Name: hox loci focused crispr/sgrna library screening identifying critical ctcf boundaries
Uploaded: 2019-03-31T14:00:00
Description: Watch this Scientific Journal Video about HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries at JoVE.com

作者还感谢尼古拉斯·塞萨里编辑了手稿。这项工作得到了国家卫生研究所 (s. h., R01DK110108, R01DK110108) 的赠款。

1. 使用在线工具进行库设计
<ol><li>利用遗传摄动平台 (GPP) 设计工具 (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) 设计了人类Hox位点 Ctcf 结合位点的 sgRNA 靶向。</li>
	<li>合成了总共 1, 070 Sgrna, 包括针对303个随机靶向基因的 Sgrna、60个阳性对照、500个非人类靶向对照和 207个 CTCF 元素或靶向基因的 lncRNA (图 1,表 1)。每个靶向 DNA 元素都有5-10 种不?...

<ol>
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蛋白质编码基因相关的 sgrna 文库已应用于功能筛选系统, 通过 sgrna 富集 24,25,26 来识别调节特定细胞功能的基因和网络,27,28。几个与非编码区域相关的 sgRNA 文库也显示在远端和近端调节元素的基因特异性功能屏幕中, 包括bcl11a、 tgf1a和耐药调节基因...

最近的基因组相互作用研究表明, 人类核基因组形成稳定的拓扑关联域 (Tad), 这些位点在细胞类型和物种之间是保守的。将基因组组织成不同的领域, 促进和限制了监管要素 (例如增强剂和促进剂) 之间的相互作用。ccctc 结合因子 (CTCF) 与 TAD边界结合在一起, 在约束位于邻近 tad 1 的 dna 元素的相互作用方面发挥着至关重要的作用。然而, 全基因组 CTCF 结合数据显示, 尽管 CTCF 在不同细胞类型中大多与相同的 dna 位点相互作用, 但它通常在一种细胞类型的特定部位起到染色质屏障的作用, 但在另一种细胞类型中却不起作用, 这表明 CTCF 的功能与其他活动一起形成染色质边界2。目前尚不清楚的是, 边界元素 (ctcf 结合位点) 是否与 CTCF 的生物功能直接相关, 以及这些联系是如何发生的。因此, 我们假设基因组中特定的 CTCF 结合位点直接调节 Tad 的形成和控制启动者在这些领域内或相邻域之间的相互作用。人类和小鼠基因组测序项目的完成以及随后的表观遗传分析发现了基因组的新的分子和遗传特征。然而, 特定签名修饰在基因调控和细胞功能中的作用及其分子机制尚未完全了解。
多行证据支持 ctcf 介导的 tad 代表功能染色质域3,4,5。虽然 CTCF 主要与不同细胞类型的相同 dna 位点相互作用, 但全基因组范围的 CTCF ChIP-seq 数据显示, CTCF 在一种细胞类型中通常起到染色质屏障的作用, 但在另一种细胞类型中却没有。ctcf 通过调解基因组组织 4、6、7在发育过程中发挥着至关重要的作用。CTCF 边界的破坏损害了增强剂的相互作用和基因表达, 导致发育障碍。这表明 ctcf 介导的 tad 不仅是结构成分, 也是适当的增强剂作用和基因转录所需的调控单位 5,8,9.
hox基因在胚胎发育过程中起着至关重要的作用, 它们的表达模式受到时间和空间的限制。Hoxa位点形成两个稳定的 tad, 通过在 HESCS 和 IMR90 细胞1中的 ctcf 相关边界元素分离前、后基因。最近的报告表明, HoxBlinc, 一个与 Hoxb相关的 incrna 位点, 介导了在HOXB位点中 Ctcf 定向 tad 和 hancer 类似物的形成。这导致前hoxb基因激活在 esc 承诺和分化10。此外, 在包括Hoxa位点在内的特定基因位点, ctcf 介导的 tad 域的改变改变了谱系特异性基因表达谱, 并与疾病状态11,12的发展有关。这些证据支持 CTCF 在协调基因转录和确定细胞身份方面的主要功能, 通过将基因组组织成功能域。
尽管 Hox 基因在胚胎发育过程中的作用,但它调节造血干细胞和祖细胞 (hsspc) 的功能。这是通过控制增殖和分化之间的平衡来实现的, 10、13、14、15。hox基因的表达在造血细胞的整个规格和分化过程中都受到严格的调控, hsp 中的表达最高, hox基因的表达在成熟过程中逐渐下降, 达到最低水平发生在分化的造血细胞16。hox基因失调是白血病转化的主要机制, 它通过对 hsp pc 的自我更新和分化特性的失调, 导致白血病转化17,18.然而, 建立和维持正常与肿瘤表达模式的机制以及相关的调控网络仍然不清楚。
CRISPR-CAS9 sgRNA 库筛选已被广泛用于检测蛋白质编码基因19以及非编码基因, 如 Ncrna20和 mirna21在不同的物种。然而, 使用 CRISPR-CAS9 sgRNA 文库来确定新的基因组目标的成本仍然很高, 因为高通量基因组测序通常用于验证 sgRNA 库筛选。我们的 sgRNA 筛选系统侧重于特定的基因组位点, 并根据标记基因表达 (如 Hoxa9), 通过一步 RT-PCR 评估靶向 sgRNA。此外, Sanger 测序证实 sgRNA 已被整合到基因组中, 并且可以检测到 Indel 突变以识别 sgRNA 靶向位置。通过 lci 特异性 Crispr-cas9 基因筛选, CBSVI/9 染色质边界被确定为建立致癌染色质域和维持 aml 发病机制中异位Hox基因表达模式的关键调节器12. 该方法不仅可广泛应用于确定 CTCF 边界在胚胎发育、造血、白细胞生成中的特定功能, 而且可作为未来表观遗传治疗的潜在治疗靶点。

