Name: genome editing in mammalian cell lines using crispr-cas
Uploaded: 2019-04-11T12:30:00
Description: Watch this Scientific Journal Video about Genome Editing in Mammalian Cell Lines using CRISPR-Cas at JoVE.com

M. h. t. 得到科学技术和研究机构联合理事会办公室赠款 (1431AFG103)、国家医学研究委员会赠款 (ofirg额 0017 2016)、国家研究基金会赠款 (NRF2013-THE001-0046 和 NRF2013 THEE001-001-00-93) a a 的支持。教育部一级赠款 (rgfa17 (s)), 来自南洋理工大学的创业补助金, 以及南洋理工大学为国际基因工程机 (iGEM) 竞赛提供的资金。

1. Sgrna 的设计
<ol><li>选择合适的 Crispr-ca 系统。<ol>
<li>首先, 检查所有 Cas9 和 Cas12a 核酸酶的 PAM 序列的目标区域, 这些核酸酶已被证明在哺乳动物细胞16、21-32 中具有功能。表 1给出了五种常用酶及其各自的 pm。 注: 除了表 1所列的内切酶外, 还有其他不太常用的 cas 酶也已成功部署在哺乳动物细胞中, 例如来自嗜热链球菌(st1cas9) 的 cas9 核酸酶,承认 PAM NNAGAO...

Crispr-卡斯系统是一种强大的革命性技术, 用于设计植物和动物的基因组和转录体。许多细菌物种已被发现含有 Crispr-卡斯系统, 这可能被用于基因组和转录组工程目的44。虽然来自化脓性链球菌(spcas9) 的 cas9 内切酶是第一个成功地在人体细胞 21,22,23,24的酶从其他细菌中应用物种,...

