方法文章

改进CAR T细胞中CRISPR-Cas9筛选：一种优化的文库构建方法

DOI：

10.3791/69721

⸱

2026年1月2日

 ,  ,  ,  ,  ,  ,  ,  , 
1Hemato-Oncology Program, Cima Universidad de Navarra, IdiSNA, 2Centro de Investigacion Biomedica en Red de Cancer (CIBERONC), 3Computational Biology and Translational Genomics Program, Cima Universidad de Navarra, IdiSNA, 4Cancer Center Clinica Universidad de Navarra (CCUN), 5Data Science and Artificial Intelligence Institute (DATAI), Universidad de Navarra, 6Hematology and Cell Therapy Department, Clinica Universidad de Navarra, IdiSNA

* These authors contributed equally

本文内容

摘要

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我们开发了一种针对原代CAR T细胞的优化CRISPR-Cas9基因敲除筛选方案。通过酶消化减少基因组DNA残留，并结合sgRNA表达盒的富集，该方法可最大限度地降低PCR假象，确保sgRNA的准确检测以及功能基因靶点的可靠鉴定。

摘要

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嵌合抗原受体（CAR）T细胞疗法在多种血液系统恶性肿瘤中已显示出显著疗效，但在实体瘤中的应用尚未取得同等成功。此外，即使在血液系统肿瘤中，CAR T细胞输注后仍存在复发问题，影响长期治疗效果。这些挑战凸显了迫切需要开发能够增强CAR T细胞疗效、持久性，并克服肿瘤及其微环境介导的耐药性的策略。基于成簇规律间隔短回文重复序列（CRISPR）-Cas9的筛选平台为系统性鉴定调控CAR T细胞功能的基因提供了有力手段。通过将遗传扰动与表型结果相联系，这类检测方法能够发现调控T细胞活化、增殖、记忆形成及细胞毒性的关键通路。标准操作流程通常包括：使用单导向RNA（sgRNA）文库转导大量细胞、Cas9介导的基因编辑、筛选已编辑细胞，以及在测序前从基因组DNA（gDNA）中通过PCR扩增sgRNA片段。然而，使用大量gDNA进行PCR扩增面临显著技术挑战，常常难以选择性地有效扩增和回收sgRNA。本文中，我们描述了一种经过优化的CRISPR-Cas9基因敲除筛选方案，并已在原代人源CAR T细胞中进行了验证。该方法在sgRNA文库制备过程中引入了一个中间步骤，通过酶消化和对sgRNA片段的选择性富集，减少gDNA残留，从而提高首轮PCR扩增的效率。这一改进使我们能够在CAR T细胞筛选实验中成功回收sgRNA信息，而此前采用传统的一步法和两步法PCR扩增方案均未能实现。综上所述，该优化的工作流程有助于在具有挑战性的样本中完成CRISPR筛选文库的制备，推动识别可用于提升治疗效果的关键遗传决定因素。

引言

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免疫疗法彻底改变了癌症治疗，提供了利用和调节免疫系统的新策略。其中，嵌合抗原受体（CAR）T细胞疗法已成为最具变革性的方法之一。该疗法涉及对患者T细胞的基因工程改造 离体 通过设计用于识别特定肿瘤抗原的合成受体，从而增强抗肿瘤活性1CAR T细胞在血液系统恶性肿瘤中已显示出显著疗效，接受靶向CD19或BCMA治疗的患者中，疗效范围从短暂缓解到长期无病生存，且毒性反应轻微。2迄今为止，美国食品药品监督管理局（FDA）已批准七种CAR T细胞产品用于血液系统肿瘤的治疗3尽管取得了这些进展，仍存在显著的局限性。问题可能源于CAR T细胞产品本身，通常受到制备工艺或患者T细胞功能状态的影响，而后者可能因疾病进展、既往治疗或年龄因素而受损。肿瘤内在的耐药机制，例如抗原下调，也可能削弱治疗效果。4此外，免疫抑制性的肿瘤微环境构成了另一重障碍，严重限制了CAR T细胞的存续时间与功能，尤其是在实体瘤患者中。5因此，迫切需要提高CAR T细胞疗法的初始疗效和长期有效性。

