Name: pooled crispr-based genetic screens in mammalian cells
Uploaded: 2019-09-04T13:00:00
Description: Watch this Scientific Journal Video about Pooled CRISPR-Based Genetic Screens in Mammalian Cells at JoVE.com

这项工作得到了加拿大基因组、安大略省研究基金和加拿大卫生研究所（MOP-142375，PJT-148802）的支持。

下面概述的实验应遵循该研究所的环境健康和安全办公室指南。
1. 集合CRISPR sgRNA慢病毒库质粒扩增
<ol><li>将现成的CRISPR sgRNA质粒DNA库稀释至TE（例如TKOv3）中的50纳克/μL。</li>
	<li>使用电能电池电化库。设置总共四个电穿孔反应，如下所述。<ol>
<li>将 2 μL 的 50 纳克/μL TKO 库添加到 25 μL 的解冻电能电池中，以预冷却的比色皿 （1.0 mm） 在冰上。</li>
		<li>使用制造商协议建议的...

<ol>
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R., 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=Optimized+libraries+for+CRISPR-Cas9+genetic+screens+with+multiple+modalities.">Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities.</a> Nature Communications. 9 (1), 5416 (2018).</li><li>Hart, T., 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=Evaluation+and+Design+of+Genome-Wide+CRISPR/SpCas9+Knock-out+Screens.">Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knock-out Screens.</a> G3: Genes|Genomes|Genetics. 7 (8), 2719-2727 (2017).</li><li>Mair, B., Tomic, J., 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=Essential+gene+profiles+for+human+pluripotent+stem+cells+identify+uncharacterized+genes+and+substrate+dependencies.">Essential gene profiles for human pluripotent stem cells identify uncharacterized genes and substrate dependencies.</a> Cell Reports. 27 (2), 599-615 (2019).</li><li>Shalem, O., 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-scale+CRISPR-Cas9+knock-out+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knock-out screening in human cells.</a> Science. 343 (6166), 84-87 (2014).</li><li>Sharma, S., Petsalaki, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+CRISPR-Cas9+Based+Genome-Wide+Screening+Approaches+to+Study+Cellular+Signalling+Mechanisms.">Application of CRISPR-Cas9 Based Genome-Wide Screening Approaches to Study Cellular Signalling Mechanisms.</a> International Journal of Molecular Sciences. 19 (4), (2018).</li><li>Steinhart, 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=Genome-wide+CRISPR+screens+reveal+a+Wnt-FZD5+signaling+circuit+as+a+druggable+vulnerability+of+RNF43-mutant+pancreatic+tumors.">Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.</a> Nature Medicine. 23 (1), 60-68 (2017).</li><li>Wang, T., 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=Gene+Essentiality+Profiling+Reveals+Gene+Networks+and+Synthetic+Lethal+Interactions+with+Oncogenic+Ras.">Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras.</a> Cell. 168 (5), 890-903 (2017).</li><li>Zimmermann, 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=CRISPR+screens+identify+genomic+ribonucleotides+as+a+source+of+PARP-trapping+lesions.">CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.</a> Nature. 559 (7713), 285-289 (2018).</li><li>Deans, R. 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=Parallel+shRNA+and+CRISPR-Cas9+screens+enable+antiviral+drug+target+identification.">Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification.</a> Nature Chemical Biology. 12 (5), 361-366 (2016).</li><li>Trinh, T. J. J., Bloom, F., Hirsch, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STBL2:+an+Escherichia+coli+strain+for+the+stable+propagation+of+retroviral+clones+and+direct+repeat+sequences.">STBL2: an Escherichia coli strain for the stable propagation of retroviral clones and direct repeat sequences.</a> Focus. 16, 78-80 (1994).</li><li>Hart, T., Moffat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BAGEL:+a+computational+framework+for+identifying+essential+genes+from+pooled+library+screens.">BAGEL: a computational framework for identifying essential genes from pooled library screens.</a> BMC Bioinformatics. 17, 164 (2016).</li><li>Wang, G. Z. 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=Identifying+drug-gene+interactions+from+CRISPR+knock-out+screens+with+drugZ.">Identifying drug-gene interactions from CRISPR knock-out screens with drugZ.</a> bioRxiv. , (2017).</li><li>Pedregosa, F. V., G, , 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=Scikit-learn:+Machine+Learning+in+Python.">Scikit-learn: Machine Learning in Python.</a> Journal of Machine Learning Research. 12, 2825-2830 (2011).</li><li>Ketela, T., 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=A+comprehensive+platform+for+highly+multiplexed+mammalian+functional+genetic+screens.">A comprehensive platform for highly multiplexed mammalian functional genetic screens.</a> BMC Genomics. 12, 213 (2011).</li><li>Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Am+I+ready+for+CRISPR?+A+user's+guide+to+genetic+screens.">Am I ready for CRISPR? A user's guide to genetic screens.</a> Nature Review Genetics. 19 (2), 67-80 (2018).</li><li>Hartenian, E., Doench, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+and+functional+genomics+using+CRISPR/Cas9+technology.">Genetic screens and functional genomics using CRISPR/Cas9 technology.</a> FEBS Journal. 282 (8), 1383-1393 (2015).</li><li>Li, W., 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=MAGeCK+enables+robust+identification+of+essential+genes+from+genome-scale+CRISPR/Cas9+knock-out+screens.">MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knock-out screens.</a> Genome Biology. 15 (12), 554 (2014).</li><li>Sheel, A., Xue, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+Amplifications+Cause+False+Positives+in+CRISPR+Screens.">Genomic Amplifications Cause False Positives in CRISPR Screens.</a> Cancer Discovery. 6 (8), 824-826 (2016).</li><li>Meyers, R. 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CRISPR技术以其使用简单、可靠性高，被广泛采用为精确基因组编辑的首选工具。集合CRISPR筛查提供了一种在单个实验中询问数千种基因扰动的方法。在池化屏幕中，sgRNA库充当分子条形码，因为每个序列都是唯一的，并映射到目标基因。通过将基因组DNA与细胞群分离，可以通过下一代测序量化sgRNA丰度来确定引起感兴趣的表型的基因。大量并行测序方法用于量化样本中的sgRNA，这意味着多个独立的?...