设计任何生物基因组的能力都有许多生物医学和生物技术应用, 例如纠正致病突变、构建准确的疾病研究细胞模型或农业发电具有理想性状的作物。自世纪之交以来, 哺乳动物细胞的基因组工程开发了各种技术, 包括美加努丁 1、2、3、锌指核酸酶4、5或转录激活物样效应酶 (talens)6,7,8,9。然而, 这些早期的技术要么难以编程, 要么繁琐组装, 从而阻碍了它们在研究和行业中的广泛采用。
近年来, 聚集定期间隔的短回文重复 (CRISPR)-crispr 相关 (Cas) 系统已成为一种强大的新的基因组工程技术 10,11。它最初是细菌中的适应性免疫系统, 已成功地用于植物和动物 (包括人类) 的基因组修饰。Crispr-ca 在如此短的时间内获得如此广泛的欢迎的一个主要原因是, 将关键的 Cas 内切酶 (如 Cas9 或 Cas12a) 带到基因组中正确位置的元素只是一个简短的嵌套单导 RNA (sgRNA), 设计简单, 合成便宜。在被招募到目标位点后, 卡斯酶起到了分子剪刀的作用, 并将其与鲁夫 c、hnh 或 nok 域 12、13、14 的结合 dna 裂解。由此产生的双链断裂 (DSB) 随后由细胞通过非同源端部连接 (NHEJ) 或同源定向修复 (HDR) 途径进行修复。在没有修复模板的情况下, DSB 通过容易出错的 NHEJ 途径进行修复, 这可能会导致在切割部位伪随机插入或删除核苷酸 (indels), 从而有可能导致蛋白质编码基因的帧-裂隙突变。然而, 在存在包含所需 DNA 变化的捐献者模板的情况下, DSB 通过高保真 HDR 途径进行修复。常见的供体模板类型包括单链寡核苷酸 (Ssodn) 和质粒。前者通常用于预期的 DNA 变化较小 (例如, 单个碱基对的变化), 而后者通常用于如果你希望插入一个相对较长的序列 (例如, 绿色荧光蛋白的编码序列或GFP) 进入目标位点。
卡斯蛋白的内切酶活性要求目标地点15 上存在一个基旋的相邻母题 (pam)。Cas9 的 PAM 位于原相的 3 ' 末端, 而 Cas12a 的 PAM (也称为 Cpf1) 位于 5 ' 端, 而不是16 '.如果 pam 不存在 17, 则 Cas-gue RNA 复合体无法引入 DSB.因此, PAM 对特定的 Cas 核酸酶能够裂解的基因组位置施加了约束。幸运的是, 来自不同细菌种类的 Cas 核酸酶通常表现出不同的 PAM 要求。因此, 通过将各种 CRISPR-Cas 系统集成到我们的工程工具箱中, 我们可以扩大可能在基因组中针对的站点的范围。此外, 可以设计或进化天然 cas 酶来识别替代 pam 序列, 从而进一步扩大了可用于操作18、19、20的基因组目标的范围。
虽然有多个 Crispr-ca 系统可用于基因组工程目的, 但由于多种原因, 该技术的大多数用户主要依赖来自化脓性链球菌(spcas9) 的 cas9 珠。首先, 它需要一个相对简单的 NGG PAM, 不像许多其他的钙蛋白, 只能在更复杂的 Pam 存在的情况下裂解。其次, 这是第一个成功部署在人的牢房21、22、23、24 的卡斯内切酶。第三, SpCas9 是迄今为止最有特色的酶。如果研究人员希望使用另一个 Cas 核酸酶, 他或她往往不清楚如何最好地设计实验, 以及与 SpCas9 相比, 其他酶在不同的生物环境中的表现如何。
为了清楚地了解不同 Crispr-ca 系统的相对性能, 我们最近对五个 Cas 内切酶--SpCas9、来自金黄色葡萄球菌的 cas9 酶 (sacas9)、cas9 酶进行了系统的比较。脑膜炎奈瑟菌 ( nmcas9), 来自 BV3L6 (ascas12a) 的 cas12a 酶, 以及来自Lachnospiraceae Nd2006 (LbCas12a 25 的 cas12a 酶. 为了进行公平的比较, 我们使用同一组靶点和其他实验条件对各种 Ca 核酸酶进行了评估。这项研究还描述了每个 Crispr-卡斯系统的设计参数, 这将为该技术的用户提供有用的参考。在这里, 为了使研究人员能够更好地利用 Crispr-卡斯系统, 我们提供了一个逐步协议, 使用不同的 Cas9 和 Cas12a 酶优化基因组工程 (参见图 1)。该协议不仅包括实验细节, 而且还包括重要的设计考虑, 以最大限度地提高在哺乳动物细胞中成功的基因组工程结果的可能性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59086/59086fig1.jpg"> 图 1: 生成经过基因组编辑的人类细胞系的工作流程概述.<a href="https://www.jove.com/files/ftp_upload/59086/59086fig1large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>