CRISPR-Cas9 基因编辑技术的出现，为通过大规模筛选方法解析基因功能提供了强有力的工具。在这些实验中，细胞被转导单导向 RNA（sgRNA）文库，通常由慢病毒载体递送，确保每个细胞仅含一个 sgRNA 并实现稳定的基因组整合。在 Cas9 介导的编辑及转导细胞筛选完成后，提取基因组 DNA（gDNA），并通过 PCR 扩增 sgRNA 串联序列以构建文库。随后的高通量测序可定量分析不同表型中 sgRNA 的分布情况，从而鉴定出对所研究过程起正向或负向调控作用的基因6。

CRISPR筛选已被广泛应用于探索T细胞生物学，最近还被用于增强CAR T细胞的功能。全基因组筛选已鉴定出调控T细胞基本过程的因子，包括活化、增殖和分化。例如，FAM49B被鉴定为T细胞受体信号通路的调控因子7，而SOCS1、TCEB2、RASA2和CBLB被证明在刺激后对增殖至关重要8。除了这些核心通路外，CRISPR筛选还揭示了参与T细胞记忆和耗竭的基因。体内研究发现Fli1是一个可增强效应反应但不破坏记忆或耗竭前体的候选因子9，而染色质重塑因子Arid1a的缺失可减少T细胞耗竭10。利用该方法还表征了调控辅助性T细胞2型（Th2）分化的因子11。通过使用靶向25种激酶的定制sgRNA文库，发现p38可促进扩增、记忆形成以及对氧化应激和基因组应激的防护12。类似地，在CD8+ T细胞中敲除REGNASE-1可赋予其长效效应表型，从而在黑色素瘤和白血病模型中改善肿瘤控制13。其他全基因组筛选鉴定出Dhx37为调控T细胞活化和细胞毒性的因子14，同时验证了LTBR及其他基因在CAR T细胞和γδ T细胞中作为T细胞功能增强因子的作用15。最近，CRISPR筛选已直接应用于CAR T细胞，揭示了新的靶点，例如PRODH2——一种参与脯氨酸代谢并增强CAR T抗肿瘤活性的酶16，以及TLE4和IKZF2，其失活在胶质母细胞瘤模型中提高了CAR T的疗效17。

CRISPR 筛选中的一个关键步骤是从极大量的基因组 DNA（gDNA）中精确地选择性扩增 sgRNA 表达盒，这一挑战在 CRISPR 筛选实验中因使用大量细胞而难以避免。从如此大量的 DNA 中进行 PCR 扩增会带来多项技术难题，包括分子拥挤效应限制了 DNA 和聚合酶分子的物理扩散、引物与聚合酶在基因组 DNA 背景上的瞬时非特异性结合、非靶标扩增导致引物耗竭，以及 DNA 磷酸骨架对 Mg2+ 的螯合作用等其他问题18,19,20,21。这些问题常导致 PCR 失败和/或扩增偏差。为缓解上述问题，已提出多种策略，包括将 sgRNA 扩增与接头添加分为两个独立的 PCR 步骤22，使用生物素标记的寡核苷酸进行靶标富集并结合磁珠捕获23,24，或设计在 sgRNA 表达盒两侧含有限制性酶切位点的质粒以实现片段富集25。

本研究中，我们介绍了一种针对原代人源CAR T细胞优化的CRISPR-Cas9基因敲除筛选方案。所使用的CRISPR文库为Brunello Kinome 1，该文库包含3052条独特的20nt长度的sgRNA，靶向763个人类激酶基因（每个靶点4条sgRNA）。T细胞通过磁珠阳性分选法从健康供体血液样本中富集的外周血单个核细胞（PBMC）组分中分离获得，分选标记为CD4+和CD8+。经抗CD3和抗CD28抗体激活后，T细胞通过离心转导（spinfected）方式导入CRISPR筛选文库，随后转导CAR慢病毒。细胞扩增后，通过核转染（nucleofection）方式导入Cas9蛋白。携带CRISPR的细胞使用嘌呤霉素进行筛选，并分选获得CAR阳性的T细胞。提取基因组DNA后，使用限制性内切酶（RE）进行消化，含有sgRNA的片段通过生物素探针-链霉亲和素捕获法富集。随后，通过两轮连续PCR选择性扩增并添加索引，对所得文库进行测序。通过引入中间步骤以减少基因组DNA残留，我们能够选择性地回收并扩增目标sgRNA，从而研究激酶在CAR T细胞中的功能作用。

方案

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This protocol was performed in accordance with Universidad de Navarra guidelines. All subjects provided written informed consent. The reagents and the equipment used in this study are listed in the Table of Materials.

