Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究
Summary
本文介绍了CRISPR/Cas9系统对鼠血干细胞和祖细胞（HSPC）进行高效基因组编辑的协议，以快速开发具有造血系统特异性基因修饰的小鼠模型系统。
Abstract
使用传统的转基因方法在造血干细胞中操作基因可能非常耗时、昂贵且具有挑战性。得益于基因组编辑技术和慢病毒介导的转基因传递系统的进步，这里描述了一种高效、经济的方法，用于建立在造血干细胞中专门操作基因的小鼠。Lenti病毒用于转导Cas9表达的纯骨髓细胞，用引导RNA（gRNA）靶向特定基因和红色荧光报告基因（RFP），然后将这些细胞移植到致命辐照的C57BL/6小鼠中。移植了表达非靶向gRNA的慢病毒的小鼠作为对照。通过对外周血的RFP阳性白细胞的流式细胞分析，对转导造血干细胞的移植进行评价。使用这种方法，在移植后4周内，可以实现骨髓细胞+90%的转导和+70%的淋巴细胞转导。基因组DNA从RFP阳性血细胞中分离出来，目标位点DNA的某些部分被PCR扩增，以验证基因组编辑。该协议提供对造血调控基因的高通量评估，并可扩展到与造血细胞参与的各种小鼠疾病模型。
Introduction
血液学和免疫学的许多研究都依赖于转基因小鼠的可用性，包括利用造血系统特异性Cre驱动器的常规和有条件的转基因/敲除小鼠，如Mx1-Cre、Vav-Cre等1，2，3，4，5。这些战略要求建立新的小鼠菌株，这可能既费时又造成财政负担。虽然基因组编辑技术的革命性进展使新的小鼠菌株在3-4个月内产生，并具备了适当的技术专长6，7，8，9，在进行实验之前，需要更多的时间来放大小鼠群。此外，这些程序成本高昂。例如，杰克逊实验室列出了目前每株16，845美元的淘汰小鼠发电服务价格（截至2018年12月）。因此，比传统的鼠转基因方法更经济、更高效的方法更有利。
定期聚集的短片段短回溯重复/CRISPR相关蛋白9（CRISPR/Cas9）技术已导致开发新的工具，用于快速和高效的基于RNA的序列特异性基因组编辑。CRISPR/Cas9系统最初被发现为一种细菌适应性免疫机制，用于摧毁入侵的病原体DNA，它被用作提高真核细胞和动物模型中基因组编辑有效性的工具。已采用多种方法将CRISPR/Cas9机械输送到造血干细胞（即电穿孔、核化、脂肪化、病毒传递等）。
在这里，利用慢病毒系统来转化细胞，因为该系统能够有效感染Cas9表达的小鼠造血干细胞，并将指导RNA表达结构、启动子、调控序列和编码基因打包在一起荧光报告蛋白（即GFP、RFP）。利用这种方法，小鼠造血干细胞的体外基因编辑已经实现，随后在致命辐照小鼠中成功重组骨髓10。本研究使用的慢病毒载体表达来自共同核心EF1a启动子的Cas9和GFP报告基因，其内部核糖体进入位点从报告基因上游。导引RNA序列由单独的U6启动子表达。该系统然后用于在候选克隆血泊驱动基因Tet2和Dnmt3a10中创建插入和删除突变。然而，该方法的转导效率相对较低（±5%-10%）由于载体插入（13 Kbp）的尺寸大，限制了转导效率，并减少了生产过程中的病毒性粒度。
在其他研究中，已经表明，更大的病毒RNA大小对病毒的产生和转导效率都有负面影响。例如，据报道，插入尺寸增加1 kb可使病毒产生量减少±50%，小鼠造血干细胞11的转导效率将降至50%以上。因此，尽可能减小病毒插入的大小，以提高系统的效率是有利的。
这一缺陷可以通过采用Cas9转基因小鼠来克服，其中Cas9蛋白以构成性或诱导方式表达12。构成性CRISPR/Cas9敲鼠以无处不在的方式表达来自CaG启动子的Cas9内分酶和EGFP。因此，在核心EF1a启动子控制下，在U6启动子和RFP检测器基因控制下，使用慢病毒载体进行sgRNA构建，实现基因组编辑。通过该系统，成功编辑了造血干细胞的基因，显示转导效率为+90%。因此，该协议提供了一种快速有效的方法来创建小鼠，其中靶向基因突变被引入造血系统。我们的实验室主要使用这种类型的技术来研究克隆血型在心血管疾病过程13、14、15中的作用，但它也适用于血液学的研究恶性肿瘤16.此外，该协议可扩展到分析HSPC中的DNA突变如何影响造血系统的其他疾病或发育过程。
为了建立一个强大的慢病毒载体系统，需要高端点病毒储存和优化条件，以转导和移植造血细胞。在协议中，在第1节中提供了关于制备高丁酸病毒的指令，在第2节中优化了鼠造造干细胞的培养条件，第3节中的骨髓移植方法，以及评估第4节的补体。
Protocol
弗吉尼亚大学机构动物护理和使用委员会（IACUC）已批准所有涉及动物受试者的程序。 1. 慢病毒颗粒的生成和纯化 注： 含有优化导引RNA的Lenti病毒颗粒可以通过Addgene提供的详细协议产生：。关于高蒂特扁豆病毒制备和储存的优化方法，讨论在其他地方17，18。<sup class=…
Representative Results
使用上述协议，每只小鼠获得大约0.8-1.0 x 108个骨髓细胞。我们获得的系系阴性细胞数量约为每只鼠标3 x 106个细胞。通常，骨髓系阴性细胞的产量是骨髓核细胞总数的4%-5%。 转导细胞（RFP阳性）的奇血性通过外周血的流式细胞测定（图2A，B）。血液从逆轨静脉中分离出来，并?…
Discussion
与传统的小鼠转基因方法相比，该协议的优点是以快速且高成本效益的方式创建动物模型，以快速且极具成本效益的方式在造血细胞中预感特定突变。结果发现，这种方法使小鼠在1个月内进行造血细胞基因操作。此协议中有几个关键步骤需要进一步考虑。
gRNA序列的筛选
建议在体外测试gRNA，以评估在体内实验前的编辑效率。使用无细胞体外?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
S.S.得到了美国心脏协会博士后奖学金17POST33670076的支持。K. W. 得到 NIH 授予 R01 HL138014、R01 HL141256 和 R01 HL139819 的支持。
Materials
| 1/2 cc LO-DOSE INSULIN SYRINGE | EXELINT | 26028 | general supply |
| 293T cells | ATCC | CRL-3216– | Cell line |
| APC-anti-mouse Ly6C (Clone AL-21) | BD Biosciences | 560599 | Antibodies |
| APC-Cy7-anti-mouse CD45R (RA3-6B2) | BD Biosciences | 552094 | Antibodies |
| BD Luer-Lok disposable syringes, 10 ml | BD | 309604 | general supply |
| BD Microtainer blood collection tubes, K2EDTA added | BD Bioscience | 365974 | general supply |
| BD Precisionglide needle, 18 G | BD | 305195 | general supply |
| BD Precisionglide needle, 22 G | BD | 305155 | general supply |