<table border="0" cellpadding="0" cellspacing="0" style="width:1424px;" width="1424">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T4 Polynucleotide Kinase (PNK)</td>
			<td style="width:175px;">NEB</td>
			<td style="width:119px;">M0201</td>
			<td style="width:688px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Shrimp Alkaline Phosphatase (rSAP)</td>
			<td>NEB</td>
			<td style="width:119px;">M0371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-Acetate-EDTA (TAE) Buffer, 50X</td>
			<td>1st Base</td>
			<td>BUF-3000-50X4L</td>
			<td>Dilute to 1X before use. The 1X solution contains 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-EDTA (TE) Buffer, 10X</td>
			<td>1st Base</td>
			<td>BUF-3020-10X4L</td>
			<td>Dilute to 1X before use. The 1X solution contains 10 mM Tris (pH 8.0) and 1 mM EDTA.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">BbsI</td>
			<td>NEB</td>
			<td>R0539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">BsmBI</td>
			<td>NEB</td>
			<td>R0580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T4 DNA Ligase</td>
			<td>NEB</td>
			<td style="width:119px;">M0202</td>
			<td>400,000 units/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Quick Ligation Kit</td>
			<td>NEB</td>
			<td style="width:119px;">M2200</td>
			<td>An alternative to T4 DNA Ligase.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Rapid DNA Ligation Kit</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">K1423</td>
			<td>An alternative to T4 DNA Ligase.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Zero Blunt TOPO PCR Cloning Kit</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">451245</td>
			<td>The salt solution comes with the TOPO vector in the kit.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">NEBuilder HiFi DNA Assembly Master Mix</td>
			<td>NEB</td>
			<td style="width:119px;">E2621L</td>
			<td>Kit for Gibson assembly.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">One Shot Stbl3 Chemically Competent E.Coli</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">C737303</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">LB Broth (Lennox), powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">L3022</td>
			<td>Reconstitute in ddH20, and autoclave before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">LB Broth with Agar (Lennox), powder</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">L2897</td>
			<td>Reconstitute in ddH20, and autoclave before use</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:443px;">SOC media</td>
			<td>-</td>
			<td style="width:119px;">-</td>
			<td>2.5 mM KCl, 10 mM MgCl2, 20 mM glucose in 1 L of LB Broth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ampicillin (Sodium), USP Grade</td>
			<td>Gold Biotechnology</td>
			<td style="width:119px;">A-301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">REDiant 2X PCR Mastermix</td>
			<td>1st Base</td>
			<td style="width:119px;">BIO-5185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Agarose</td>
			<td>1st Base</td>
			<td style="width:119px;">BIO-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">T7 Endonuclease I</td>
			<td>NEB</td>
			<td style="width:119px;">M0302</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Plasmid DNA Extraction Miniprep Kit</td>
			<td>Favorgen</td>
			<td style="width:119px;">FAPDE 300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Dulbecco&#39;s Modified Eagle Medium (DMEM), High Glucose</td>
			<td>Hyclone</td>
			<td>SH30081.01</td>
			<td>4.5 g/L Glucose, no L-glutamine, HEPES and Sodium Pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">L-Glutamine, 200mM</td>
			<td>Gibco</td>
			<td style="width:119px;">25030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Penicillin-Streptomycin, 10, 000U/mL</td>
			<td>Gibco</td>
			<td style="width:119px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">0.25% Trypsin-EDTA, 1X</td>
			<td>Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:443px;">Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td style="width:119px;">SV30160</td>
			<td>FBS is heat inactivated before use at 56 oC for 30 min</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Phosphate Buffered Saline, 1X</td>
			<td>Gibco</td>
			<td style="width:119px;">20012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">jetPRIME transfection reagent</td>
			<td>Polyplus Transfection</td>
			<td style="width:119px;">114-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">QuickExtract DNA Extraction Solution, 1.0</td>
			<td>Epicentre</td>
			<td style="width:119px;">LUCG-QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">ISOLATE II Genomic DNA Kit</td>
			<td>Bioline</td>
			<td style="width:119px;">BIO-52067</td>
			<td>An alternative to QuickExtract</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Q5 High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td style="width:119px;">M0491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Deoxynucleotide (dNTP) Solution Mix</td>
			<td>NEB</td>
			<td style="width:119px;">N0447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">6X DNA Loading Dye</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td>10 mM Tris-HCl (pH 7.6) 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol, 60 mM EDTA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Protease Inhibitor Cocktail, Set3</td>
			<td>Merck</td>
			<td style="width:119px;">539134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Nitrocellulose membrane, 0.2&#181;m</td>
			<td>Bio-Rad</td>
			<td style="width:119px;">1620112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-glycine-SDS buffer, 10X</td>
			<td>Bio-Rad</td>
			<td style="width:119px;">1610772</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris, 192 mM glycine, and 0.1% SDS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tris-glycine buffer, 10X</td>
			<td>1st base</td>
			<td style="width:119px;">BUF-2020</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris and 192 mM glycine.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Ponceau S solution</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P7170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">TBS, 20X</td>
			<td>1st base</td>
			<td style="width:119px;">BUF-3030</td>
			<td>Dilute to 1X before use. The 1x solution contains 25 mM Tris-HCl (pH 7.5) and 150 mM NaCl.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Tween 20</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P9416</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Skim Milk for immunoassay</td>
			<td>Nacalai Tesque</td>
			<td style="width:119px;">31149-75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">WesternBright Sirius-femtogram HRP</td>
			<td>Advansta</td>
			<td style="width:119px;">K12043</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Antibody for &#946;-actin (C4)</td>
			<td>Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-47778</td>
			<td>Lot number: C0916</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">MiSeq system</td>
			<td>Illumina</td>
			<td style="width:119px;">SY-410-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">NanoDrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">Qubit fluorometer</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">Q33226</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">EVOS FL Cell Imaging System</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">AMF4300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pSpCas9(BB)-2A-GFP (PX458)</td>
			<td>Addgene</td>
			<td style="width:119px;">48138</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA</td>
			<td>Addgene</td>
			<td style="width:119px;">61591</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: xCas9 3.7</td>
			<td>Addgene</td>
			<td style="width:119px;">108379</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: pX330-U6-Chimeric_BB-CBh-hSpCas9</td>
			<td>Addgene</td>
			<td style="width:119px;">42230</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: hCas9</td>
			<td>Addgene</td>
			<td style="width:119px;">41815</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: eSpCas9(1.1)</td>
			<td>Addgene</td>
			<td style="width:119px;">71814</td>
			<td>Single vector system: The gRNA is expressed from the same plasmid.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:443px;">CRISPR plasmid: VP12 (SpCas9-HF1)</td>
			<td>Addgene</td>
			<td style="width:119px;">72247</td>
			<td>Dual vector system: The gRNA is expressed from a different plasmid.</td>
		</tr>
	</tbody>
</table>

