使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变
Summary
这里介绍的是sgRNA / CAS9内切酶和下一代测序方案，可用于鉴定与CD4启动子附近的双链断裂修复相关的突变。
Abstract
DNA中的双链断裂（DSBs）是最具细胞毒性的DNA损伤类型。由于无数的侮辱可导致这些病变（例如，复制应激，电离辐射，未修复的紫外线损伤），DSB每天都会发生在大多数细胞中。除了细胞死亡之外，未修复的DSB还会降低基因组完整性，由此产生的突变会驱动肿瘤发生。这些风险和DSB的患病率促使人们研究细胞修复这些病变的机制。下一代测序可以与电离辐射诱导DSB配对，以提供一种强大的工具来精确定义与DSB修复缺陷相关的突变。然而，这种方法需要计算上具有挑战性且成本高昂的全基因组测序来检测与电离辐射相关的随机发生的DSB的修复。罕见的切割内切酶，如I-Sce1，提供了产生单个DSB的能力，但它们的识别位点必须插入到感兴趣的基因组中。因此，修复部位本质上是人为的。最近的进展允许引导RNA（sgRNA）将Cas9内切酶引导到任何感兴趣的基因组位点。这可以应用于DSB修复的研究，通过允许下一代测序专注于Cas9诱导的DSB侧面的DNA，使下一代测序更具成本效益。该手稿的目标是通过提出一种方案来证明这种方法的可行性，该协议可以定义源自CD4基因上游DSB修复的突变。该方案可以调整以确定与外源性因素相关的DSB诱变潜力的变化，例如修复抑制剂，病毒蛋白表达，突变和环境暴露，计算要求相对有限。一旦对生物体的基因组进行了测序，这种方法理论上可以在任何基因组位点和可以转染的生物体的任何细胞培养模型中使用。对这种方法的类似改编可以比较同一遗传背景中不同位点之间的修复保真度。
Introduction
保持基因组稳定性对所有生物体都至关重要。准确的DNA复制和强大的DNA损伤反应（DDR）对于忠实地繁殖遗传物质1，2是必要的。DNA损伤在大多数细胞中定期发生2，3。当这些损伤被感知时，细胞周期进展停止，DNA修复机制被激活。DNA或DSB中的双链断裂是毒性和致突变性最强的DNA损伤类型3，4。
虽然几种DDR信号通路可以修复这些病变，但研究最彻底的DSB修复途径是同源重组（HR）和非同源末端连接（NHEJ）。HR是一种基本上无差错的途径，它使用姐妹染色单体作为同源模板来修复DSB。这往往发生在细胞周期5，6，7的S期和G2期。NHEJ更容易出错，但它可以在整个细胞周期8，9中发生。已经开发了各种报告物测定法来测量特定修复机制10，11，12的效率。这些测定倾向于依靠流式细胞术，使用GFP或mCherry作为读数11，13对DSB修复途径活性进行高通量测量。虽然效率很高，但它们依赖于在人为引入的DSB上进行的规范修复。
还有许多其他方法可用于研究DSB修复。其中许多依赖于免疫荧光（IF）显微镜1，14。IF显微镜检测在DSB暴露于遗传毒性化学物质或电离辐射15，16诱导后代表修复复合物的离散核病灶。跟踪这些病灶的形成和消退分别提供了修复开始和完成的指示，分别为14，17。然而，这些DSB诱导方法（即化学物质或电离辐射）不会在基因组中定义的位置引起DSB。在功能上也不可能使用它们来始终如一地诱导少量（例如，2-4个）的DSB。结果，最常用的诱导DSBs的方法导致大量病变随机分布在整个基因组18中。通过插入罕见切割内切内切酶的识别位点并表达相关的内切酶（例如I-Sce119），可以引入少量DSB。不幸的是，目标位点的必要整合阻止了在内源性基因组位点检查DSB。
本手稿描述了一种检测与在用户定义的位点处生成的DSB修复相关的突变的方法。我们提供了一个代表性的例子，说明该方法适用于评估病毒蛋白增加与DSB相关的突变数量的能力。具体而言，本手稿描述了使用单一引导 RNA （ sgRNA ）来指导 CAS9 内切酶在人包皮角质形成细胞表达载体对照（ HFK LXSN ）和表达人瘤病毒 8 型（ HFK 8E6 ）的 E6 蛋白的人类 CD4 开放阅读框处诱导 DSB 。断裂周围区域的靶向下一代测序（NGS）允许严格定义与病变修复相关的突变。这些数据表明，病毒蛋白在DSB修复过程中导致突变增加约20倍。它还提供了DSBs在单个位点的诱变后果的无偏表征，而无需进行全基因组测序。原则上，该方案可以很容易地进行调整，以比较基因组位点或细胞系之间突变的相对风险。
Protocol
Representative Results
Discussion
除了提供的信息深度之外，此方法还具有几个优点。首先，DSB修复，从理论上讲，可以在任何基因组位点进行评估，而无需修改目标细胞的基因组。其次，通过制作和分析针对定义区域的单个DSB，降低了成本和计算工作量，从而增加了对修复的NGS分析的访问。最后，随着其他生物体的基因组常规可用，并且多个出版物证明成功转染了不同的哺乳动物和非哺乳动物细胞系，这种方法的效用预计将广?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
本手稿中报告的研究得到了美国国立卫生研究院国家普通医学科学研究所（P20GM130448）（NAW和RP）的支持;美国国立卫生研究院国家癌症研究所（NCI R15 CA242057 01A1）;堪萨斯州立大学约翰逊癌症研究中心;和美国国防部（CMDRP PRCRP CA160224 （NAW））。我们感谢KSU-CVM共聚焦核心和Joel Sanneman的免疫荧光显微镜。内容完全由作者负责，并不一定代表这些资助机构的官方观点。
Materials
| 6 Well Tissue Culture Plate | Celltreat | 229106 | Cell culture plate |
| BCA Kit | VWR | 89167-794 | BCA assay kit |
| Centrifuge 5910 R | Eppendorf | 2231000772 | Tabletop Centrifuge |