1. T cell isolation and activation

  1. PBMC isolation
    1. Collect blood samples from donors on ethylenediaminetetraacetic acid (EDTA) tubes, transfer blood samples to 50 mL conical tubes, and dilute blood with PBS (dilution 1:1).
    2. Add 15 mL of density gradient medium to the bottom of a 50 mL conical tube (density for PBMCs: 1.07). Then, add carefully 25 mL of diluted blood over the density gradient medium layer (slowest speed of the pipette controller). Centrifuge for 30 min at 800 × g without brake.
    3. Collect PBMCs ring/layer with a Pasteur pipette into a 50 mL conical tube and add PBS to a total volume of 50 mL to wash. Centrifuge at 650 × g for 8 min. Discard the supernatant.
      NOTE: Check the turbidity of the supernatant to be sure that there are no cells there. If in doubt, divide the suspension into twice the original number of 50 mL conical tubes, add PBS until 50 mL, and centrifuge again.
    4. Resuspend in 50 mL of PBS. Count with acridine orange-propidium iodide (AOPI) in a cell counter. Centrifuge at 650 × g for 8 min.
  2. CD4 and CD8 magnetic selection
    1. Prepare fluorescence-activated cell sorting (FACS) buffer: PBS, EDTA 2.5 µM, BSA 0.5% and filter through 0.2 µm.
    2. Resuspend PBMCs in 80 µL of FACS buffer sterile per 10 × 106 cells and add 10 µL of microbeads CD4 and 10 µL of microbeads CD8 per 10 × 106 cells. Incubate 20 min at 4 °C.
    3. Add 10 mL of FACS buffer and centrifuge for 8 min at 650 × g. Discard the supernatant completely.
    4. Resuspend in 500 µL per 1 × 108 cells of FACS Buffer in a 15 mL conical tube.
      NOTE: A run can accommodate 200 x 106 cells.
    5. Use the Possel program for AutoMacs (high-speed magnetic cell sorter) isolation.
    6. Add up to 10 mL of PBS, count with AOPI in a fluorescent cell counter, and centrifuge at 650 × g for 8 min.
    7. Resuspend cells 1 x 106/mL in T cell growth medium containing 3% human serum (HS), 1% penicillin/streptomycin (P/S), 625 IU/mL interleukin (IL)-7, 85 IU/mL IL-15. Add 10 µL of T cell stimulation reagent per 1 mL and mix well. Plate 2 mL per well in a 24 well plate (p24w) and incubate for 24 h at 37 °C.

