Name: generation of defined genomic modifications using crispr-cas9 in human pluripotent stem cells
Uploaded: 2019-09-25T14:00:00
Description: Watch this Scientific Journal Video about Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells at JoVE.com

这项研究得到了国家心脏、肺和血液研究所（NHLBI）、国家卫生研究院的资助，资助了U01HL099656（P.G.和D.L.F.）和U01HL134696（P.G.和D.L.F.）。

1. 导引RNA（gRNA）的设计和结构
注：每个gRNA由两个60基对（bp）寡核苷酸组成，这些基质退火产生100 bp双绞合（ds）寡核苷酸（图1A-C）。gRNA 设计、生成和测试切割效率的时间表约为 2 周（图 2）。
<ol><li>选择要编辑基因组的DNA区域，并确定符合格式的3-4 23 bp序列，5'-G（N 19）NGG-3'。这些序列应位于感兴趣区域?...

<ol>
	<li>Srivastava, D., Dewitt, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+In+Vivo+Cellular+Reprogramming:+The+Next+Generation.">Review In Vivo Cellular Reprogramming: The Next Generation.</a> Cell. 166 (6), 1386-1396 (2016).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+Modeling+Development+and+Disease+with+Organoids.">Review Modeling Development and Disease with Organoids.</a> Cell. 165 (7), 1586-1597 (2016).</li><li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+Embryonic+Stem+Cells+to+Relevant+Populations:+Lessons+from+Embryonic+Development.">Differentiation of Embryonic Stem Cells to Relevant Populations: Lessons from Embryonic Development.</a> Cell. 132 (4), 661-680 (2008).</li><li>Liu, C., Oikonomopoulos, A., Sayed, N., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+human+diseases+with+induced+pluripotent+stem+cells:+from+2D+to+3D+and+beyond.">Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond.</a> Development. 145 (5), 1-6 (2018).</li><li>Avior, Y., Sagi, I., Benvenisty, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotent+stem+cells+in+disease+modelling+and+drug+discovery.">Pluripotent stem cells in disease modelling and drug discovery.</a> Nature Reviews Molecular Cell Biology. 17 (3), 170-182 (2016).</li><li>Tiyaboonchai, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATA6+Plays+an+Important+Role+in+the+Induction+of+Human+Definitive+Endoderm,+Development+of+the+Pancreas,+and+Functionality+of+Pancreatic+&#946;+Cells.">GATA6 Plays an Important Role in the Induction of Human Definitive Endoderm, Development of the Pancreas, and Functionality of Pancreatic &#946; Cells.</a> Stem Cell Reports. 8 (3), 589-604 (2017).</li><li>Guo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+hESCs+to+Probe+the+Interaction+of+the+Diabetes-Associated+Genes+CDKAL1+and+MT1E.">Using hESCs to Probe the Interaction of the Diabetes-Associated Genes CDKAL1 and MT1E.</a> Cell Reports. 19 (8), 1512-1521 (2017).</li><li>Zeng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Isogenic+Human+ESC+Platform+for+Functional+Evaluation+of+Genome-wide-Association-Study-Identified+Diabetes+Genes+and+Drug+Discovery.">An Isogenic Human ESC Platform for Functional Evaluation of Genome-wide-Association-Study-Identified Diabetes Genes and Drug Discovery.</a> Cell Stem Cell. 19 (3), 326-340 (2016).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+of+human+embryonic+stem+cells+and+induced+pluripotent+stem+cells+with+zinc+finger+nucleases+for+cellular+imaging.">Genome editing of human embryonic stem cells and induced pluripotent stem cells with zinc finger nucleases for cellular imaging.</a> Circulation Research. 111, 1494-1503 (2012).