seed between 3.5 and 4 × 10⁶ HEK293T cells (see the Table of Materials) in 10 mL of Dulbecco's modified Eagle medium (DMEM) containing high glucose, sodium pyruvate, L-glutamine, 10% fetal bovine serum (FBS), and penicillin/streptomycin in a 10 cm cell culture dish. Move the 10 cm plate gently backward and forward, then from right to left (repeat this sequence 5x) to distribute cells homogeneously over the culture dish. Incubate cells at 37 °C in a cell incubator with 5% CO₂.
   NOTE: All procedures related to the handling of cultured cells and Nanoblades should be performed under a cell culture laminar flow hood to avoid their contamination.
2. Day 2: Plasmid transfection
   1. Cells should be 70-80% confluent 24 h after plating (Figure 1A). Replace the medium with 10 mL of fresh DMEM containing high glucose, sodium pyruvate, L-glutamine, 10% FBS (penicillin and streptomycin can be omitted although it is not mandatory) before transfection.
      NOTE: At this step, it is important that the cells are not confluent. Otherwise, transfection efficiency as well as particle production could be reduced.
   2. For each 10 cm plate, prepare the following quantities of plasmids in a 1.5 mL tube: 0.3 µg pCMV-VSV-G, 0.7 µg pBaEVRless, 2.7 µg MLV Gag/Pol, 1.7 µg BIC-Gag-Cas9, 4.4 µg of BLADES or SUPERBLADES plasmid encoding the cloned sgRNA (or 2.2 µg each if using two sgRNAs).
   3. Add 500 µL of transfection buffer (see the Table of Materials), vortex for 10 s, and then centrifuge for 1 s. Add 20 µL of the transfection reagent (see the Table of Materials), vortex the tube for 1 s, and then centrifuge for 1 s.
   4. Incubate for 10 min at room temperature, and add the entire solution dropwise to the cells in DMEM medium using a P1000 pipettor. Move the 10 cm plate gently backward and forward, then from right to left (repeat this sequence 5x) to uniformly distribute the transfection reagent over the cells. Incubate cells at 37 °C for at least 40 h in a cell incubator with 5% CO₂.
      NOTE: If desired, medium can be changed 4 h after transfection.
3. On Day 3, check the morphology of the transfected cells under the microscope.
   NOTE: Producer cells will begin to fuse. This is a normal occurrence due to the expression of fusogenic viral envelopes (Figure 1B,C).
4. Day 4: Harvesting Nanoblades
   NOTE: At least 40 h after transfection, the cells would have fused together because of expression of the fusogenic viral envelopes, and sometimes, the cells are completely detached from the plate support (Figure 1D).
   1. Collect 9 mL of the culture medium supernatant using a 10 mL pipette.
      NOTE: Nanoblades are VLPs capable of delivering the Cas9 protein and its associated sgRNA into primary cells and in vivo. Although they are not considered genetically modified organisms as they are devoid of genetic material, they can induce genetic changes. Therefore, they must be manipulated with caution to avoid any contact with users (especially if they are programmed to target tumor suppressor genes). Users are advised to follow their local safety guidelines for the manipulation of retroviral vectors and work in a BSL-2 level laboratory when preparing VLPs and performing transduction experiments. Nanoblades can be inactivated with 70% ethanol or 0.5% of sodium hypochlorite. It is also advisable to treat all plastic waste (pipette tips, tissue culture plates, centrifugation tubes) with 0.5% sodium hypochlorite for at least 10 min to inactivate the Nanoblades.
   2. Centrifuge the collected supernatant at 500 × g for 5 min to remove cellular debris and recover the supernatant without disturbing the cell pellet.
      ​NOTE: If Nanoblades are meant to be used on primary cells, filter the supernatant using a 0.45 µm or 0.8 µm filter. Be aware that this step drastically reduces the Nanoblade titer as a significant fraction will be blocked in the filter membrane.
   3. Pellet the Nanoblades overnight (12-16 h) in a swinging bucket rotor at 4,300 × g or at 209,490 × g in an ultracentrifuge for 75 min at 4 °C (see the Table of Materials).
      ​NOTE: If target cells can grow in DMEM, it is possible to incubate them directly with the supernatant obtained after step 3.4.2 without concentrating the Nanoblades.
5. Day 5: Resuspension and storage of Nanoblades
   1. After centrifugation, slowly aspirate the medium and resuspend the white pellet with 100 µL of cold 1x phosphate-buffered saline (PBS). Cover the tube with parafilm, and incubate for 1 h at 4 °C with gentle agitation before resuspending the pellet by pipetting up and down.
      NOTE: A white viscous material may appear upon resuspension; this is normal and does not significantly affect the efficiency of transduction.
   2. Store the Nanoblades at 4 °C if planning on using them within four weeks. Otherwise, snap-freeze the Nanoblades in liquid nitrogen and store them at -80 °C.
      ​NOTE: Wear protection goggles and cryogenic gloves when manipulating liquid nitrogen. Snap-freezing and storage at -80 °C leads to a significant decrease in Nanoblade efficiency. Moreover, thawed Nanoblades should not be frozen again. The protocol can be paused here.