CRISPR-Cas 是定期聚类短纤维重复和 CRISPR 相关核酸酶的简称，由单个核酸酶蛋白（例如 Cas9）与合成导引RNA（sgRNA）复合体组成。这种核糖核蛋白复合物以Cas9酶为目标，在特定的基因组位点1诱导双链DNA断裂。双绞线断裂可以通过同源定向修复 （HDR） 修复，或者更常见的是，通过非同源端连接 （NHEJ），一种容易出错的修复机制，导致插入和/或删除 （INDELS），经常破坏基因功能1.CRISPR的效率和简单性使以前无法实现的基因组靶向水平远远超过以前的基因组编辑技术，即锌指核酸酶（ZNF）或转录活化剂样效应核酸酶（TALENS），两者都受到更高的设计复杂性，较低的转染效率，并在多路基因编辑2的局限性。
CRISPR单导RNA基因组编辑的基础研究应用使科学家能够高效、廉价地询问单个基因的功能和遗传相互作用网络的拓扑结构。使用CRISPR-Cas系统大大提高了执行功能性全基因组屏幕的能力，特别是与早期的基因扰动技术（如RNA干扰（RNAi）和基因陷阱诱变相比。特别是，RNAi受到高脱靶效果和不完全击倒，导致与CRISPR3，4，5相比，灵敏度和特异性较低，而基因陷阱方法只有在单倍体中才可行细胞功能丧失，限制了细胞模型的范围，可以询问6。CRISPR能够产生完整的基因敲除，提供了一个更具生物学上强大的系统来询问突变表型，低噪声，最小的脱靶效应和在试剂5上的一致活性。CRISPR-Cas9 sgRNA库，针对整个人类基因组现已广泛可用，允许在一个单一的实验3，7，8，9同步产生数千个基因敲除.
我们开发了独特的CRISPR-Cas9全基因组sgRNA慢病毒库，称为多伦多敲除（TKO）库（可通过Addgene获得），这些库结构紧凑，经过序列优化，便于高分辨率功能基因组学屏幕。最新的库，TKOv3，以18，000个人类蛋白质编码基因为目标，71，090个指南，利用经验数据10优化了编辑效率。此外，TKOv3 可作为单组分库（LCV2：：TKOv3、Addgene ID #90294）在单个载体上表达Cas9和sgRNA，从而减轻了生成稳定的Cas9表达细胞的需求，从而在广泛的范围内实现全基因组的敲除哺乳动物细胞类型。TKOv3也可用于没有Cas9（pLCKO2：：TKOv3，Addgene ID= 125517）的载体中，可用于表达Cas911的细胞。
全基因组CRISPR-Cas9编辑的细胞群可以暴露在不同的生长条件下，随着时间的推移，通过下一代测序量化了sgRNA的丰度，为评估具有可追溯遗传的细胞的辍学或富集提供了读出扰动。CRISPR 敲除库可用于识别在扰动时导致细胞健康缺陷、中度药物敏感性（例如敏感或耐药基因）、调节蛋白质表达（例如，报告人）或特定基因所需的基因通路功能和细胞状态12，13，14。例如，癌细胞系中的差分健身屏可以识别肿瘤基因的消耗或减少，并富集或增加肿瘤抑制基因3，14，15。同样，使用中间剂量的治疗药物可以揭示耐药性和致敏基因16，17。
此处提供了详细的基因组规模CRISPR-Cas9功能丧失筛选方案，使用多伦多敲除库（TKOv1或v3）在哺乳动物细胞从库生成、筛选性能到数据分析。尽管此协议已针对使用多伦多挖空库进行筛选进行了优化，但可以应用它并扩展到所有 CRISPR sgRNA 集合库。

