Name: dissection of enhancer function using multiplex crispr-based enhancer interference in cell lines
Uploaded: 2018-06-02T15:00:00.000+00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines at JoVE.com

这项工作得到了 nih/NHGRI R00 HG006922 和 nih/NHGRI R01 HG008974 和猎人癌症研究所的支持。J.B.C. 在遗传学 T32GM007464 的 NIH 培训计划中得到了支持。

<ol>
	<li>Consortium, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+encyclopedia+of+DNA+elements+in+the+human+genome.">An integrated encyclopedia of DNA elements in the human genome.</a> Nature. 489 (7414), 57-74 (2012).</li><li>Roadmap Epigenomics, C., 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=Integrative+analysis+of+111+reference+human+epigenomes.">Integrative analysis of 111 reference human epigenomes.</a> Nature. 518 (7539), 317-330 (2015).</li><li>Andersson, 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=An+atlas+of+active+enhancers+across+human+cell+types+and+tissues.">An atlas of active enhancers across human cell types and tissues.</a> Nature. 507 (7493), 455-461 (2014).</li><li>Perry, M. W., Boettiger, A. N., Bothma, J. P., Levine, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shadow+enhancers+foster+robustness+of+Drosophila+gastrulation.">Shadow enhancers foster robustness of Drosophila gastrulation.</a> Curr Biol. 20 (17), 1562-1567 (2010).</li><li>Lam, D. D., 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=Partially+redundant+enhancers+cooperatively+maintain+Mammalian+pomc+expression+above+a+critical+functional+threshold.">Partially redundant enhancers cooperatively maintain Mammalian pomc expression above a critical functional threshold.</a> PLoS Genet. 11 (2), e1004935 (2015).</li><li>Patwardhan, R. P., 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=Massively+parallel+functional+dissection+of+mammalian+enhancers+in+vivo.">Massively parallel functional dissection of mammalian enhancers in vivo.</a> Nat Biotechnol. 30 (3), 265-270 (2012).</li><li>Savic, D., 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=Promoter-distal+RNA+polymerase+II+binding+discriminates+active+from+inactive+CCAAT/+enhancer-binding+protein+beta+binding+sites.">Promoter-distal RNA polymerase II binding discriminates active from inactive CCAAT/ enhancer-binding protein beta binding sites.</a> Genome Res. 25 (12), 1791-1800 (2015).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Cheng, A. 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=Multiplexed+activation+of+endogenous+genes+by+CRISPR-on,+an+RNA-guided+transcriptional+activator+system.">Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system.</a> Cell Res. 23 (10), 1163-1171 (2013).</li><li>Hilton, I. B., 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=Epigenome+editing+by+a+CRISPR-Cas9-based+acetyltransferase+activates+genes+from+promoters+and+enhancers.">Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers.</a> Nat Biotechnol. , (2015).</li><li>Kearns, N. 