4. Concentration of Nanoblades on a sucrose-cushion

NOTE: As an alternative to overnight centrifugation or ultracentrifugation (step 3.4.3), the Nanoblades can be concentrated on a sucrose cushion. This yields a purer fraction of Nanoblades, although the total amount recovered will be lower.

1. Prepare a 10% sucrose solution (weight to volume) in 1x PBS, and filter it through a 0.2 µm syringe filter (see the Table of Materials).
2. Begin the process of concentrating the Nanoblades on the sucrose cushion.
   1. Place 9 mL of VLP-containing sample (from step 3.4.3) into an ultracentrifuge tube (see the Table of Materials). Using a 3 mL syringe and cannula, slowly layer 2.5 mL of the 10% sucrose under the sample, trying not to mix the VLP-containing sample and the sucrose solution.
   2. Alternatively, place 2.5 mL of 10% sucrose into an ultracentrifuge tube (see the Table of Materials). Tilt the tube and slowly add the 9 mL of VLP-containing sample (from step 3.4.3) with a low-speed pipettor. During this operation, progressively raise the tube to a vertical position.
3. Centrifuge the samples at 209,490 × g in an ultracentrifuge for 90 min at 4 °C.
   NOTE: This technique can be adapted for low-speed centrifugation (4,300 × g) overnight as described in ¹².
4. After centrifugation, remove the supernatant carefully and place the tube upside down on tissue paper to remove any remaining liquid. After 1 min, add 100 µL of 1x PBS and place the tube at 4 °C with a parafilm cover in a tube holder on an agitation table for 1 h (see the Table of Materials) before resuspending the pellet by pipetting up and down.
   ​NOTE: The protocol can be paused here.

5. Monitoring Cas9 loading within Nanoblades by dot-blot

1. Prepare the dilution buffer by adding 1 volume of lysis buffer containing a non-ionic surfactant (see the Table of Materials) in 4 volumes of 1x PBS. Dilute 2 µL of concentrated Nanoblades in 50 µL of dilution buffer, vortex briefly, and transfer 25 µL of this mixture into a new tube containing 25 µL of dilution buffer. Repeat this operation to have 4 tubes of Nanoblade dilutions (2-fold dilution steps).
2. For the standard controls, dilute 2 µL of recombinant Cas9 nuclease (see the Table of Materials) into 50 µL of dilution buffer, vortex briefly, and proceed to make eight serial dilutions (2-fold dilution for each step).
3. Carefully spot 2.5 µL of each VLP dilution and 2.5 µL of each standard onto a nitrocellulose membrane with a multichannel pipet (a larger volume may result in overlapping spots).
   NOTE: A methanol-treated polyvinyldifluoride membrane may also be used.
4. Once the particles are absorbed onto the membrane, block the membrane with 1x Tris-buffered saline containing a non-ionic surfactant (TBS-T) supplemented with non-fat dry-milk (5% w/v) for 45 min at room temperature.
   NOTE: The protocol can be paused here, and the membrane stored at 4 °C in 1x TBS-T.
5. Discard the 1x TBST supplemented with non-fat dry-milk, and incubate the membrane overnight at 4 °C with the Cas9-horseradish peroxidase antibody (1/1000 dilution in 1x TBST, 5% milk). Wash the membrane 3x with TBS-T, and visualize the signal using an enhanced chemiluminescent substrate kit.
6. Quantify the dot intensity for the Nanoblades and recombinant Cas9 standard dilutions using the proprietary software provided with the gel imaging station or imageJ¹³. Define a linear curve linking dot intensity to the Cas9 concentration. Using the function of the obtained curve, extrapolate the Cas9 content in each preparation.
   NOTE: The amount of recombinant Cas9 protein control can saturate the reading for the most concentrated samples of the standard dilution set (Figure 2). It is therefore advised, when defining the linear curve, to remove the reading from the undiluted samples (and sometimes that of the first dilution steps) if they are not in the linear range with respect to the known concentration of Cas9 that was spotted. Similarly, when extrapolating the amount of Cas9 within the Nanoblade samples, only use the readings that are within the linear range of the standard curve.