| CLC Genomic Workbench | Qiagen | 832001 | deep sequence data analysis software/indel caller/variant caller |
| Digital Microplate Genie pulse | Scientific industries | SI-400A | Plate shaker |
| DYKDDDDK Tag Monoclonal Antibody (FG4R) | ThermoFisher Scientific | MA191878 | Anti-FLAG antibody |
| Epilife CF Kit | ThermoFisher Scientific | MEPICF500 | Cell cultrue media and supplements |
| Fetal Bovine Serum (FBS) | VWR | 89510-194 | Cell culture supplement |
| Goat anti-Rabbit IgG | ThermoFisher Scientific | A-11012 | Secondary antibody |
| HighPrep PCR Clean-up system | MagBio | AC-60005 | Bead-based PCR cleanup kit |
| KAPA HiFi HotStart ReadyMix PCR Kit | KAPA Biosystems | KK2600 | PCR mastermix/PCR assay |
| MagAttract HMW DNA kit | Qiagen | 67563 | High Molecular Weight DNA extraction kit |
| Magnetic Stand-96 | Thermo Fisher Scientific | AM10027 | 96-Well Magnetic Rack |
| MiniAmp Thermal Cycler | Applied Biosystems | A37834 | Thermal Cycler |
| Miseq | Illumina | SY-410-1003 | Sequencer |
| Miseq v2 300 cycle reagent kit | Illumina | MS-102-2002 | 300-cycle cartridge/sequencing reagents |
| Nextera XT DNA Library Prep kit | Illumina | FC-131-1024 | Library preparation kit |
| Nextera XT Kit v2 Set A | Illumina | 20027215 | Indexes |
| Nunc 96-well polypropylene DeepWell Stroage plates | Thermo Fisher Scientific | 260251 | deep well 96-well plates |
| Penicillin-Streptomycin Solution (100X) | Calsson Labs | PSL02-6X100ML | Antibiotics for cell culture |
| Phosphate Buffered Saline (PBS) | Bio Basic | PD8117 | PBS |
| px330-CD4 | Addgen | 136938 | SgRNA/CAS9 plasmids targeting 5’- GGCGTATCTGTGTGAGGACT |
| QIAxcel Advanced System | Qiagen | 9001941 | capillary electrophersis machine |
| QIAxcel DNA screening kit | Qiagen | 929004 | DNA buffer/ capillary electrophersis tubes |
| Qubit 1x ds HS Assay Kit | ThermoFisher Scientific | Q23851 | Fluorometer reagents/1x dsDNA solution |
| Qubit 4 Fluorometer | ThermoFisher Scientific | Q33238 | Fluorometer |
| Qubit Assay Tubes | Thermo Fisher Scientific | Q32856 | Fluorometer assay tubes |
| RIPA Lysis Buffer | VWR | VWRVN653-100ML | Lysis buffer for protein extraction |
| Trypsin-EDTA (0.05%), phenol red | ThermoFisher Scientific | 25300054 | Trypsin |
| Vortex-Genie 2 | Scientific industries | SI-0236 | Vortex |
| Xfect Transfection Reagent | Takara Bio | 631318 | Transfection reagent |
| genomic data analysis software | QIAGEN | CLC Workbench v21.0. | Data analysis software |
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