| BV510-anti-mouse CD8a (Clone 53-6.7) | Biolegend | 100752 | Antibodies |
| BV711-anti-mouse CD3e (Clone 145-2C11) | Biolegend | 100349 | Antibodies |
| Collagen from calf skin | Sigma-Aldrich | 9007-34-5 | general supply |
| Corning Costar Ultra-Low Attachment Multiple Well Plate, 6 well | Millipore Sigma | CLS3471 | general supply |
| CRISPR/Cas9 knock-in mice | The Jackson Laboratory | 028555 | mouse |
| DietGel 76A | Clear H2O | 70-01-5022 | general supply |
| Dulbecco’s Modified Eagle’s Medium (DMEM) – high glucose | Sigma Aldrich | D6429 | Medium |
| eBioscience 1X RBC Lysis Buffer | Thermo fisher Scientific | 00-4333-57 | Solution |
| Falcon 100 mm TC-Treated Cell Culture Dish | Life Sciences | 353003 | general supply |
| Falcon 5 mL round bottom polystyrene test tube | Life Sciences | 352054 | general supply |
| Falcon 50 mL Conical Centrifuge Tubes | Fisher Scientific | 352098 | general supply |
| Falcon 6 Well Clear Flat Bottom TC-Treated Multiwell Cell Culture Plate | Life Science | 353046 | general supply |
| Fisherbrand microhematocrit capillary tubes | Thermo Fisher Scientific | 22-362566 | general supply |
| Fisherbrand sterile cell strainers, 70 μm | Fisher Scientific | 22363548 | general supply |
| FITC-anti-mouse CD4 (Clone RM4-5) | Invitrogen | 11-0042-85 | Antibodies |
| Fixation Buffer | BD Bioscience | 554655 | Solution |
| Guide-it Compete sgRNA Screening Systems | Clontech | 632636 | Kit |
| Isothesia (Isoflurane) solution | Henry Schein | 29404 | Solution |
| Lenti-X qRT-PCR Titration Kit | Takara | 631235 | Kit |
| Lineage Cell Depletion Kit, mouse | Miltenyi Biotec | 130-090-858 | Kit |
| Millex-HV Syringe Filter Unit, 0.45 mm | Millipore Sigma | SLHV004SL | general supply |
| PBS pH7.4 (1X) | Gibco | 10010023 | Solution |
| PE-Cy7-anti-mouse CD115 (Clone AFS98) | eBioscience | 25-1152-82 | Antibodies |
| PEI MAX | Polysciences | 24765-1 | Solution |
| Penicillin-Streptomycin Mixture | Lonza | 17-602F | Solution |
| PerCP-Cy5.5-anti-mouse Ly6G (Clone 1A8) | BD Biosciences | 560602 | Antibodies |
| pLKO5.sgRNA.EFS.tRFP | Addgene | 57823 | Plasmid |
| pMG2D | Addgene | 12259 | Plasmid |
| Polybrene Infection/Transfection Reagent | Sigma Aldrich | TR-1003-G | Solution |
| Polypropylene Centrifuge Tubes | BECKMAN COULTER | 326823 | general supply |
| psPAX2 | Addgene | 12260 | Plasmid |
| RadDisk – Rodent Irradiator Disk | Braintree Scientific | IRD-P M | general supply |
| Recombinant Murine SCF | Peprotech | 250-03 | Solution |
| Recombinant Murine TPO | Peprotech | 315-14 | Solution |
| StemSpan SFEM | STEMCELL Technologies | 09600 | Solution |
| TOPO TA cloning kit for sequencing with One Shot TOP10 Chemically Competent E.coli | Thermo fisher Scientific | K457501 | Kit |
| Zombie Aqua Fixable Viability Kit | BioLegend | 423102 | Solution |
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Citer Cet Article
Sano, S., Wang, Y., Evans, M. A., Yura, Y., Sano, M., Ogawa, H., Horitani, K., Doviak, H., Walsh, K. Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models. J. Vis. Exp. (152), e59977, doi:10.3791/59977 (2019).