genome editing in mammalian cell lines using crispr-cas

聚集定期间隔短回文重复 (CRISPR) 系统在细菌适应性免疫中自然发挥作用, 但已成功地将其重新用于许多不同生物的基因组工程。最常见的是, 与通配的通配型 CRISPR (Cas9) 或 Cas12a 内切酶用于裂解基因组中的特定位点, 之后 DNA 双链断裂通过非同源端连接 (NHEJ) 途径或同源定向修复 (HDR) 路径取决于捐献者模板是否分别不存在或存在。到目前为止, 来自不同细菌物种的 CRISPR 系统已被证明能够在哺乳动物细胞中进行基因组编辑。然而, 尽管该技术表面上很简单, 但仍需要考虑多个设计参数, 这往往会让用户对如何最好地进行基因组编辑实验感到困惑。在这里, 我们描述了一个完整的工作流程, 从实验设计到检测携带所需 DNA 修饰的细胞克隆, 目的是促进在哺乳动物细胞系中成功执行基因组编辑实验。我们强调了用户需要注意的关键注意事项, 包括 CRISPR 系统的选择、间隔长度以及单链寡核苷酸 (ssODN) 供体模板的设计。我们设想, 这种工作流程将有助于基因敲除研究、疾病建模工作或记者细胞系的生成。

为了进行基因组编辑实验, 需要克隆一个表达针对感兴趣位置的 sgRNA 的 Crispr 质粒。首先, 质粒被消化与限制性酶 (通常类型的 IIs 酶), 使其线性化。建议在1% 琼脂糖凝胶上解决消化产物, 同时使用未消化的质粒, 以区分完全消化和部分消化。由于未被消化的质粒是超卷的, 它们的运行速度往往比线性化的等离子体快 (参见图 7a)。其次, sgRNA 的两个寡核苷酸必须退火?...