2. CAR T cell production and CRISPR screening

  1. Library spinfection and CAR transduction
    1. Add the corresponding amount of lentivirus CRISPR library (e.g., Brunello Kinome 1 library. See Supplementary Table 1), determined in a titration assay, and 8 µg/mL polybrene.
    2. Spinfect by centrifugation at 700 x g for 1 h 30 min at 32 °C.
    3. Replace media with fresh T cell growth medium containing 3% HS, 1% P/S, 625 IU/mL IL-7, 85 IU/mL IL-15 (final [cell]= 106 cells/mL).
    4. After 6 h, add the corresponding amount of CAR lentiviral vector to a multiplicity of infection (MOI) of 3 and incubate for 4 days.
  2. Cas 9 electroporation
    1. Collect CAR T cells, wash once with PBS, and count cells with AOPI in a fluorescent cell counter.
    2. Prepare 4 µM Cas9 in electroporation buffer.
    3. Resuspend CAR T cells in 50 µL per 5 × 106 cells. Mix and carefully add 50 µL of cell suspension in each well of a multiwell cuvette strip without forming bubbles. Then, using a pipette tip, remove any possible bubbles and run the tip along the edges of the well.
      NOTE: If any well in a cuvette is left without a sample, add the same volume of electroporation Buffer. This is a critical step: if the machine detects a bubble or volume difference, it will give an error.
    4. Use the program EXPAND T CELL 3 to nucleofect CAR T cells.
    5. Leave the cells in the cuvettes in an incubator at 37 °C for 40 min.
    6. Resuspend the nucleofected cells in T cell growth medium containing 3% HS, 1% P/S, 625 IU/mL IL-7, 85 IU/mL IL-15 (final [cell]= 106 cells/mL) and incubate at 37 °C for 3 days.
  3. Puromycin selection
    1. Count with AOPI in a fluorescent cell counter.
    2. Adjust to 1 x 106 cells/mL with T cell growth medium containing IL7 and IL15.
    3. Add puromycin (2.5 µg/mL) to the media.
    4. Count cells every 2 days for 6 days and adjust to 1 x 106 cells/mL with cell growth medium containing 3% HS, 1% P/S, 625 IU/mL IL-7, 85 IU/mL IL-15, and 2.5 µg/mL puromycin.
    5. Check % CAR.
  4. Sort CAR T cell populations and store samples (dry cell pellet) at -80 °C.

3. Isolation of gDNA

NOTE: Avoid any cell or gDNA losses throughout the process, as this may compromise the representativity of the sgRNAs. Aim for a sgRNA coverage of at least 500x.

  1. Extract gDNA from samples using the cells and tissue DNA extraction kit. Elute twice to maximize gDNA recovery.
    NOTE: Do not use more than 4 x 106 cells/column in order to recover the gDNA more efficiently.
  2. Measure gDNA concentration with the dsDNA quantification kit.
    NOTE: If the concentration is too low for subsequent steps in the protocol, use solid-phase reversible immobilization (SPRI) bead-based clean-up reagent to concentrate the DNA.

4. Enrichment of sgRNA cassette (and elimination of gDNA carryover)

  1. Restriction enzyme digestion
    1. Digest a maximum of 5 µg gDNA with 20 U of the enzyme/s (e.g., for lentiGuide-puro backbone for Brunello Kinome 1 library use the combination NdeI and PspXI) in a total volume of 50 µL. Scale up the number of reactions to achieve the desired sgRNA representativity.
      ​NOTE: Select the restriction enzymes to cut in the flanking regions of the cassette, but bear in mind that some of the sgRNAs might contain a restriction site. These sgRNAs will be lost in this step, and they will not show up in the sequencing. Absence of this guide serves as a control of digestion. Supplementary Table 1 shows a list of Kinome 1 sgRNAs that are cleaved by PspXI and NdeI.
    2. Incubate overnight at 37 °C in a thermocycler.
    3. Perform a 2x SPRI clean-up.
      1. Prepare 10 mL of Elution Buffer (EB) (10 mM Tris-HCl, pH 8.0) and 50 mL of fresh 70% ethanol.
      2. Vortex the beads and add 100 µL to the product of the digestion.
      3. Mix thoroughly by pipetting and incubate for 5 min. Magnetize for another 5 min.
      4. While on the magnet, add 200 µL of 70% ethanol without disturbing the pellet. Wait 30 s, then discard the supernatant.
      5. Repeat step 4.1.3.3 for a total of 2 washes. Allow the beads to dry for 2 min.
      6. Remove from the magnet, add 40 µL of elution buffer, and resuspend the beads by pipetting. Incubate for 2 min.
      7. Magnetize for 2 min and transfer the supernatant to a new tube.
        NOTE: When setting up this protocol for the first time, verify the digestion through (capillary) electrophoresis. Ensure that a smear is observed.
  2. Pulldown of the sgRNA cassette.
    1. Preparation of streptavidin magnetic beads.
      1. Prepare Wash/Binding Buffer 2x: 10 mM Tris-HCl pH 7.5, 2 M NaCl, 1 mM EDTA. Dilute half of the volume with H2O to 1x.
      2. Vortex the beads for 1 min.
      3. Transfer the desired volume of beads to a tube (typically 1 mg will suffice), add an equal volume of Wash/Binding Buffer 1x, or at least 1 mL, and resuspend.
      4. Place the tube on a magnet for 1 min and discard the supernatant.
      5. Remove the tube from the magnet and resuspend the washed beads in the same volume of 1x Wash/Binding Buffer as the initial volume of beads taken from the vial.
      6. Repeat for a total of 3 washes.
      7. Resuspend beads in 2x Wash/Binding Buffer.
    2. Add 5 µL of 10 µM pulldown primers listed in Table 1 to the purified digestion from step 4.1.3.6.
    3. Incubate in a dry bath at 96 °C for 5 min, then immediately bring to ice for 5 min.
    4. Add 1 mg of pre-washed streptavidin magnetic beads.
      NOTE: Biotin-streptavidin binding works best at 1 M NaCl. Make sure to mix equal volumes of beads and biotinylated DNA.
    5. Incubate for 20 min while resuspending the mix every 4 min.
    6. Magnetize for 5 min.
    7. Wash the beads 3 times with 1× Wash/Binding Buffer.
    8. Resuspend the beads in 50 µL of EB 8.0.
      NOTE: The sgRNA cassettes used here are bound to the streptavidin beads. Do not discard them.