<table border="0" cellpadding="0" cellspacing="0" style="width:689px;" width="689">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Lipofectamine 3000 reagent</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td style="width:100px;">L3000-008</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Thermo Fisher Scientific</td>
			<td>25530049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stbl3 cells&#160;</td>
			<td>Life Technologies</td>
			<td>&#160;C737303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEK293T</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MOLM-13</td>
			<td>DSMZ</td>
			<td>ACC 554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">lentiCRISPRv2</td>
			<td>Addgene</td>
			<td>52961</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td>A137A</td>
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			<td>&#160;M0202S</td>
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			<td height="21" style="height:21px;">QIAquick Gel Extract kit</td>
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			<td>28106</td>
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			<td>Bio-Rad Laboratories</td>
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			<td>Thermo Fisher Scientific</td>
			<td style="width:100px;">09-528-178</td>
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</table>

hox loci focused crispr/sgrna library screening identifying critical ctcf boundaries

ccctc 结合因子 (CTCF) 介导的稳定拓扑关联域 (Tad) 在约束位于邻近 Tad 的 DNA 元素的相互作用方面起着至关重要的作用。CTCF 在调节 hox 基因的时空表达方面发挥着重要作用, 这些基因控制胚胎发育、身体模式、造血和白血病发生。然而, 它在很大程度上是未知的是否和如何乡相关的 CTCF 边界调节染色质组织和hox基因表达。在目前的协议中, 生成了一个针对 Hoxa/bcp 位点中所有 CTCF 结合位点的特定 sgrna 集合库, 以检查破坏 ctcf 相关染色质边界对 tad 形成和hox基因的影响表达。通过 CRISPR-CAS9 基因筛选, 位于hoxaeeehoxa9基因 (CBS7/9) 之间的 CTCF 结合位点已被确定为致癌染色质域的关键调节剂, 对维持异位hox基因也很重要。mll 重组急性髓系白血病 (AML) 的表达模式。因此, 这种 sgRNA 库筛选方法为特定基因位点中 CTCF 介导的基因组组织提供了新的见解, 也为注释遗传调控元素的功能表征提供了基础, 包括编码和编码。在人类基因组计划时代的正常生物过程中, 不编码。

Crispr-cas9 技术是功能基因组研究的有力研究工具。它正在迅速取代传统的基因编辑技术, 在全基因组和以基因为中心的个人应用中都具有很高的用途。在这里, 第一个单独克隆的 lace 特异性 Crispr-casr9 排列的 sgRNA 库包含 1, 070 SgRNA, 包括针对303随机靶向基因的 SgRNA、60个阳性对照、500个非人类靶向对照和 207个 CTCF 元素或 lncRNA在四个Hox位点中靶向基因 (图...