<table>
	<tbody>
		<tr>
			<td>0.22 micron filter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30&#176;C plate incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C shaking incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37&#176;C, 5% CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>Promega</td>
			<td>V4221</td>
			<td></td>
		</tr>
		<tr>
			<td>50X TAE buffer</td>
			<td>BioShop</td>
			<td>TAE222.4</td>
			<td></td>
		</tr>
		<tr>
			<td>6 N Hydrochloric acid solution</td>
			<td>BioShop</td>
			<td>HCL666.500</td>
			<td></td>
		</tr>
		<tr>
			<td>95% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar blue</td>
			<td>ThermoFisher Scientific</td>
			<td>DAL1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue-light transilluminator</td>
			<td>ThermoFisher Scientific</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin,Heat Shock Isolation, Fraction V. Min. 98%, Biotechnology grade</td>
			<td>Bioshop</td>
			<td>ALB001.250</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modification of Eagles Medium</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td>Cel culture media</td>
		</tr>
		<tr>
			<td>Electroporation cuvettes</td>
			<td>BTX</td>
			<td>45-0134</td>
			<td></td>
		</tr>
		<tr>
			<td>Electroporator</td>
			<td>BTX</td>
			<td>45-0651</td>
			<td></td>
		</tr>
		<tr>
			<td>Endura electrocompetent cells</td>
			<td>Lucigen</td>
			<td>90293</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>GIBCO</td>
			<td>12483-020</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T packaging cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>recommend passage number &#60;15</td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide (Polybrene)</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>Cationic polymer to enhance transduction efficiency</td>
		</tr>
		<tr>
			<td>Hexadimethrine Bromide (Polybrene)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB agar plates with carbenicillin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium with carbenicillin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low molecular weight DNA ladder</td>
			<td>New England Biolabs</td>
			<td>N3233S</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBNext Ultra II Q5 Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0544L</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life Technologies</td>
			<td>31985-070</td>
			<td>Reduced serum media</td>
		</tr>
		<tr>
			<td>Plasmid maxi purification kit</td>
			<td>Qiagen</td>
			<td>12963</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G (envelope plasmid)</td>
			<td>Addgene</td>
			<td>Plasmid #12259</td>
			<td>lentiviral system</td>
		</tr>
		<tr>
			<td>psPAX2 (packaging plasmid)</td>
			<td>Addgene</td>
			<td>Plasmid #12260</td>
			<td>lentiviral system</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Wisent</td>
			<td>400-160-UG</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR assay</td>
			<td>ThermoFisher Scientific</td>
			<td>Q32853</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Q33226</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse A</td>
			<td>Invitrogen</td>
			<td>12091021</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C recovery medium</td>
			<td>Invitrogen</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>SYRB Safe DNA gel stain</td>
			<td>ThermoFisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Toronto KnockOut CRIPSR library (TKOv3) - Cas9 included</td>
			<td>Addgene</td>
			<td>Addgene ID #90203</td>
			<td>Genome-wide CRISPR library , includes Cas9, 71,090 sgRNA</td>
		</tr>
		<tr>
			<td>Toronto KnockOut CRIPSR library (TKOv3) - non-cas9</td>
			<td>Addgene</td>
			<td>Addgene ID #125517</td>
			<td>Genome-wide CRISPR library, non-Cas9, 71,090 sgRNA</td>
		</tr>
		<tr>
			<td>Tris-EDTA (TE) solution, pH8.0</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure agarose</td>
			<td>ThermoFisher Scientific</td>
			<td>16500500</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard genomic DNA purification kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE 9 DNA transfection reagent</td>
			<td>Roche</td>
			<td>06 365 809 001</td>
			<td>Lipid based transfection reagent</td>
		</tr>
	</tbody>
</table>