5. sgRNA library preparation

  1. PCR1
    1. Prepare PCR1 mix with 20 µL of Herculase 5x buffer, 1 µL of dNTP (100 mM), 1 µL of DNA polymerase, 2.5 µL of 10 µM of PCR1 forward and reverse primers listed in Table 1, and 23 µL of PCR grade water. Pipette mix and add PCR1 mix to the beads from step 4.2.8.
      NOTE: Table 1 lists PCR1 primers designed for the Brunello Kinome Library 1.
    2. Place the tubes in a thermocycler and run the program for PCR1 described in Table 2.
      NOTE: Make sure the thermocycler can accommodate 100 µL reactions. Otherwise, aliquot in 50 µL reactions. Scale as needed.
    3. Pool all PCR1 from the same samples in a single tube.
    4. SPRI clean-up PCR1 using 150 µL (1.5x) of beads (follow steps 4.1.3.3-4.1.3.7). Resuspend each PCR reaction in 100 µL of EB 8.0.
      NOTE: If setting up this protocol for the first time, verify the size and quality of the PCR product by running 2 µL of a 30-cycle PCR product in a capillary electrophoresis system (e.g., Agilent's TapeStation Systems).
  2. PCR2
    1. Prepare PCR2 mix with 10 µL of the product of PCR1, 10 µL of 5X buffer, 0.5 µL of dNTP (100 mM), 1 µL of DNA polymerase, 1.25 µL of 10 µM PCR2 primers forward and reverse listed in Table 1 (use a different rP7-i7-BC and P5-i5-BC combination for each sample), and 26 µL of PCR grade water for a total volume of 50 µL.
      NOTE: To maintain library representation, perform one PCR2 reaction per 104 constructs in the library.
    2. Place the tubes in a thermocycler and run the program for PCR2 described in Table 2.
    3. SPRI clean-up PCR2 using 75 µL (1.5x) of beads (follow steps 4.1.3.3-4.1.3.7). Resuspend each PCR reaction in 50 µL of EB 8.0.
  3. Quantify PCR2 (i.e., the sgRNA library) using the dsDNA quantification kit and verify the amplicon size by capillary electrophoresis (e.g., TapeStation Systems). Pool the samples.
  4. Sequence on an Illumina instrument.
    NOTE: For the 3052 sgRNA Brunello Kinome 1 library here, a sequencing depth of 100-200 million reads per sample is typically enough.

6. Analysis

  1. Demultiplex samples using bcl2fastq.
  2. Check the quality of the fastq files generated with FastQC.
  3. Extract the sequence of the sgRNA using as a pattern the cassette sequences flanking the sgRNA, as well as the length of the guide (20 nt).
  4. Align the sequence extracted to the library reference using Bowtie2 to check how many of the hypothetical guides are authentic sgRNA from the library. Use samtools to convert the SAM file with information about the alignment into BAM format and generate summary files.
  5. Count and normalize the number of reads for each sgRNA in the edited and control samples with the count function from MAGecK software.
  6. Compare the counts with the test function of MAGecK to obtain the RRA scores and the list of candidate genes.