6. Transduction of target cells with Nanoblades (procedure for transduction in a 12-well plate)

1. In a 12-well plate, seed 100,000-200,000 cells (either primary or immortalized adherent cells) per well in 1 mL of the appropriate cell culture medium. Allow the cells to adhere to the plate surface before transduction.
2. In a 1.5 mL microcentrifuge tube, add 5-20 µL of concentrated Nanoblades (from step 3.5.1 or 4.4) to 500 µL of cell culture medium, and mix by pipetting up and down with a P1000 pipettor. Remove the medium from cells, and replace it with the 500 µL of this Nanoblade mixture.
   NOTE: Transduction must be optimized for each cell type. It is important to use the smallest possible volume of medium (while avoiding drying of the target cells) so that the Nanoblades remain highly concentrated. Adherent cells must be transduced directly while attached to the plate (do not transduce in suspension as this will significantly decrease transduction efficiency). Some cells tolerate prolonged exposure to Nanoblades (24-48 h) while others are very sensitive and may form small syncytia. In this case, Nanoblades must be incubated with cells only for 4-6 h before replacing the medium. Spinoculation¹⁴ can also improve transduction for cells grown in suspension. Adjuvants such as cationic polymers (see the Table of Materials) can also improve transduction efficiency in some cell types.
3. After 4-6 h of cell incubation in a low volume of medium containing Nanoblades, increase the volume of medium to the normal amount (1 mL if working with a 12-well plate), or replace it with fresh medium if the cells are sensitive to VLPs.
   NOTE: Cell medium containing Nanoblades must be inactivated with 0.5% sodium hypochlorite for 10 min before discarding it. Use gloves and protective goggles when manipulating sodium hypochlorite. If Nanoblades induce cell death, adapt the amount and total time of exposure to reduce cell mortality.

7. Measuring CRISPR efficiency at the targeted locus by T7 endonuclease assay

1. Design PCR primers to amplify a 400-700 base-pair (bp) region encompassing the CRISPR-cleavage site.
   NOTE: The cleavage site should be distant from the amplicon edge by at least 200 bp and should be slightly shifted from the center of the amplicon so that upon T7 endonuclease cleavage, 2 fragments of different sizes will be released.
2. Extract genomic DNA from cells treated with Nanoblades targeting the gene of interest and from control cells treated with Nanoblades programmed with a control sgRNA (see the Table of Materials).
   ​NOTE: The protocol can be paused here.
3. Using 150 ng of genomic DNA as a template, program a PCR reaction of 30 µL volume (final volume) by following the manufacturer's protocol. Check that the PCR amplification yields a single amplicon of the expected size by running a 2% agarose gel stained with 5 µg/mL of ethidium bromide (or a safer alternative DNA gel stain).
   ​NOTE: Wear appropriate protection gear when manipulating ethidium bromide, which is suspected of causing genetic defects. The protocol can be paused here.
4. Heteroduplex generation and digestion
   1. In a 0.2 mL PCR tube, add 5 µL of the enzyme buffer (provided with the T7 endonuclease I), 20 µL of water, and 24 µL of the PCR product from step 7.3. Allow heteroduplex formation by heating the samples to 94 °C over 3 min and then by decreasing the temperature (2 °C per min) to reach 40 °C.
   2. Add 0.5 µL of T7-endonuclease I at room temperature to each heteroduplex tube, including the control. Incubate at 37 °C for 15 min. Load the resulting reaction in a 2.5% (weight/volume) agarose gel stained by ethidium bromide. After migration, image the gel on a UV transilluminator.
      NOTE: Wear appropriate protection gear when manipulating ethidium bromide, which is suspected of causing genetic defects. Use UV-protection goggles when using the UV transilluminator.
   3. Measure cleavage efficiencies by analyzing the image resulting from the digestion reaction to quantify the intensity of each band with appropriate software (see the Table of Materials).

