Name: using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells
Uploaded: 2018-01-05T15:00:00
Description: Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

这项工作得到了 NIH (R01HL131835), 一个达蒙罗扬临床研究员奖和斯塔尔癌症财团的支持。

1. sgRNA 设计使用在线工具13
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设计 sgRNAs 的目标基因的兴趣使用免费的在线工具。
	<ol>
<li>复制并粘贴感兴趣基因的 NCBI 参考序列到宽广的学院 sgRNA 设计师网页工具: (http://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design)。 注意: 此工具可识别显内的 sgRNA 序列和跨越内含子/外显子边界但仍在外显子内进行劈裂的那些。</li>
		<li>下载并打开 excel 中的文本输出...

<ol>
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在这里, 我们展示了使用 CRISPR/Cas9 基因编辑研究的生物学功能的CALR突变的造血细胞。该协议的成功与否高度依赖于多个因素。首先, 重要的是要知道什么类型的突变可能是负责的表型, 是需要的。在本协议中, 读数是 Ba/F3 细胞向 mIL-3 独立的转化, 而突变的类型在CALR的外显子9中 indels。但是, 如果所需的突变是单个基对替代, 那么 HDR 介导的修复是首选方法, 因为它可以引入精确点突变或...

CRISPR/Cas9 技术最近在活细胞和生物体中彻底改变了目标基因组的编辑。它已成为生物医学研究的一个非常强大的工具, 目前正被用作治疗遗传性疾病的潜在途径1。所有基因组编辑工具的基础是建立一个核酸诱导的 DNA 双链断裂 (争端) 在基因组所在地要修改。DSBs 可以通过同源结束连接 (NHEJ) 或同源定向修复 (HDR)2,3进行修复。Cas9 核酸优于其他基因组工程核酸, 如锌指核酸 (ZFNs) 和转录激活剂样效应核酸 (TALENs) 的优势是它依赖于 RNA 的目标核酸到一个理想的 DNA 序列, 比较到 ZFNs 和 TALENs 中发现的蛋白质-DNA 相互作用2,3。
在原核细胞 CRISPR/Cas9 核酸通路作为自适应免疫系统的发现后4,5, 许多努力已经适应了哺乳动物细胞系和模型生物体的使用途径2,3. 作为基因编辑的工具, CRISPR/Cas9 通路利用两个主要组成部分:链球菌化脓(Sp) 衍生 Cas9 核酸和 sgRNAs 的目标基因的兴趣2,3。sgRNA 由20核苷酸组成, 通过 RNA-DNA 基对互补2,3, 直接 Cas9 到基因组上的特定站点。sgRNA 的目标位置必须位于相邻的 protospacer 相邻主题 (PAM) 的位置, 其形式为 5 "NGG, 这是 SpCas9 核酸所认可的。有了这些工具, Cas9 可以通过设计 sgRNAs 的目标区域来引导到任何 DNA 序列。除了 Sp 派生的 Cas9, 还有其他的变体, Cas9 具有不同的功能, 这取决于特定的应用程序。例如, 有 Cas9 的变种具有更高的特异性为在目标编辑或单分裂容量为脱氧核糖核酸偷6,7。此外, 催化的非活动 Cas9 最近已经发展为转录调节8。科学家们现在已经使用 CRISPR/Cas9 系统的各种应用, 如基因敲除和击倒研究的生物学功能的基因9, 功能和功能库屏幕10和遗传模型有机体的工程学11。
在本协议中, 我们将 CRISPR/Cas9 方法与 Ba/F3 细胞转化法结合起来, 以了解CALR突变的生物学功能。Ba/F3 细胞是一种小鼠 IL-3 依赖的造血细胞系, 可以 IL-3 独立于某些致癌基因的表达, 如 BCR 的12。为了了解突变体钙是否能将 Ba/F3 细胞转化为细胞因子的独立生长, 我们针对内生的Calr轨迹的外显子9使用 CRISPR/Cas9 引入 indel 突变, 然后从细胞中撤回 IL-3, 以应用积极的选择压力, 与目标综述功能CALR突变发现 MPN 患者。该协议包括 sgRNAs 的设计、克隆和交付、稳定 Cas9 表达细胞的开发和 CRISPR 靶基因编辑的筛选。该协议可以应用于不同的基因和各种细胞因子相关的兴趣, 在模型和研究癌症相关基因的生物学功能方面特别有价值。

<table>
	<tbody>
		<tr>
			<td>BsmBI</td>
			<td>New England Biolabs</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Blasticidin</td>
			<td>Sigma Aldrich</td>
			<td>15025</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Life Technologies</td>
			<td>A1113803</td>
			<td></td>
		</tr>
		<tr>
			<td>Stbl3 cells</td>
			<td>Life Technologies</td>
			<td>C737303</td>
			<td></td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Thermo Fisher Scientific</td>
			<td>MIR2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>51985034</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Thermo Fisher Scientific</td>
			<td>MT10040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>10-017-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Omega Scientific</td>
			<td>FB-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin/L-glutamine</td>
			<td>Life Technologies</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>mIL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-VSV-G</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>pLX_TRC311-Cas9</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma Aldrich</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>pXPR-011</td>
			<td>Addgene</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Genessee Scientific</td>
			<td>25-507</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERB49</td>
			<td></td>
		</tr>
		<tr>
			<td>LentiGuide-Puro</td>
			<td>Addgene</td>
			<td>Plasmid #52963</td>
			<td></td>
		</tr>
		<tr>
			<td>PNK</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
	</tbody>
</table>

using crispr/cas9 gene editing to investigate the oncogenic activity of mutant calreticulin in cytokine dependent hematopoietic cells

聚类定期 interspaced 短复发重复 (CRISPR) 是一种自适应免疫系统的原, 已被科学家用来产生 RNA 引导核酸, 如 CRISPR 相关 (Cas) 9 site-specific 真核基因组编辑.Cas9 的基因组工程被用来有效、容易和有力地改变许多生物相关的哺乳动物细胞系和生物体内源基因。在这里, 我们展示了一个如何利用 CRISPR/Cas9 方法来理解特定基因突变的生物学功能的例子。我们模型钙 (CALR) 突变的小鼠 interleukin-3 (mIL-3) 依赖亲 B (Ba/F3) 细胞通过交付单一指南 rna (sgRNAs) 的目标内源性CALR所在地在特定地区的插入和/或删除 (indel) CALR突变发生在骨髓肿瘤 (MPN), 一种类型的血液癌症患者。sgRNAs 在目标区域创建双链断裂 (DSBs), 由同源端连接 (NHEJ) 修复, 以提供各种尺寸的 indels。然后, 我们采用标准 Ba/F3 细胞转化法, 以了解生理水平表达的Calr突变对造血细胞转化的影响。这种方法可应用于其他基因, 以研究其生物学功能的各种哺乳动物细胞系。

采用本文所概述的方法, 本实验的目的是研究在造血细胞转化过程中引入 indel 突变对内源性Calr轨迹的功能效应。CRISPR/Cas9 系统被用作在 Ba/F3 细胞中创建内源性的Calr突变的工具。选择了两个 sgRNAs 来针对Calr (图 1) 的外显子 9, 在该区域中, 插入和/或删除 (indel) 突变通常发生在Calr变种 MPN 患者17、<sup ...

Watch this Scientific Journal Video about Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells at JoVE.com

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

有针对性的基因编辑使用 CRISPR/Cas9 极大地促进了对基因的生物功能的理解。在这里, 我们利用 CRISPR/Cas9 的方法, 模型钙突变的细胞因子依赖性造血细胞, 以研究其致癌活动。

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

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Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...