Watch this Scientific Journal Video about Genome Editing in Mammalian Cell Lines using CRISPR-Cas at JoVE.com

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

Crispr-卡斯是一种强大的技术, 用于设计复杂的植物和动物基因组。在这里, 我们详细介绍了一个使用不同的 Cas 内切酶有效编辑人类基因组的协议。我们强调重要的注意事项和设计参数, 以优化编辑效率。

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

该协议使用CRISPR-Cas9创建淘汰线或敲打线。这种技术的主要优点是，它简单和高效遵循，特别是对于新手研究人员。对于细胞传传，在37摄氏度下用每盘0.25%的两毫升2毫升的通宵培养细胞治疗两分钟。当细胞从板底提升时，用两毫升细胞培养中中和酶反应，将细胞悬浮液转移到锥形管中。通过离心收集细胞，并在五毫升新鲜细胞培养中重新供应颗粒进行计数。然后将1.8倍10至第五细胞播种到24井组织培养板的一个井中，在37摄氏度和5%的二氧化碳下进行隔夜培养。对于细胞的转染，根据实验设计和制造商的说明，在适当体积的转染试剂中加入含有适当浓度的CRISPR质粒的适当转染混合物。在室温下在推荐的一段时间内孵育转染混合物后，以滴法方式将溶液添加到细胞中。然后轻轻旋转板，将板混合并放在加湿的 37 摄氏度培养箱中，并加5%的二氧化碳。在转染结束时，添加150微升的三辛-EDTA，以分离细胞，正如刚刚演示的，通过离心收集分离的细胞。将颗粒重新在PBS中2%的胎儿牛血清中，并通过30微米网状滤网过滤细胞，进入5毫升荧光激活细胞分选或FACS管。然后使用非转染细胞在流动细胞仪上设置负控制门，根据用于转染的CRISPR质粒上的荧光标记对转染细胞进行排序，并放入含有100微升细胞培养基的管中。收集所有转染细胞后，以最大速度离心，将细胞粒粒，并在300微升细胞培养培养的微升中重新下粒。将200微升细胞放入24井组织培养板的一个井中，使细胞在37摄氏度的培养箱中恢复几天。以最大速度将其余 100 微升的分拣细胞提取 5 分钟，然后根据标准 DNA 提取协议从生成的颗粒中提取基因组 DNA。接下来，加入10微升聚合酶链反应缓冲液、1微升脱氧核苷酸混合物、2.5微升用户定义的反向底因、0.5微升DNA聚合酶、2至5微升提取的基因组DNA模板和足够的双蒸馏水，使反应最终达到50微升。在热循环器上运行混合物，根据标准协议使用 1X TAE 缓冲液解决 2% agarose 凝胶上的反应。使用干净、锋利的手术刀切除聚合酶链反应产品，并使用凝胶提取套件根据制造商的说明进行DNA纯化。使用 260 纳米吸光度分光光度计测量产品的浓度。准备含有200纳米分离DNA的测定混合物、T7内核糖酶 I 反应缓冲液的两微升，以及足够的双蒸馏水，使混合物的最终体积达到 19 微升。将聚合酶链反应产品在热循环器中以指示参数进行，将5个T7内核糖酶 I 单元与再核糖核酸产品混合，在 37 摄氏度下孵育 50 分钟。在孵育结束时，使用1X TAE缓冲液在2.5%的阿加罗斯凝胶上解析消化的DNA，并在适当的凝胶成像系统上对凝胶进行成像。打开 ImageJ 中的凝胶图像，并在带周围绘制一个尽可能靠近其边界的矩形框。单击"分析"和"设置测量"，确认已检查区域、平均灰色值和集成密度选项。单击"确定"，然后选择"分析"和"测量"。均值或原始强度密度值表示波段强度。当培养的转染细胞开始成为汇合体时，如所证明的，用三头肌-EDTA分离它们，并在100毫米组织培养皿中稀疏地播种细胞，以便给单个菌落留出足够的空间，让个体菌落在将细胞返回到细胞培养箱之前生长。当菌落开始形成时，使用四 X 放大倍率的显微镜挑选单个菌落转移到每个孔包含 500 微升细胞培养培养基的 24 孔板的单个孔中。注意您的尖端不会接触周围的殖民地，以防止个别井内菌落，或从稀疏的殖民地生长区域采摘。当所有克隆被挑选后，将板放在细胞培养培养箱中，直到培养物成为汇合。由于未消化质粒是超线圈的，它们往往比线性质粒运行得更快。为了确定寡核苷酸是否已成功克隆到CRISPR质粒骨干中，在提取质粒并送去桑格测序之前，进行聚体PCR并接种阳性克隆。成功交付质粒后，荧光信号可在显微镜下轻松可视化，从而通过流式细胞仪对转染细胞进行排序。T7内核糖酶 I 测定，以检查基因组DNA的裂解效率，根据在 agarose 凝胶上观察到的带子强度计算。此外，如果同源式修复实验设计为在目标位点合并一个限制位点，则可以使用相应的限制性酶进行限制片段长度多态性测定。为了进一步验证蛋白质编码基因是否已成功灭活，可以进行西方印迹，以确保不存在靶向蛋白。转染后对细胞进行排序可消除不合并质粒的细胞，从而增加后期筛选的细胞具有敲除或敲入基因的百分比。随着这项技术的发展，我们现在能够产生敲出细胞系来研究基因功能和敲打细胞系来模拟特定疾病，以了解其机理。