8. Measuring CRISPR efficiency at the targeted locus by Sanger sequencing and TIDE analysis

NOTE: As an alternative to the T7 endonuclease assay, CRISPR efficiency can be monitored by analysis and deconvolution of Sanger sequencing traces based on the TIDE protocol¹⁵.

1. Perform Sanger sequencing of PCR amplicons from step 7.3 (include a control condition corresponding to untreated cells) using either the forward or reverse PCR primer.
2. Analyze the Sanger sequencing traces of the control condition (untreated cells) and Nanoblade-treated samples using the TIDE server (https://tide.nki.nl) and following their analysis guidelines.

9. Nanoblade complex-formation with ssODN donors for homology-directed repair (procedure for transduction in a 12-well plate)

NOTE: Guidelines for the design of ssODN for efficient homology-directed repair mediated editing have been described previously¹⁶.

1. In a 12-well plate, seed 100,000-200,000 cells per well in 1 mL of the appropriate cell culture medium. Allow the cells to adhere to the plate surface before transduction.
2. Prepare 100 µL of a solution of the cationic polymer (see the Table of Materials) at 8 µg/mL in 1x PBS.
   1. Mix 19 µL of the cationic polymer solution with 100 pmol of the ssODN template. Add 20 µL of concentrated Nanoblades (from step 3.5.1 or 4.4), and incubate for 15 min on ice.
   2. Remove the complexed Nanoblades/ssODN from ice, and add 500 µL of cell culture medium (at 37 °C). Remove the medium from target cells (from step 9.1), and add the 500 µL of medium containing the complexed Nanoblades/ssODN. Allow the cells to proliferate for 48 h before genotyping.
3. Extract genomic DNA from a fraction of the cell population using a dedicated extraction kit (see the Table of Materials).
   1. Design PCR primers to amplify a 400-700 bp region encompassing the knock-in site.
      ​NOTE: PCR primers should not overlap with the homology arms of the ssODN to avoid false-positive results resulting from the PCR amplification of any residual ssODN still present within target cells.
   2. Using 150 ng of genomic DNAs from control cells (untreated) or Nanoblade-treated-cells as a template, program a 30 µL PCR reaction following the manufacturer's protocol.
      ​NOTE: ssODN traces may be present in the cell medium several days after transduction with the complex. This ssODN may serve as a partial template for PCR assays attempting to screen for the correct integration. Hence, it is advisable to passage the cells at least twice after transduction, to avoid eventual false-positive assays.
   3. Load 5 µL of the control and Nanoblade-treated PCR reactions in a 1% (weight/volume) agarose gel stained by ethidium bromide. After migration, image the gel on a UV-transilluminator.
      NOTE: If homology recombination is successful and corresponds to the insertion of more than 1 bp of genetic material, there should be a difference in the molecular weight of the PCR amplicons between the control and the Nanoblade-treated sample. As the efficiency of HDR does not reach 100%, two bands should be visible in the Nanoblade-treated sample (one of similar size to the control PCR amplicon corresponding to the unedited allele and one of higher molecular weight corresponding to the knock-in allele, see Figure 3B middle panel).
4. Perform Sanger sequencing of the control and Nanoblade-treated PCR amplicons.
5. Quantify knock-in efficiency using the TIDER protocol¹⁷.

10. Nanoblade delivery in vivo

1. Deliver up to 25 µL of concentrated Nanoblades from step 3.5.1 through retro-orbital injection or up to 100 µL through tail vein injection, as described in¹⁸, if working with mice.
   NOTE: All procedures involving animal experimentation (including Nanoblade injections for genome editing purposes) require an approved protocol from a local ethics committee.
2. For the generation of transgenic mice, use a micro-injector to deliver from 1 pL to 10 pL of concentrated Nanoblades from step 4.4 into the perivitelline space of mouse oocytes as described previously¹⁸.
   NOTE: For perivitelline injection, it is essential to purify and concentrate Nanoblades on a sucrose cushion to avoid clogging of the micro-injector.