Watch this Scientific Journal Video about HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries at JoVE.com

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

一个 crisprsgrna 文库已被应用于蛋白质编码基因的询问。然而, sgRNA 文库揭示 CTCF 边界在基因调控中的作用的可行性仍未得到探讨。在这里, 我们描述了一个Hox位点特定的 sgRNA 库, 以阐明在hox位点中 ctcf 边界的功能。

公司重点调查的社区图书馆筛选确定关键 CTCF 边界

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

我们的池sgRNA称为库筛选，是一种强大的遗传方法，用于识别和评估整个基因组中非编码元素的生物功能。这种技术使我们能够检查破坏相关结合位点、色边界或其他调控元素对靶向基因表达的影响。我们的方法可用于识别CTCF，长非编码RNA和增强剂的作用，在早期胚胎发育HOX基因调控中的作用，以及某些具有异常HOX基因特征的白血病。从针对 CTCF 绑定位点设计的 sgRNA 开始此过程。在细胞制备中克隆sgRNA库，如文本协议中所述。为了包装扁病毒，用20微克的纯化库载体、15微克的包装质粒和10微克的包络质粒，在采集病毒前48小时进行共传递 HEK293 T细胞。48小时后，收集病毒上清液，并通过0.45微米低蛋白结合PVDF膜进行过滤。接下来，使用浓缩器将扁病毒上经剂集中50倍。将浓缩病毒储存在零下80摄氏度的冰柜中。在12孔板的单独井中工作，将MOLM13细胞与各种剂量的浓缩扁病毒混合，共6组。立即将这些混合物以1000倍g离心，在33摄氏度下进行两小时。将12个井板在37摄氏度和5%的二氧化碳下将转回孵化器4小时。轻轻吸吸上一提液，而不干扰细胞调色板，并重新悬浮带新鲜补充的RPMI 1640中等的换能细胞。然后将重新悬浮的细胞转移到T25烧瓶中，在37摄氏度下孵育48小时，不产生紫霉素。48小时后，这些细胞分成两个烧瓶，一个实验组用每毫升一微升的紫菜素治疗5天，一个对照组5天不进行紫辛治疗。通过将用紫霉素治疗的活细胞数除以不进行普霉素治疗的细胞数，测量优化的MOI值。要通过池库对细胞进行转导，在6井板中感染150万个MOLM13细胞，在介质中感染0.3 MOI的sgRNA池扁病毒。使用没有扁病毒感染的细胞作为控制。立即将六个井板以1000倍g离心，在33摄氏度下两小时旋转细胞。然后，将板在摄氏37度的5%二氧化碳中转移回孵化器4小时。在不干扰细胞调色板的情况下轻轻吸进上一提液，用新鲜介质重新悬浮转导细胞。然后，将细胞转移到T25烧瓶中，在37摄氏度下孵育48小时，不产生霉素。48小时后，用每毫升一微克的紫菜素治疗细胞5天。两天后交换新鲜介质，并保持最佳的细胞密度。用限制稀释方法在96个井板中播种单克隆。在37摄氏度和5%的二氧化碳下孵育这些单克隆。培养他们 3 - 4 周。计算 sgRNA 集成 MOLM13 细胞与 0.4% Trypan Blue 溶液染色，然后将每井 10，000 个活细胞转移到 96 井 PCR 板。将板以 1000 次 g 离心五分钟，然后用移液器彻底取出和丢弃上清液。避免干扰单元格调色板。洗涤后，如文本中描述的，将50微升的细胞解解主混合添加到每个井中。上下移位五次以重新挂起单元格调色板。在室温下孵育混合物10分钟。然后，将混合物转移到37摄氏度5分钟，然后将75摄氏度再转5分钟。现在向含有RT qPCR反应混合物的PCR孔中加入一微升的细胞来亚酸盐。这种混合物包括标记基因向前和反向底转，逆转录酶和一步反应混合。在50摄氏度下运行逆转录反应10分钟，然后聚合激活，DNA变性在95摄氏度下运行1分钟。执行RT PCR 与 40 周期的 PCR 反应，如文本协议中详细说明。验证HOXA9通过桑格测序减少的表达克隆，并执行PCR与MOLM13基因组DNA如文本协议所述。使用 PCR 纯化套件提取和纯化 PCR 产品。使用 T4 结扎缓冲液、T 向量 DNA、PCR DNA 和 T4 连接酶将纯化 PCR 产品放入 T 向量中。