</li><li>Xue, H., Wu, J., Li, S., Rao, M. S., Liu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Modification+in+Human+Pluripotent+Stem+Cells+by+Homologous+Recombination+and+CRISPR/Cas9+System.">Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.</a> Methods in Molecular Biology. 1307, 173-190 (2014).</li><li>Huang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+gene-corrected+adult+beta+globin+protein+in+human+erythrocytes+differentiated+from+patient+iPSCs+after+genome+editing+of+the+sickle+point+mutation.">Production of gene-corrected adult beta globin protein in human erythrocytes differentiated from patient iPSCs after genome editing of the sickle point mutation.</a> Stem Cells. 33 (5), 1470-1479 (2015).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unbiased+detection+of+off-target+cleavage+by+CRISPR-Cas9+and+TALENs+using+integrase-defective+lentiviral+vectors.">Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors.</a> Nature Biotechnology. 33 (2), 175-178 (2015).</li><li>Sim, X., Cardenas-Diaz, F. L., French, D. L., Gadue, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Doxycycline-Inducible+System+for+Genetic+Correction+of+iPSC+Disease+Models.">A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models.</a> Methods in Molecular Biology. 1353, 13-23 (2015).</li><li>Hockemeyer, D., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+Induced+Pluripotent+Stem+Cells+Meet+Genome+Editing.">Review Induced Pluripotent Stem Cells Meet Genome Editing.</a> Stem Cell. 18 (5), 573-586 (2016).</li><li>Byrne, S. M., Mali, P., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+human+stem+cells.">Genome editing in human stem cells.</a> Methods in Enzymology. 546, 119-138 (2014).</li><li>Zhu, Z., Gonz&#225;lez, F., Huangfu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+iCRISPR+platform+for+rapid+genome+editing+in+human+pluripotent+stem+cells.">The iCRISPR platform for rapid genome editing in human pluripotent stem cells.</a> Methods in Enzymology. 546, 215-250 (2014).</li><li>Hou, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+genome+engineering+in+human+pluripotent+stem+cells+using+Cas9+from+Neisseria+meningitidis.">Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (39), 15644-15649 (2013).</li><li>Cong, L., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+CRISPR-Cas9+system.">Genome engineering using CRISPR-Cas9 system.</a> Methods in Molecular Biology. 1239, 197-217 (2015).</li><li>Mali, P., Esvelt, K. M., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9+as+a+versatile+tool+for+engineering+biology.">Cas9 as a versatile tool for engineering biology.</a> Nature Methods. 10 (10), 957-963 (2013).</li><li>Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L., Corn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+homology+directed+genome+editing+by+catalytically+active+and+inactive+CRISPR-Cas9+using+asymmetric+donor+DNA.">Enhancing homology directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.</a> Nature Biotechnology. 34 (3), 339-344 (2016).</li><li>Maguire, J. A., Cardenas-Diaz, F. L., Gadue, P., French, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+crispr+cas9-mediated+genome+editing+in+human+pluripotent+stem+cells.">Highly efficient crispr cas9-mediated genome editing in human pluripotent stem cells.</a> Current Protocols in Stem Cell Biology. 48 (1), 64 (2019).</li></ol>