结果

Nanoblade 制备的方案相对简单，除需要使用细胞培养罩、CO₂ 培养箱以及摆动桶离心机或超速离心机外，仅需常规实验室设备。然而，某些步骤需特别注意，例如生产细胞的来源与操作，以及转导条件。如图1A所示，接种细胞时应确保其在培养板中均匀分布，并在转染当天达到约70–80%的融合度（避免细胞成团）。转染后24小时（图1B、C），生产细胞将形成合胞体，产生体积较大且具有多个细胞核的细胞。转染后40小时（图1D），培养板中大多数细胞将形成合胞体并开始从培养板上脱落。

这是完全正常的现象，由包膜糖蛋白的表达引起，该蛋白可诱导相邻细胞之间的融合。通过离心浓缩（或直接取自生产细胞的上清液）后，可使用重组Cas9作为参照，通过在硝酸纤维素膜上进行点印迹（dot-blot）对Nanoblades内部装载的Cas9蛋白进行绝对定量（图2）。此步骤对于确定转导靶细胞时所需Nanoblades的正确用量至关重要。进行点印迹实验时，应仅考虑落在标准曲线线性范围内的读数。然而，无论Nanoblades中Cas9的含量如何，都必须通过T7核酸内切酶实验（图3）或Sanger测序，在靶细胞上直接检测基因组编辑效率。

如图3所示，Nanoblades的效率在不同批次之间可能存在差异，尽管其通常与Cas9的量相关。在图3所示的示例中，第1泳道的批次导致总体编辑效率为20%，而第3泳道的批次则达到60%的效率。在此情况下，可通过增加第1批次Nanoblades的使用体积，以实现与第3批次相似的编辑效率。图4展示了在不同类型原代细胞中使用Nanoblades所获得的最大编辑效率。需要注意的是，编辑效率可能因所用sgRNA的序列及靶点可及性而有所不同。

图 1：Nanoblade 制备过程中生产细胞的形态学特征。（A）铺板后 24 小时，汇合度为 70–80% 的 HEK293T 细胞。（B 和 C）转染后 24 小时的 HEK293T 细胞形态。（D）转染后 40 小时的 HEK293T 细胞形态。比例尺 = 400 µm。请点击此处查看该图的高清版本。

图2：通过点印迹法定量Nanoblade中Cas9的载量。（A）重组Cas9蛋白或经超速离心浓缩100倍的Nanoblade样品（#1、#2和#3）进行连续2倍梯度稀释后，点样于硝酸纤维素膜上，随后与HRP偶联的抗Cas9抗体孵育。通过增强型化学发光法显色。（B）采集并定量重组Cas9稀释样品的化学发光信号，将信号强度对已知点样量的Cas9作图。对处于线性范围内的稀释点（见蓝色叉号）计算回归曲线，排除所有超出线性范围的浓度点（见红色叉号）。（C）利用（B）中所得线性回归方程推算各Nanoblade制剂中的Cas9浓度（nM）。为此，应仅使用落在回归曲线线性范围内的Nanoblade稀释样品的定量信号进行计算。请点击此处查看该图的放大版本。

图3：转导后编辑效率的监测。（A）T7核酸酶检测法测定Nanoblade处理细胞中的切割效率。使用靶向EMX1基因的Nanoblade转导细胞，并通过T7核酸酶检测进行分析。泳道1：Nanoblade制备批次#1（切割效率20%）；泳道2：对照细胞；泳道3：Nanoblade制备批次#2（切割效率60%）。（B）在DDX3开放阅读框内敲入Flag标签序列。使用靶向DDX3位点的sgRNA编程的浓缩Nanoblade，从不同的HEK293T克隆（#1、#2）中制备，并与不同剂量的Flag-DDX3单链寡脱氧核苷酸（ssODN）模板复合，所得复合物用于转导HEK293T靶细胞。转导后，细胞培养三天，随后收集细胞以提取基因组DNA和总蛋白。使用抗Flag琼脂糖微珠对Flag-DDX3蛋白进行免疫沉淀，再用抗Flag抗体对回收蛋白进行Western印迹分析（上图）。通过PCR检测Flag标签在Ddx3位点的定点插入，PCR引物包括：跨越插入位点的上下游引物（中图），或一条识别Flag标签序列的正向引物与一条特异性识别Flag插入位点下游Ddx3位点的反向引物（下图）。缩写：EMX1 = Empty Spiracles Homeobox 1；DDX3 = DEAD-box RNA解旋酶3；PCR = 聚合酶链式反应；ODN = 寡脱氧核苷酸；ssODN = 单链ODN；sgRNA = 单导向RNA；IP = 免疫沉淀。请点击此处查看该图的放大版本。