将结扎混合物放入16摄氏度的孵化器中过夜。将结扎混合物转移到 DH5 alpha 能力细胞和 LB 安西林抗生素加糖板上的板中。在37摄氏度下孵育板。从 LB 板中选取单个克隆，然后通过基因分型和桑格测序进行验证。在 0.2 毫升 PCR 管中设置具有 200 毫微克的异质混合物组，以及 200 毫微克的测试 PCR amplicons。包括仅含有 400 纳米的参考 PCR 安普利森的同性混合物组作为控制。在充满800毫升水的一升烧杯中，在95摄氏度下单独孵育杂合和同质混合物，5分钟。然后，将混合物逐渐冷却至室温至退火，形成异质或同性。现在，用一微升的英德尔突变检测核酸酶和两个微升的核酸酶反应缓冲液，在42摄氏度下分别消化400纳米的退火杂质和同质复性混合物，60分钟。使用阿加罗斯凝胶电泳分析消化的样品。异质混合物DNA应切割成小片段，同质DNA不应切割。基于HOXA9基因的表达水平与对照细胞的比较，用RT qPCR方法确认了针对MOLM13阳性克隆的sgRNA。在筛选的528个存活克隆中，有10个克隆在HOXA9水平上表现出50%以上的降幅。与HOXA9集成的sgRNA减少，HOXA9不变，HOXA9增加克隆，进一步通过PCR扩增sgRNA和桑格序列的鉴定得到进一步确认。在显示HOXA9水平降低的10个克隆中，有6个包含针对CBS79站点的sgRNA，由桑格测序确定。集成sgRNA阳性克隆的内德尔突变由基于PCR的基因分型和核酸酶消化确定。位于HOXA7和HOXA9基因之间的CTCF位点的异质缺失由基于PCR的基因分型识别。HOXA9 减少的克隆 5、6、28 和 121 在 CBS79 边界位置显示删除。通过核酸酶消化分析分析的CBS79位点内突变也观察到类似的结果。执行此程序时，重要的是使等量的异质复性与同性，以便通过核酸酶消化分析检测 sgRNA 诱导的内德尔突变。我们的方法能够利用选定的标记，对目标基因进行下一代测序，以发现该基因对白血病生成的影响。总的来说，我们的方法可以防止RPC在正常生物过程中对著名的遗传不规则元素进行功能校正和减少，并在死后基因组计划误差中减少白血病。

Research

JoVE Journal

Genetics

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

科研

遗传学

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

支气管肺泡灌洗外来体在脂多糖致脓毒性肺损伤中的作用

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

Mating-based Overexpression Library Screening in Yeast

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

Screening Cotton Genotypes for Reniform Nematode Resistance

筛选抗内虫抗性棉基因型

A rapid non-destructive reniform nematode (Rotylenchulus reniformis) screening protocol is needed for the development of resistant cotton (Gossypium ...

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

通过高分辨率熔融在水稻种群中识别突变

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens

植物病原体分泌效应器中RNA沉默抑制器的筛选和鉴定

RNA silencing is an evolutionarily conserved, sequence-specific gene regulation mechanism in eukaryotes. Several plant pathogens have evolved proteins ...