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=Functional+annotation+of+native+enhancers+with+a+Cas9-histone+demethylase+fusion.">Functional annotation of native enhancers with a Cas9-histone demethylase fusion.</a> Nat Methods. 12 (5), 401-403 (2015).</li><li>Thakore, P. I., 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=Highly+specific+epigenome+editing+by+CRISPR-Cas9+repressors+for+silencing+of+distal+regulatory+elements.">Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements.</a> Nat Methods. 12 (12), 1143-1149 (2015).</li><li>Groner, A. C., 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=KRAB-zinc+finger+proteins+and+KAP1+can+mediate+long-range+transcriptional+repression+through+heterochromatin+spreading.">KRAB-zinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading.</a> PLoS Genet. 6 (3), e1000869 (2010).</li><li>Qi, L. S., 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=Repurposing+CRISPR+as+an+RNA-guided+platform+for+sequence-specific+control+of+gene+expression.">Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.</a> Cell. 152 (5), 1173-1183 (2013).</li><li>Fulco, C. P., 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=Systematic+mapping+of+functional+enhancer-promoter+connections+with+CRISPR+interference.">Systematic mapping of functional enhancer-promoter connections with CRISPR interference.</a> Science. 354 (6313), 769-773 (2016).</li><li>Joo, J. Y., Schaukowitch, K., Farbiak, L., Kilaru, G., Kim, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulus-specific+combinatorial+functionality+of+neuronal+c-fos+enhancers.">Stimulus-specific combinatorial functionality of neuronal c-fos enhancers.</a> Nat Neurosci. 19 (1), 75-83 (2016).</li><li>Xie, S., Duan, J., Li, B., Zhou, P., Hon, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Engineering+and+Analysis+of+Combinatorial+Enhancer+Activity+in+Single+Cells.">Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.</a> Mol Cell. 66 (2), 285-299 (2017).</li><li>Carleton, J. B., Berrett, K. C., Gertz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+Enhancer+Interference+Reveals+Collaborative+Control+of+Gene+Regulation+by+Estrogen+Receptor+alpha-Bound+Enhancers.">Multiplex Enhancer Interference Reveals Collaborative Control of Gene Regulation by Estrogen Receptor alpha-Bound Enhancers.</a> Cell Syst. 5 (4), 333-344 (2017).</li><li>Gilbert, L. 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=CRISPR-mediated+modular+RNA-guided+regulation+of+transcription+in+eukaryotes.">CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes.</a> Cell. 154 (2), 442-451 (2013).</li><li>Ayer, D. E., Laherty, C. D., Lawrence, Q. A., Armstrong, A. P., Eisenman, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad+proteins+contain+a+dominant+transcription+repression+domain.">Mad proteins contain a dominant transcription repression domain.</a> Mol Cell Biol. 16 (10), 5772-5781 (1996).