在此协议中，CRISPR-CAS9 与两个ssODN修复模板一起，在人类多能干细胞中证明具有特定的异构基因组变化或同源基因组变化。该方法成功地生成了异源细胞系，以接近10%的效率表达异质基因组变化。该协议已针对人类ESC和iPSCs在辐照MEF上生长，支持细胞生长和存活后培养细胞在低密度细胞排序后。通过维持具有10纳克/mL bFGF和Y-27632二盐的细胞，可以最大限度地减少细胞死亡。该协议可能适用于馈送自?...

人类胚胎干细胞 （hESCs） 和诱导多能干细胞 （iPSC） 是模拟人类疾病的宝贵工具，因为它们具有更新能力，同时保持生成不同谱系细胞类型的能力1，2 ，3，4.这些模型开启了询问基因功能的可能性，并了解特定突变和表型如何与各种疾病5、6相关。然而，为了理解特定改变如何与特定的表型相关联，使用成对同源对照和突变细胞系对于控制线变异性7、8非常重要。转录活化剂样效应素核酸酶（TALEN）和锌指核酸酶已用于在各种基因模型（包括原发细胞）中生成插入或删除（indels）突变;但这些核酸酶可能使用繁琐，而且价格昂贵9，10，11，12，13，14。集群定期间隔短节重复（CRISPR）-CAS9核酸酶的发现，由于在基因组的几乎任何区域的印德尔形成效率、使用简单性以及成本降低15，使该领域发生了革命性的变化。,16,17,18,19.
使用CRISPR-CAS9基于基因组编辑技术的挑战是生成或校正一个等位基因的特定突变，而不在第二个等位基因20中产生indel突变。该协议的主要目标是通过使用两个单链寡核苷酸 （ssODN） 修复模板来克服这一挑战，以减少第二个等位基因的形成。两种ssODN都设计包含无声突变，以防止CAS9核酸酶的重新切割，但只有一个包含利息的改变。该方法提高了产生特定杂性基因修饰的效率，而不会在第二个等位基因中诱导indel形成。利用该协议，在六个独立基因组位置进行基因编辑实验，证明在一个等位基因中精确引入所需的基因组变化，而第二个等位基因形成无indel形成，总效率为±10%。所述议定书改编自马奎尔等人21日。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">5-ml polystyrene round-bottom tube with cell-strainer cap</td>
			<td style="width:145px;">Corning</td>
			<td style="width:124px;">352235</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-well polystyrene tissue culture dishes</td>
			<td>Corning</td>
			<td style="width:124px;">353046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AflII restriction endonuclease</td>
			<td>New England Biolabs</td>
			<td style="width:124px;">R0520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>VWR</td>
			<td style="width:124px;">N605</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 medium</td>
			<td style="width:145px;">ThermoFisher</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:375px;">dNTPs</td>
			<td style="width:145px;">Roche</td>
			<td style="width:124px;">11969064001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence-activated cell sorter (FACS) apparatus</td>
			<td style="width:145px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gel extraction kit</td>
			<td>Macherey-Nagel</td>
			<td>740609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibson Assembly Kit</td>
			<td>New England Biolabs</td>
			<td>E2611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gRNA_Cloning Vector</td>
			<td>Addgene</td>
			<td style="width:124px;">41824</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB agar plates containing 50 &#956;g/ml kanamycin</td>
			<td style="width:145px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lipofectamine Stem Reagent</td>
			<td style="width:145px;">ThermoFisher</td>
			<td style="width:124px;">(STEM00001)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel Growth Factor Reduced (GFR)</td>
			<td>Corning</td>
			<td style="width:124px;">354230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Murine embryonic fibroblasts (MEFs)</td>
			<td style="width:145px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nucleospin Gel Extraction and PCR Clean-up Kit</td>
			<td>Macherey-Nagel</td>
			<td style="width:124px;">740609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital shaking incubator</td>
			<td style="width:145px;"></td>
			<td style="width:124px;"></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:19px;">pCas9_GFP vector</td>
			<td style="width:145px;">Addgene</td>
			<td style="width:124px;">44719</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR strip tubes</td>
			<td style="width:145px;">USA Scientific</td>
			<td>1402-2900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phusion High Fidelity DNA Polymerase and 5&#215; Phusion buffer</td>
			<td>New England Biolabs</td>
			<td>M0530</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PurelinkTM Quick Plasmid Miniprep Kit</td>
			<td>Invitrogen</td>
			<td style="width:124px;">K210011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td style="width:145px;">Qiagen</td>
			<td style="width:124px;">Qiagen 19133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StellarTM electrocompetent Escherichia coli cells</td>
			<td>Takara</td>
			<td style="width:124px;">636763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SOC medium</td>
			<td>New England Biolabs</td>
			<td>B9020S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypLE Express Enzyme</td>
			<td>ThermoFisher</td>
			<td style="width:124px;">12605036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Y-27632 dihydrochloride/ROCK inhibitor (ROCKi)</td>
			<td>Tocris</td>
			<td style="width:124px;">1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of defined genomic modifications using crispr-cas9 in human pluripotent stem cells

人类多能干细胞为研究基因功能和模拟与疾病相关的特定突变提供了强大的系统。由于CRISPR-CAS9介导在第二个等位基因的形成，精确异构基因修饰的生成具有挑战性。在这里，我们演示了一个协议，以帮助克服这一困难，通过使用两个修复模板，其中只有一个表示所需的序列变化，而两个模板包含无声突变，以防止重新切割和indel形成。该方法对DNA的基因编辑编码区域产生异源对照和突变人类干细胞系，研究人类疾病和生物学最为有利。此外，还优化了转染和筛选方法，以减少基因编辑实验的人工和成本。总体而言，该方案广泛适用于利用人类多能干细胞模型进行的许多基因组编辑项目。

生成 gRNA 和针对印子的筛查
每个gRNA将被克隆成质粒载体，并使用U6启动子表达。AflII限制酶用于使质粒（添加基因#41824）线性化，位于 U6 启动子之后。两个 60 bp 寡核糖退火后产生的 100 bp 波段使用 DNA 程序集克隆到 gRNA 表达载体中。一旦gRNA质粒生成，它们与CRISPS-CAS9 GFP质粒（添加基因#44719）一起转染成hESC或iPSC。GFP® 细胞在2天后进行分拣，以丰富转染细胞...

Watch this Scientific Journal Video about Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells at JoVE.com