结果

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如方案部分所述，CAR T细胞的制备遵循我们既定的方案26。对于CRISPR筛选，我们采用了Wang等人17开发的工作流程（图1A）。简言之，分别从两位独立供体中分离T细胞并激活24小时，随后依次转导CRISPR sgRNA文库和CAR慢病毒载体，在第5天时CAR阳性细胞比例分别达到67%和72%。部分细胞通过核转染导入Cas9蛋白以诱导基因敲除，其余细胞则作为sgRNA表达水平的基线对照。转导后的细胞通过抗生素筛选，并根据所研究的表型对不同细胞群进行分选。在整个筛选过程中，通过基于AOPI的细胞计数监测CAR T细胞的增殖情况，并以倍数变化表示（图1B）。在第8、10和12天加入嘌呤霉素后，我们证实了两位供体中转导了CRISPR文库的CAR T细胞均成功富集，表现为持续存活和增殖。未转导T细胞（UTD）以及未转导CRISPR文库的CAR T细胞作为抗生素筛选的对照。

基因组DNA提取后，通过PCR扩增制备sgRNA文库。为解决gDNA过度残留带来的问题，我们采用了一种结合限制性内切酶消化和sgRNA片段的生物素-链霉亲和素富集的方案（图2A，B）。使用限制性内切酶NdeI和PspXI对gDNA进行消化，这两种酶的识别位点位于sgRNA片段两侧（图2C）。设计了可结合在PCR1引物结合位点上游的生物素标记寡核苷酸，用于捕获包含sgRNA的片段，随后通过链霉亲和素磁珠进行富集，以去除残留的gDNA。随后选择性扩增含有sgRNA的片段（图2D），并通过两轮连续PCR进行加标签（indexing）。

使用下一代测序技术（NGS）对每个样本的文库进行测序。在完成去卷积、质量控制和sgRNA鉴定后，利用MAGeCK分析流程对数据进行分析。首先，通过count命令对对照组（基础状态）和Cas9编辑样本中的sgRNA读数进行量化和标准化（图3A、B）。Pearson和Spearman相关性分析表明，在实验过程中sgRNA的代表性在供体基因组DNA中得以保持，与质粒文库相比具有良好的一致性（图3C）。接着，使用test命令比较两种条件，为每个基因分配稳健秩聚合（RRA）评分（图3D）。采用显著性阈值p < 0.05 和对数倍数变化（LFC）> 0.5 来识别显著富集或耗竭的基因（图3E）。最后，利用Metascape对候选基因进行通路富集分析，揭示与所鉴定调控因子相关的主要基因本体（Gene Ontology）术语（图3F）。

CRISPR 基因编辑流程图、T 细胞修饰时间线、表型变异图、倍数变化图表。
图 1：CAR T 细胞中的 CRISPR 筛选。（A）CAR T 细胞中 CRISPR 筛选的示意图。（B）使用嘌呤霉素（P）筛选转导细胞。质粒文库包含嘌呤霉素抗性基因用于阳性筛选。在第 8 天向未转导（UTD）样本、CAR T 细胞以及转导的 CAR T 细胞培养基中加入抗生素。未添加嘌呤霉素的样本用作增殖对照。数据显示了两个独立供体的结果。请点击此处查看该图的放大版本。

基因编辑过程；A：消化→下拉→PCR，B：gRNA 示意图，C/D：电泳结果。
图 2：用于 sgRNA 富集分析的文库制备。（A）文库制备流程示意图。（B）限制性内切酶切割位点（剪刀图标）与 PCR1 引物（箭头）及生物素化引物（带绿色圆点的箭头）结合区域相对位置的示意图。（C）基因组 DNA 在限制性内切酶消化前后的电泳结果。（D）PCR1 后 sgRNA 盒式结构的特异性富集。结果显示了两种表型的基底样本和 Cas9 样本中两个独立供体的数据。请点击此处查看该图的放大版本。