图 4：使用 Nanoblades 在不同原代细胞类型中实现的编辑效率。 缩写：PBL = 外周血淋巴细胞；IL = 白细胞介素；CD = 分化簇；iPSC = 诱导性多能干细胞。 请点击此处查看此图的放大版本。

讨论

Nanoblades 可实现 Cas9/sgRNA RNP 复合物在细胞系和原代细胞中的快速且剂量依赖性递送。与传统的转染方法和其他病毒载体递送方式不同，Nanoblades 与蛋白质电穿孔类似，具有以无转基因方式瞬时递送 Cas9/sgRNA RNP 的优势。Nanoblades 提供了一个高度通用、简便且经济的蛋白质递送平台，可轻松快速地适配于不断扩展的 CRISPR 变体家族。Nanoblades 可在 HEK293T 细胞系或其衍生细胞系中生产。本研究中使用的 HEK293T 细胞系经过优化，以最大化逆转录病毒和慢病毒颗粒的产量（参见材料表）。然而，尽管其他来源的 HEK293T 细胞也可能适用，用户必须对来自不同来源的 HEK293T 细胞进行测试和比较，因为不同来源的 HEK293T 细胞在病毒颗粒产量上已观察到显著差异。此外，细胞还需频繁检测支原体污染，并每三天传代一次（通常按 ⅛ 稀释），以避免过度汇合，后者会对病毒颗粒的产量产生不利影响。

细胞传代次数不应超过20代。细胞培养使用添加了葡萄糖、青霉素/链霉素、谷氨酰胺和10%灭活胎牛血清的DMEM培养基。由于血清来源可能影响Nanoblade制备的质量，因此在大规模生产前应测试不同批次的血清。转染后的第二天，Nanoblade也可在其他培养基中高效生产，例如RPMI或可替代DMEM的无血清最低必需培养基改良配方。如下所述，尽管使用某些DNA转染试剂进行转染后更换培养基是可选步骤，但调整病毒样颗粒（VLPs）释放所处的培养基可能具有优势，特别是有助于减少颗粒制备中的血清残留。然而，目前尚未尝试在转染前一天将细胞培养于低血清最低必需培养基中。

如前所述，Nanoblades 是通过在生产细胞中过表达一组质粒混合物而产生的。过表达似乎是实现高效生产所必需的。事实上，本实验室曾构建了一株生产细胞系，其中携带 Gag-Pol 表达结构的质粒通过抗生素筛选得以稳定存在；然而，该系统未能产生可观测量的 Nanoblades。当将编码 sgRNA 的结构稳定整合至生产细胞基因组时，也观察到了类似结果。正如其他颗粒生产系统所报道的那样，构建一株稳定表达至少部分参与 Nanoblades 生成的组分的细胞系可能是可行的；但这种方法必然需要处理大量上清液，并采用适当的技术来纯化颗粒。上述方案描述了生产 Nanoblades 的首选方法，该方法利用了特定的转染试剂（参见材料表）。

尽管其他厂商的转染试剂也已成功测试使用，但本课题组绝大多数关于Nanoblade的实验结果均遵循本文所述的操作流程。使用磷酸钙试剂可实现低成本转染，并获得良好的生产效率；然而，该方法必须在转染后第二天更换培养基，且可能在沉淀的病毒颗粒制备物中残留磷酸钙。与生产细胞中Nanoblade组分需要高水平表达的要求一致，已有观察表明，与Cas9蛋白结合的sgRNA数量可能是高效基因组编辑的限制因素。为提高sgRNA的装载效率，近期已有两个独立研究团队利用类似于Nanoblade的蛋白递送载体开发了两种技术方法。这些方法依赖于T7聚合酶介导的sgRNA在细胞质中的表达⁶，或在sgRNA序列中引入逆转录病毒包装信号以介导其与Gag多蛋白的结合⁶。这些策略确实有可能改善Nanoblade中sgRNA的装载，但目前尚未经过验证。