pooled crispr-based genetic screens in mammalian cells

使用CRISPR-Cas系统的基因组编辑已经大大提升了精确编辑各种生物体的基因组的能力。在哺乳动物细胞的背景下，这项技术代表了一种用于功能基因组学研究的全基因组基因筛选的新手段。面向所有开放阅读框架的指南RNA（sgRNA）库允许在单个细胞池中方便地产生数千个基因扰动，这些细胞池可以筛选特定表型，以暗示基因功能和细胞过程。公正和系统的方式。CRISPR-Cas屏幕为研究人员提供了一种简单、高效和廉价的方法来揭示细胞表型的基因蓝图。此外，对不同细胞系和不同癌症类型的屏幕进行鉴别分析可以识别肿瘤细胞中上下文必要的基因，揭示特定抗癌治疗的潜在靶点。在人类细胞中执行全基因组屏幕可能令人望而生畏，因为这需要处理数千万个细胞，并且需要分析大量数据。这些屏幕的细节，如细胞系表征、CRISPR 库注意事项，以及分析过程中了解 CRISPR 技术的局限性和功能，经常被忽视。此处提供了一个详细的协议，用于成功执行基于全基因组 CRISPR-Cas9 的屏幕。

基因组规模CRISPR筛选工作流程概述
图 1概述了池 CRISPR 筛选工作流程，从低 MOI 下目标细胞感染 CRISPR 库慢病毒开始，以确保单个集成事件和足够的库表示（通常为 200 到 1000-折叠）。感染后，细胞用抗生素紫霉素进行治疗，以选择转导细胞。选择后，收集基线T0细胞颗粒，以评估筛选开始时的...

Watch this Scientific Journal Video about Pooled CRISPR-Based Genetic Screens in Mammalian Cells at JoVE.com

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

CRISPR-Cas9技术提供了一种有效的方法，可以精确编辑任何细胞类型的哺乳动物基因组，并代表一种执行全基因组基因筛选的新方法。此处提供了一个详细的协议，讨论成功执行池全基因组 CRISPR-Cas9 屏幕所需的步骤。