基因表达比较分析：表格、图表、相关性图、对数倍数变化、显著性分析。
图 3：使用 MAGeCK 进行引导序列富集分析。（A）MAGeCK count 功能输出结果的汇总。（B）读段计数的分布情况。（C）供体质粒 gDNA 与质粒文库计数之间相关性的 Pearson 和 Spearman 相关系数。（D）通过改进的稳健排序整合方法（RRA 评分）排序的潜在富集/耗竭基因。（E）火山图显示显著差异基因（p 值 < 0.05，LFC > 0.5），以红色标出。（F）对富集的显著基因进行 Metascape 通路分析后得到的前导 Gene Ontology（GO）条目。请点击此处查看该图的放大版本。

表1：引物序列。 请点击此处下载该表格。

表2：PCR条件。 请点击此处下载该表格。

补充表 1：激酶组 1 文库中的 sgRNA 列表以及在 NdeI 和 PspXI 消化过程中丢失的 sgRNA 请点击此处下载该文件。

讨论

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CRISPR筛选依赖于单导向扰动原理，即每个细胞中应仅整合一条sgRNA。为实现这一点，sgRNA通常通过慢病毒载体递送，且感染复数（MOI）必须严格控制。MOI应为 约 0.3 通常为推荐值，可使 25% 至 30% 的细胞被单个病毒颗粒转导6因此，准确的滴定对于确定所需病毒颗粒体积至关重要。另一个关键步骤是实验过程中单导向RNA（sgRNA）覆盖度的丢失。细胞收获过程中的细胞损失、基因组DNA（gDNA）提取效率低下（如柱过载或DNA洗脱不完全）以及PCR扩增时模板过量，均可能导致sgRNA覆盖度下降，从而影响sgRNA分析结果的准确性。关于生物素标记的寡核苷酸，建议采用高效液相色谱（HPLC）纯化，以避免合成过程中残留的游离生物素饱和链霉亲和素磁珠。此外，强烈建议在寡核苷酸3'端引入氨基修饰，以防止后续PCR中生物素化引物的非特异性扩增。在设计这些寡核苷酸时，应避免其序列与PCR1引物发生重叠，以防扩增过程中产生竞争。此外，生物信息学分析需格外谨慎：sgRNA提取模式依赖于质粒骨架（如lentiGuide-puro或lentiCRISPR v2），比对严格性可从完全匹配到允许一定错配容忍度不等。另外，由于本方案包含酶切步骤，部分含有限制性酶切位点的sgRNA可能丢失，需相应调整参考文库。

本研究提出了一种优化的CRISPR筛选文库制备方案，通过酶切，更重要的是通过富集步骤，富集含有sgRNA的片段。此前已有研究建议使用限制性内切酶消化来减少基因组DNA（gDNA）投入量并富集sgRNA表达盒27。类似地，生物素标记的探针已在分子生物学中被广泛用于捕获特定核酸或蛋白质-核酸复合物28,29,30,31。

本方案基于这些原则，并将其改进应用于原代CAR T细胞中的CRISPR筛选，但需承认存在若干局限性。首先，原代CAR T细胞对筛选规模存在限制，因其扩增能力有限。为维持足够的覆盖深度和sgRNA代表性，需要大量起始细胞。当此条件不可行时，可使用较小的定制文库12，但这会降低遗传筛选的广度。生物学变异性是另一项挑战；不同供体之间的差异不可避免，且显著筛选结果的重叠程度通常较低17。其次，富集策略依赖于限制性内切酶切割位点的存在及其位置。在极少数情况下，这些位点可能出现在sgRNA序列内部，对于冗余度较低的小型文库而言，这一问题更为突出。然而，大多数文库（尤其是全基因组设计的文库）针对每个靶点包含多个sgRNA，从而最大限度地降低了向导序列丢失的整体影响。最后，在限制性内切酶消化不适用的情况下，基于片段化的替代富集策略（如超声破碎联合探针捕获24）可能提供一定的灵活性。