靶细胞的转导是该实验流程中的关键步骤。在大多数永生化细胞系中，使用Nanoblade进行转导几乎不会产生细胞病变效应。然而，在原代细胞中，可能存在细胞毒性问题。因此，必须针对每种细胞类型优化转导条件。具体而言，在优化转导方案时，Nanoblade的处理时间是一个需要调整的重要因素。对于原代神经元或骨髓细胞等敏感细胞，使用Nanoblade孵育4–6小时后更换培养基，可在最大限度降低细胞毒性的同时实现Cas9蛋白的有效递送。此外，某些佐剂（如阳离子聚合物等）可显著提高部分细胞的转导效率（参见材料表）。需要注意的是，Nanoblade属于病毒样颗粒（VLPs），可能引发免疫原性反应。当使用某些类型的原代细胞（例如巨噬细胞或树突状细胞）时，这一特性可能成为限制因素，因为与Nanoblade共孵育可能引起细胞基因表达和表型的显著改变。如果巨噬细胞和树突状细胞来源于造血干细胞前体（如小鼠骨髓细胞），建议在细胞完全分化之前进行Nanoblade转导，以避免诱导针对Nanoblade的细胞免疫反应。否则，在处理已分化的免疫细胞时，Cas9蛋白的电穿孔递送可作为一种可行的替代方案。

Nanoblade 可用于体内转导小鼠受精卵或胚胎，以生成转基因动物。与经典的逆转录病毒或慢病毒载体类似，它们也可直接注射到成年动物的组织中。然而，Nanoblade（与逆转录病毒和慢病毒载体相似）可能被宿主动物的免疫反应灭活；因此，注射剂量需针对每种应用进行优化。这种免疫反应还可能限制功能性病毒样颗粒（VLPs）向注射部位附近组织的分布。最后，与慢病毒载体不同，Nanoblade 不含外源转基因，且 Cas9 的递送具有时间限制性。因此，它们无法用于需要在细胞筛选后对 sgRNA 进行高通量测序的全基因组功能筛选。当需要快速、剂量依赖且无外源转基因的基因组编辑时，Nanoblade 具有重要应用价值²⁰。此外，与蛋白质电穿孔类似，相较于通过 DNA 转染或经典病毒载体实现的 Cas9/sgRNA 长期表达，Nanoblade 引起的脱靶效应更少³。未来对 Nanoblade 的开发将聚焦于整合适用于不同技术应用的 Cas9 变体，例如碱基编辑和 RNA 靶向。