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

CRISPR筛选的辍学屏幕为研究人员提供了一种简单、高效、廉价的方法来在全基因组水平上检测链功能。CRISPR 的优点在于只需更改引导序列即可编辑任何基因。CRISPR指南库使研究人员能够以公正、系统的方式在一个实验中对任何生物体的整个基因组进行询问。目前，CRISPR屏幕正被用于识别数百种人类癌症的基本基因，并绘制基因相互作用图。屏幕还可以全面描述药物，以揭示药物的行动机制。此处描述的 CRISPR 库以人类细胞为目标。但是，针对其他物种（如小鼠）的指南库可用，也可以进行类似的筛选。在人类细胞中执行全基因组筛选在实践中可能令人望而生畏，因为它涉及处理数千万个细胞，并且需要分析大量数据。在启动屏幕之前，请确保对单元格系进行仔细描述。这包括了解细胞系的纯度、加倍的时间、扁病毒转导效率以及抗生素选择剂的敏感性。可视化演示将提供有关如何实际处理屏幕中所需的数百万个单元格的图片，并系统地跟踪数千个扰动。演示这个程序的将是安德里亚·哈布西德、卡马尔迪普·奥拉赫和瑞安·克利米，他们都是我实验室的技术人员。首先将现成的CRISPR sgRNA质粒转化为电镀细胞，然后根据手稿说明进行生长。当准备收获菌落时，在每个盘子中加入七毫升的 LB 与卡贝尼西林，用细胞扩散器刮掉菌落。使用 10 毫升移液器将刮过的细胞转移到无菌的一升锥形烧瓶中，然后用五毫升 LB 用卡贝尼西林冲洗盘子。再次，将冲洗溶液转移到烧瓶中。根据手稿方向将细胞离心，然后确定湿颗粒的重量。使用 Maxi 或巨型质粒纯化试剂盒纯化质粒 DNA。通过播种293个T细胞并孵育过夜，为转染细胞做好准备。第二天，为15厘米的板准备三个转染质粒混合物。计算一次转染所需的质粒量，并混合质粒，使要转染的板数加上一个质粒。接下来，为每种转染和等分减少血清介质准备一个基于脂质的转染试剂，并放入单独的1.5毫升微离质管中，用于要转染的板数。加入转染试剂，轻轻混合，在室温下孵育五分钟。孵育后，以转染试剂与DNA复合物微克的三比一的比例将脱氧核糖核酸加入转染试剂。轻轻混合溶液，在室温下离开30分钟。将转染混合物加入包装细胞，根据手稿说明孵育。在病毒收获当天，检查细胞的异常和融合形态，作为良好病毒产生的迹象，通过收集上根液并转移到无菌离心管中来收获扁病毒。首先选择要在整个屏幕中保持的 CRISPR 指南 RNA 库覆盖率。根据库覆盖率，确定每个引导RNA维持该细胞所需的细胞数和MOI 0.3感染所需的细胞数。接下来，确定设置感染所需的板数，然后，收获细胞并播种到每个板。将六溴甲基溴添加到所有板块中，并将所需的病毒量添加到筛选和控制两个板块中。不要添加病毒来控制病毒。将该卷替换为介质。通过倾斜将板彻底混合，将板放入培养箱中，确保板是水平的。根据手稿方向收集受感染的细胞，从池状细胞中收集三个细胞颗粒，用于基因组 DNA 提取。将细胞以500倍g离心5分钟，用PBS清洗。标记管子，并在零下 80 摄氏度处将细胞颗粒冷冻干燥。将受感染的细胞池拆分为三个复制组，确保在每个复制中保持库覆盖率。以膨胀时通常使用的密度为细胞播种。对每个复制使用相同数量的单元格，并在复制之间使用相同的单元格总数。继续通过细胞，从池状受感染细胞的每个复制中收获三个细胞颗粒，最多15至20个细胞翻倍。在每个通道，收获细胞从每个板之间的所有细胞相互复制。用时间标记每个颗粒并复制指定。根据手稿方向设置 PCR1，每 50 微升反应 3.5 微克基因组 DNA，总共 100 微克基因组 DNA，并设置相同的 50 微升反应，以实现所需的覆盖。根据稿稿说明设置一个PCR管反应。使用池 PCR1 产品的五个微升作为模板，并每个样本使用唯一的索引底像组合，以允许对测序库样本进行池化。完成 PCR 后，在低电压下在 2%的加糖凝胶上运行 PCR 管产品 1 至 1 1~2 小时。在蓝光透光器上可视化产品，并切除 200 基对带。使用分光光度计和荧光计净化DNA并测量其数量和质量。理想的导向RNA库应以相似数量表示每个引导RNA。下一代测序可用于确认库具有紧密的指南RNA分布。精密召回分析可用于评估屏幕性能。高性能屏幕应以大于 6 的基因子恢复大量基本基因，且误发现率小于 5% 精度-召回曲线应具有锋利的肘部和一条直线到终点。贝叶斯因子表示基因敲除导致健身缺陷的置信度。高分表明信心增强，而低分表示基因敲除具有生长优势。全基因组的敲除池可以在过量的药物代理存在的情况下进行培养，以寻找抑制剂抗药性基因。为了对胸腺素块的抑制器执行正选择屏幕，T0 上所有引导 RNA 的规范化读取计数根据胸腺素处理样品的均等规范化读取计数绘制。成功执行CRISPR屏幕的一个关键细节是保持每个引导RNA的良好分布，从质粒的转染到细胞的转导。这将最大限度地减少随机效应的机会，这些随机效应可能扭曲引导RNA表示，并导致误报或阴性结果。在屏幕验证之后，应进行实验以确认命中，因为主屏幕只识别潜在的命中。根据命中的生物学，可以使用各种方法。CRISPR编辑与下一代测序相结合，使各种人类模型系统中的功能屏幕的基因组尺度丢失。由于CRISPR的简单性，这些类型的屏幕现在可供所有研究人员广泛访问，使更多的科学家能够利用功能性遗传方法来研究生物过程和疾病。

Research

JoVE Journal

Genetics

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

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 ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

在非活动 X 染色体上重新激活 MeCP2 的池 shRNA 屏幕

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

蛋白质组学与染色质相关蛋白杂交捕获对哺乳动物细胞的适应性研究

The hybridization capture of chromatin-associated proteins for proteomics (HyCCAPP) technology was initially developed to uncover novel DNA-protein ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

甲醛辅助分离测定哺乳动物细胞染色质可及性的调控元素

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

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

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 ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

人牙小泡干细胞的分离、鉴定及微 rna 遗传修饰

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

Genetic Engineering of Dictyostelium discoideum Cells Based on Selection and Growth on Bacteria

基于细菌选择和生长的盘状细胞基因工程

Dictyostelium discoideum is an intriguing model organism for the study of cell differentiation processes during development, cell signaling, and other ...