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

该协议提供了一种方法，促进在人类多能干细胞中使用CRISPR-CAS9生成定义的异质核苷酸或同源核苷酸变化。

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

基因功能和疾病的研究受到人类近亲繁殖性质的阻碍。患者特异性和控制 iPS 细胞系的不同遗传背景会影响给定功能测定中的分化效率和表型。使用基因相同或等基因线，其中唯一的区别是特定的兴趣突变是克服这些问题的关键。许多人类疾病是由异质突变引起的，这些突变很难用CRISPR-Cas9系统产生。这是因为非靶向等位基因的内德尔突变的产生，这种突变可能以非常高的频率发生。我们的技术克服了这个问题，使用两个修复模板，其中只有一个包含感兴趣的突变。任何尝试这种技术的调查员都应该熟练掌握人类多能干细胞培养。殖民地采摘的视频演示将有助于显示正确的大小和形态以及用于采摘和筛选的技术。证明这个程序将是利奥卡德纳斯。首先，在6井板中辐照的 MEF 上镀上人类 ESC。准备转染主组合。通过移液和在室温下孵育15分钟。当细胞达到70至80%的汇合，然后，添加转染主混合下降明智的每个井与细胞和孵育在37摄氏度48小时。每 24 小时更换一次介质。为了收获细胞，首先，在室温下用三重E孵育细胞三分钟，以酶去除 MEF。加入0.5毫升的 HESC 介质与 10 微摩尔 ROCK 抑制剂。用300倍g的颗粒细胞3分钟，用10微摩尔 ROCK抑制剂在0.5毫升的人类ESC介质中重新悬浮。通过 35 微米细胞滤株盖将悬浮液过滤成 5 毫升管。使用荧光激活细胞排序，对活细胞进行闸门，对绿色荧光蛋白阳性细胞进行排序。接下来，将最多1.5倍10的细胞直接转移到一个10厘米的培养皿中，该培养皿涂有1至3个基底膜基质和辐照 MEF 和含有 ROCK 抑制剂的人类 ESC 介质。每天使用无 ROCK 抑制剂的人类 ESC 介质更换介质。10到15天后，菌落的直径约为1至2毫米。在显微镜下，使用P200移液器小心刮单个克隆，将细胞吸引到移液器中。将细胞分散在96井板的井中，用菌落绘制的介质中轻轻移液三到四次。然后，为每个引导RNA选择20个菌落，并分配到PCR条管中进行筛选。在10，000次G下离心，将细胞进行5分钟的离心。现在，在20微升蛋白质酶K缓冲液中孵育细胞颗粒，以大力分离DNA和涡流。在10，000次G下离心5分钟。接下来，对编辑的DNA进行PCR的筛选。将主混合物的20微升加入管中，包括用于放大感兴趣区域的前进和反向底剂，以及5个微升的蛋白质酶K消化。在执行筛选 PCR 时，使用从控制 iPSC 线路分离的基因组 DNA 确认清洁安培。评估 PCR 产品在 70 至 90 伏特的电泳后，按体积 agarose 凝胶 2.5% 的重量评估 PCR 产品的大小变化。任何大小差异都表示裂痕。要转染单链寡合DNA和CRISPR-Cas9质粒，在辐照的 MEF 上 6 井盘上将目标细胞系进行板状，经过一夜的孵育后达到 70 至 80% 的汇合。设置上述转染反应。通过移液混合反应，在室温下孵育15分钟。然后，将转染反应混合物滴入细胞中。48 小时后，将单元格准备为单元格排序，如上。电镀单细胞后约10天，使用200微升移液器将菌落采摘到PCR条管中，并放入预涂明胶和 MEF 的 12 井板井中。将100微升的细胞转移到24或48井板的每个井中，以前用一种用于岩石抑制剂的人类ESC介质中涂有明胶和辐照的 MEF。使用剩余的100微升进行DNA分离。为了检查单链寡聚DNA的整合成功，请从每个菌落中分离出五微升DNA，使用先前设计的筛选底剂执行PCR。使用单股寡糖DNA中产生的独特酶位点，净化PCR产品，并准备40微升的限制性酶消化。通过移液混合反应，并在制造商推荐的温度下孵育一到三个小时。然后，将用负载染料添加的 1.5% 重量（由溴化钾 agarose 凝胶）添加的消化 PCR 产品可视化，电泳电压为 80 至 100 伏，使用 40 分钟。如果成功整合单链寡核DNA，则使用嵌套底转序列特定的突变。在这项研究中，为了指导RNA构造，从1.5%的加糖凝胶中切除了100个碱基对带。细胞转染后，使用2.5%的加糖凝胶对导导RNA切割进行验证。180 基对 PCR 产品显示了未切割控制和带移指示内德尔形成的不同克隆。不同的指南 RNA 具有不同的切割效率。为了避免CRISPR-Cas9重新切割编辑的等位基因，使用G到A无声突变修改了PAM序列。PAM 序列中的无声突变生成了唯一的限制位点，这有助于筛选成功集成到一个或两个等位基因中。基因组编辑的干细胞系可用于下游分化和功能测定，以研究给定突变对人类发展和疾病的影响。这种方法允许生成具有特定异质或同源突变的等源细胞系，这些突变涉及各种人类疾病。这些疾病模型为开发新的细胞疗法铺平了道路，也为药物发现提供了平台。

Research

JoVE Journal

Genetics

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

科研

遗传学

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

DNA 修饰的免疫: 共焦图像的计算分析

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...