genome-editing-in-mammalian-cell-lines-using-crispr-cas

Research

JoVE Journal

Genetics

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

通过DNA含量测定的哺乳动物复制时序的全基因组的测定

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

染色质免疫沉淀（ChIP）在小鼠T细胞系

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these enhancers combine to produce a transcriptional response. As millions of enhancers have been identified, high-throughput tools are needed to determine enhancer function on a genome-wide scale. Current methods for studying enhancer function include making genetic deletions using nuclease-proficient Cas9, but it is difficult to study the combinatorial effects of multiple enhancers using this technique, as multiple successive clonal cell lines must be generated. Here, we present Enhancer-i, a CRISPR interference-based method that allows for functional interrogation of multiple enhancers simultaneously at their endogenous loci. Enhancer-i makes use of two repressive domains fused to nuclease-deficient Cas9, SID and KRAB, to achieve enhancer deactivation via histone deacetylation at targeted loci. This protocol utilizes transient transfection of guide RNAs to enable transient inactivation of targeted regions and is particularly effective at blocking inducible transcriptional responses to stimuli in tissue culture settings. Enhancer-i is highly specific both in its genomic targeting and its effects on global gene expression. Results obtained from this protocol help to understand whether an enhancer is contributing to gene expression, the magnitude of the contribution, and how the contribution is affected by other nearby enhancers.

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target gene.

基于多重 CRISPR 增强器的细胞系干扰增强功能解剖

Multiple enhancers often regulate a given gene, yet for most genes, it remains unclear which enhancers are necessary for gene expression, and how these ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

转化、基因组编辑和拟合氮的热带大麻树帕拉斯波尼亚安德森

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

使用基因组-规模CRISPR/Cas9基因屏幕进行细胞表面受体识别

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing in mammalian cells. Here we describe detailed procedures to seamlessly genome edit the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus as an example in human pluripotent stem cells. Combining a piggyBac-based donor plasmid and the CRISPR-Cas9 nickase mutant in a two-step genetic selection, we demonstrate correct and efficient targeting of the HNF4&#945; locus.

Here, we describe a detailed method for seamless gene editing in human pluripotent stem cells using a piggyBac-based donor plasmid and the Cas9 nickase mutant. Two point mutations were introduced into exon 8 of the hepatocyte nuclear factor 4 alpha (HNF4&#945;) locus in human embryonic stem cells (hESCs).

使用无缝基因组编辑将点突变引入人类多能干细胞

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

CRISPR-Cas9-中切基因组编辑在纤维阿莫西特 亨蒂埃拉阿曼尼西斯

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...