材料

本文使用的材料清单

姓名	公司	目录编号	评论
13.2 mL，薄壁聚丙烯管，14 x 89 mm - 50支装	Beckman Coulter Life Sciences	331372	用于Nanoblades纯化的超速离心管
Amersham Protran Premium 蛋白印迹膜，硝酸纤维素膜	Merck	GE10600004	用于定量纯化Nanoblades中Cas9水平的硝酸纤维素膜
BIC-Gag-CAS9	Addgene	119942	编码GAG（F-MLV）-CAS9(sp)融合蛋白。可在生产细胞中与过表达的gRNA(s)及适当的包膜蛋白共同作用，产生GAG-CAS9类病毒颗粒
BICstim-Gag-dCAS9-VPR	Addgene	120922	编码GAG-dCAS9-VPR融合蛋白，用于靶向转录激活
BLADE	Addgene	134912	用于在Nanoblades系统中克隆sgRNA序列的空骨架载体
BsmBI-v2	New England Biolabs	R0739S	限制性内切酶，用于消化BLADE和SUPERBLADES载体以进行sgRNA克隆
Cas9 (7A9-3A3) 小鼠单克隆抗体（HRP偶联）#97982	Cell Signaling Technology	97982S	用于点印迹法定量Cas9的抗Cas9抗体
Cas9 核酸酶，S. pyogenes	New England Biolabs	M0386T	重组Cas9蛋白，用作Nanoblades中Cas9载量绝对定量的参考标准
溴化乙锭溶液（10 mg/mL，溶于H2O）	Sigma-Aldrich	E1510-10ML	用于琼脂糖凝胶染色及DNA可视化
Fisherbrand Wave Motion 摇床	Fisher Scientific	88-861-028	离心后重悬Nanoblades的振荡平台
gelAnalyzer			http://www.gelanalyzer.com；用于酶切后条带强度的定量分析
Gesicle Producer 293T	Takara	632617	Nanoblades生产细胞系
Gibco DMEM，高糖，含丙酮酸钠	ThermoFisher Scientific	41966052	Gesicle Producer 293T细胞的培养基
GoTaq G2 DNA聚合酶	Promega	M7848	T7核酸酶检测前用于基因组DNA扩增的Taq聚合酶
jetPRIME 转染试剂盒（用于DNA及DNA/siRNA）	Polyplus	POL114-15	用于在Gesicle Producer 293T细胞中生产Nanoblades的转染试剂
Millex-AA，0.80 µm，针头滤器	Millipore	SLAA033SS	在浓缩Nanoblades前去除细胞碎片的针头滤器
Millex-GS，0.22 µm，针头滤器	Millipore	SLGS033SS	用于蔗糖垫溶液灭菌的针头滤器
Millex-HP，0.45 µm，聚醚砜，针头滤器	Millipore	SLHP033RS	在浓缩Nanoblades前去除细胞碎片的针头滤器
Monarch DNA凝胶回收试剂盒	New England Biolabs	T1020L	用于BsmBI酶切后纯化pBLADES或pSUPERBLADES质粒片段的DNA凝胶回收试剂盒
NEB Stable 感受态E. coli（高效率）	New England Biolabs	C3040I	用于质粒转化与扩增的感受态细菌
NucleoBond Xtra Midi 试剂盒（用于转染级质粒DNA）	Macherey-Nagel	740410.50	从培养细菌中纯化质粒DNA的大提试剂盒
Nucleospin 基因组DNA提取试剂盒	Macherey-Nagel	740952.50	从转导细胞中提取基因组DNA
NucleoSpin 质粒DNA小提试剂盒	Macherey-Nagel	740588.50	从培养细菌中纯化质粒DNA的小提试剂盒
NucleoSpin 组织DNA小提试剂盒（适用于细胞与组织）	Macherey-Nagel	740952.5	基因组DNA提取试剂盒
Optima XE-90	Beckman Coulter Life Sciences	A94471	超速离心机
pBaEVRless	Els Verhoeyen (Inserm U1111)	个人请求需发送至：els.verhoyen@ens-lyon.fr	见Girard-Gagnepain A. 等人发表于Blood 124, 1221–1231 (2014) 的研究：狒狒内源性逆转录病毒Rless包膜蛋白。狒狒包膜假型慢病毒在向早期细胞因子刺激及静息状态造血干细胞进行基因转移时优于VSV-G假型慢病毒
pBS-CMV-gagpol	Addgene	35614	编码小鼠白血病病毒（Murine Leukemia Virus）的gag和pol基因
pCMV-VSV-G	Addgene	8454	用于生产慢病毒和MuLV逆转录病毒颗粒的包膜蛋白
磷酸盐缓冲液（PBS）	ThermoFisher Scientific	14200091	10X PBS，需用Millipore水稀释
Polybrene 转染试剂	Millipore Sigma	TR-1003-G	阳离子聚合物，可增强特定哺乳动物细胞中逆转录病毒转导效率。也可促进寡脱氧核苷酸（ODN）通过病毒依赖性方式进入细胞，实现同源定向修复
蔗糖，分子生物学级，≥99.5%（GC）	Sigma-Aldrich	S0389-5KG	用于制备超速离心纯化Nanoblades时的蔗糖垫
SUPERBLADE5	Addgene	134913	用于在Nanoblades系统中克隆sgRNA序列的空骨架载体（经Chen B等人，2013年优化，以提高基因组编辑效率）
SuperSignal West Dura 增强型化学发光底物	ThermoFisher Scientific	34076	用于Cas9点印迹检测的增强型化学发光（ECL）HRP底物
SW 41 Ti 摆动桶转子	Beckman Coulter Life Sciences	331362	超速离心用转子
SYBR Safe DNA凝胶染料	ThermoFisher Scientific	S33102	溴化乙锭的替代品，用于琼脂糖凝胶染色及DNA可视化
T4 DNA连接酶	New England Biolabs	M0202S	用于将BLADE或SUPERBLADES载体与对应sgRNA可变区的双链DNA寡核苷酸连接的DNA连接酶
T7 内切酶 I	New England Biolabs	M0302S	T7内切酶I可识别并切割不完全匹配的DNA，用于监测特定基因座上的基因组编辑程度
含Triton的裂解缓冲液	Promega	E291A	用于裂解Nanoblades并实现Cas9定量的裂解缓冲液
TWEEN 20	Sigma-Aldrich	P9416	用于TBST缓冲液的配制