总体而言，在CRISPR编辑的细胞中，sgRNA片段仅占总基因组DNA的极小部分，因此高效的富集对于成功的文库构建至关重要。与标准方案相比，我们优化的工作流程减少了基因组DNA（gDNA）的残留，并更有利于目标sgRNA的PCR扩增。尽管将gDNA片段化与生物素标记探针结合链霉亲和素磁珠进行富集的技术在概念上并非全新，但将其整合到CRISPR筛选中可避免常见的PCR偏差，提高CRISPR筛选的可重复性。此外，在每个关键步骤引入标准化的质量控制指标（例如分选后T细胞纯度、嘌呤霉素抗性细胞比例、gDNA完整性以及CAR转导效率），将进一步增强该工作流程的稳健性和可重复性。总之，尽管该方案是在原代CAR T细胞背景下开发的，且仅使用了两位供体（尽管每位供体使用了多个样本，这可能仍存在一定局限性），但此优化后的实验流程可能广泛适用于其他CRISPR筛选及存在大量gDNA残留的NGS工作流程。

致谢

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本研究由 MICIU/AEI /10.13039/501100011033 和 FEDER、欧盟资助的 PID2022-137914OB-I00 项目支持。本研究还得到卡洛斯三世健康研究所（Instituto de Salud Carlos III，ISCIII）通过先进疗法网络 TERAV 和 TERAV+（RD21/0017/0009 和 RD24/0014/0010）以及癌症生物医学研究中心网络 CIBERONC（CB16/12/00489）的支持。本研究同时获得纳瓦拉政府卫生部门（Gobierno de Navarra Salud，GN2023/08 和 GN2024/04）以及战略项目（DIAMANTE 0011-1411-2023-000105 和 0011-1411-2023-000074）的支持。图1B 和 图2A 使用 BioRender.com 制作。

材料

本文使用的材料清单
姓名公司目录编号评论
4200 TapeStationAgilentG2991A
吖啶橙InvitrogenA1301
AMPure XP Beckman CoulterA63881
autoMACS NEO 分离仪Miltenyi130-120-327高速磁性细胞分选仪
生物素化寡核苷酸IDT
BSASigmaA9647
Cellometer K2 荧光细胞计数仪RevvityCMT-K2-MX-150荧光细胞计数仪
CHT4 计数室RevvityCHT4-SD100-002
CytoSinct CD4 纳米磁珠GenScriptL00863-1
CytoSinct CD8 纳米磁珠GenScriptL00864-1
dNTPAgilent200418-51
Dynabeads M-280 链霉亲和素磁珠Invitrogen11206D
DynaMag-2 磁力架Invitrogen12321D
EDTA 0.5 M pH 8.0Invitrogen15575-038
乙醇Merck1,00,98,31,000
ExPERT Atx 电穿孔仪MaxCyteExPERT ATx
Ficoll-PaqueCytiva17544203
Filtropur S 0.2 µmSarstedt83,18,26,001
GenCRISPR Ultra NLS-Cas9-ResearchGenScriptZ03621-1
Herculase Buffer 5XAgilent600675-525X 缓冲液
Herculase II 融合酶Agilent600679-51DNA 聚合酶
IL-15Miltenyi130-095-765
IL-7Miltenyi130-095-362
Mastercycler X50aEppendorf6313HG006059
MaxCyte 电穿孔缓冲液CytivaEPB1
NdeINew England BiolabsR0111L
Nucleospin 组织提取试剂盒Macherey-Nagel74,09,52,250
PBSGibco14190-094
聚凝胺MerckTR-1003-6
引物ThermoFisher
碘化丙啶SigmaP4864
PspXINew England BiolabsR0656S
嘌呤霉素GibcoA11138-03
Qubit 3.0 荧光计InvitrogenQ33216
Qubit HS DNA 检测试剂盒InvitrogenQ32851
R-50x3 无菌孔板处理组件MaxCyteLM243882
T Cell TransAct 人源Miltenyi130-111-160T 细胞刺激试剂
Tape Station HSD1000Agilent5067-5585
TEXMACSMiltenyi130-097-196T 细胞生长培养基
Tris 缓冲液，1.0 M，pH 8.0，分子生物学级EMD Milipore648314
分子生物学级水，无 DNA 酶、RNA 酶和蛋白酶ThermoScientific327390010

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CRISPR Cas9 sgRNA DNA

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