</li><li>Alland, L., 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=Role+for+N-CoR+and+histone+deacetylase+in+Sin3-mediated+transcriptional+repression.">Role for N-CoR and histone deacetylase in Sin3-mediated transcriptional repression.</a> Nature. 387 (6628), 49-55 (1997).</li><li>Konermann, S., 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=Optical+control+of+mammalian+endogenous+transcription+and+epigenetic+states.">Optical control of mammalian endogenous transcription and epigenetic states.</a> Nature. 500 (7463), 472-476 (2013).</li><li>Rennoll, S. A., Scott, S. A., Yochum, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+repression+of+AXIN2+and+MYC+gene+expression+using+designer+TALEs.">Targeted repression of AXIN2 and MYC gene expression using designer TALEs.</a> Biochem Biophys Res Commun. 446 (4), 1120-1125 (2014).</li><li>Stampfel, 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=Transcriptional+regulators+form+diverse+groups+with+context-dependent+regulatory+functions.">Transcriptional regulators form diverse groups with context-dependent regulatory functions.</a> Nature. 528 (7580), 147-151 (2015).</li><li>Perez-Pinera, P., 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=RNA-guided+gene+activation+by+CRISPR-Cas9-based+transcription+factors.">RNA-guided gene activation by CRISPR-Cas9-based transcription factors.</a> Nat Methods. 10 (10), 973-976 (2013).</li><li>Heigwer, F., Kerr, G., Boutros, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-CRISP:+fast+CRISPR+target+site+identification.">E-CRISP: fast CRISPR target site identification.</a> Nat Methods. 11 (2), 122-123 (2014).</li><li>Parsi, K. M., Hennessy, E., Kearns, N., Maehr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+an+Inducible+CRISPR-dCas9-KRAB+Effector+System+to+Dissect+Transcriptional+Regulation+in+Human+Embryonic+Stem+Cells.">Using an Inducible CRISPR-dCas9-KRAB Effector System to Dissect Transcriptional Regulation in Human Embryonic Stem Cells.</a> Methods Mol Biol. , 221-233 (2017).</li><li>Romanienko, P. 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=A+Vector+with+a+Single+Promoter+for+In+Vitro+Transcription+and+Mammalian+Cell+Expression+of+CRISPR+gRNAs.">A Vector with a Single Promoter for In Vitro Transcription and Mammalian Cell Expression of CRISPR gRNAs.</a> PLoS One. 11 (2), e0148362 (2016).</li><li>Smith, I., 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+of+RNAi+and+CRISPR+technologies+by+large-scale+gene+expression+profiling+in+the+Connectivity+Map.">Evaluation of RNAi and CRISPR technologies by large-scale gene expression profiling in the Connectivity Map.</a> PLoS Biol. 15 (11), e2003213 (2017).</li><li>Liu, X. S., 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=Editing+DNA+Methylation+in+the+Mammalian+Genome.">Editing DNA Methylation in the Mammalian Genome.</a> Cell. 167 (1), 233-247 (2016).</li><li>O'Geen, 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=dCas9-based+epigenome+editing+suggests+acquisition+of+histone+methylation+is+not+sufficient+for+target+gene+repression.">dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression.</a> Nucleic Acids Res. 45 (17), 9901-9916 (2017).</li><li>Amabile, 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=Inheritable+Silencing+of+Endogenous+Genes+by+Hit-and-Run+Targeted+Epigenetic+Editing.">Inheritable Silencing of Endogenous Genes by Hit-and-Run Targeted Epigenetic Editing.</a> Cell. 167 (1), 219-232 (2016).</li></ol>

作者没有什么可透露的。

本协议描述了一种简单而灵活的方法来解剖增强函数在内源基因组轨迹没有物理改变 DNA 序列。虽然在概念上与以前发布的 CRISPR 干扰协议使用 dCas9-KRAB27相似, 但增强器-i 在3主要方式上与这些协议不同。首先, 增强器-i 利用 MAD120的 SIN3A 交互域来实现增强停用。利用 HDAC 抑制剂可以抢救增强剂失活, 提示失活的主要机制是 HDAC 依赖性的。不像 CRISPR 干扰 dCas9-KRAB, 增强剂-i 不导致沉积 H3K9me3。这很可能是由于增强剂-i 依赖于瞬态引入导 rna, 而细胞在转染后3天被收割。在 CRISPR 干扰中, H3K9me3 的增加在7天后转导12中观察到。最后, 增强 i 协议提供了一个针对多个站点同时同时监控目标效率的策略。在马赛克序列17中, dCas9-KRAB 用于同时针对多个增强剂, 但这种技术依靠单细胞 RNA 测序来识别表达变化, 许多基因 (如雌激素应答基因) 由于低单细胞 RNA-下增强因子的灵敏度-i 提供了一个可靠的方法来研究促进剂单独和组合为任何基因。
增强剂的最关键的步骤-i 是转染, 这应该是优化的细胞线的兴趣。该协议依赖于嘌呤霉素处理, 以丰富的转染细胞, 但有可能的联合转染引导 rna 与荧光蛋白和分类的荧光细胞使用流式细胞术可能被证明是一个更好的浓缩方法的一些细胞类型。我们建议通过 qPCR 来监测导 rna 和 SID4x-dCas9-KRAB 的表达水平, 以诊断和确认转染。如果指南 rna 水平低 (周期阈值 > 30), 用户也可以考虑其他指南 rna 生产策略, 如体外转录28。也有可能, 尽管高 gRNA 水平, 引导 rna 靶向 SID4x-dCas9-KRAB 蛋白是低效的, 在这种情况下选择不同的指南 rna 序列可能是必要的。通过对增强 i 治疗细胞的染色质对融合蛋白进行芯片序列处理, 可以对靶向的效率进行监测。如果在感兴趣的区域有 SID4x-dCas9-KRAB 的高信号, 并且没有发现其假设的目标基因的表达变化, 那么该区域在研究的条件下可能不会对该基因的表达做出贡献。
增强器的一个潜在限制是, 如果太多的站点同时被瞄准, 则非目标效果可能会累积。然而, CRISPR 干扰策略对于击倒的目标效果要比 rna29少, 特别是当使用多克隆细胞线表示 dCas9-KRAB 时。当我们同时瞄准10个地点时, 我们已经看到了 SID4X-dCas9-KRAB 的基因组结合, 我们没有确定基因表达的变化, 因为这些绑定事件。由于某些促进剂可能会接触到多个促进剂和/或其他促进剂, 有可能许多基因可能会改变表达后, 针对单一的增强剂, 虽然不清楚这种形式的基因调控是常见的。为了确认所观察到的表达式更改是由于针对特定的增强器, 而不是目标效果, 用户可以使用两组不同的非重叠导向 rna 来针对同一区域进行增强。此外, 利用核酸酶能力的 Cas9 对该区域的遗传缺失可以进一步证实其对基因表达的影响。
由于增强剂-i 功能通过组蛋白脱乙酰, 它是可能的, 其失活能力仅限于有明显水平的组蛋白乙酰化。有各种各样的替代性压制融合, 可能更有效地针对特定的增强剂。DNA 甲基融合到 dCas9 可以用来减少基因表达时, 针对远端促进剂30, 但这种镇压往往不是短暂的。另一个压制融合使用 GATA1 (FOG1) 域的朋友, 它导致组蛋白 H3 赖氨酸 27 trimethylation 和压抑基因表达的水平类似 dCas9-KRAB 跨各种细胞系和促进者31。有趣的是, 添加更多的 FOG1 到 dCas9 减少了启动器的压制潜力, 这表明 SID 域的一个副本可能提供更多的增强失活比目前使用的4拷贝在增强剂 i。在上述 dCas9 融合的不同组合中, 某些位点可能受益于双目标。例如, 可以通过同时转导 dCas9-DNMT3a 和 dCas9-KRAB32来实现稳定的长期压制。这些压制性融合大多只针对一个单一的轨迹, 在同一时间, 它仍然不清楚哪些是最有效地操作多个促进剂同时。
增强剂 i, 虽然一个适当的方法来研究促进者的组合, 少数基因, 仍然有点有限的吞吐量, 如果用户希望研究假定促增剂为数以百计的基因。这种技术的未来应用将结合成像技术, 同时在多个样本中量化多个基因。重要的是, 这些技术与从裂解液中直接检测 rna 分子是相容的, 消除了耗时的 rna 分离的需要。这些适应将有助于审讯更大的增强剂。

大规模的排序项目 (如编码1、路线图 Epigenomics2和幽灵3 ) 在人类基因组中对数以百计的细胞类型进行了数以百万计的假定增强。据估计, 每一个启动子与平均4.9 促进剂, 每个增强剂接触平均2.4 基因3, 表明基因表达往往是多种分布式调节相互作用的综合结果。还有一个重要的挑战是, 不仅要定义个体增强剂如何促进基因表达, 而且还要确定它们是如何结合来影响表达的。遗传方法通常用于确定模型有机体中的增强剂与果蝇4到小鼠5之间的关系。然而, 这些实验是耗时和低吞吐量的研究多促进剂在多个基因。
研究增强函数的一种方法是大规模的并行报道。这些化验可以同时筛选数以千计的 DNA 序列, 使其能够驱动表达的记者基因6。虽然这些化验结果表明, DNA 序列可以单独足以传达基因调控信息7, 他们来与在本地染色质上下文之外和与异源启动子进行的警告。此外, 在大量平行报道中分析的 DNA 序列的大小通常少于 200 basepairs, 可能排除相关的周围序列。重要的是, 由于记者的化验只是一次测量一个序列的活动, 他们没有考虑到在促进剂之间可能存在的复杂关系。因此, 虽然大量平行的记者化验可以是关于 dna 序列的内在活动的信息, 但他们不一定告诉我们的功能, 该 dna 序列的范围内的基因组。
最近开发的 CRISPR/Cas9 工具8促进了基因调控的研究, 因为它们允许在内源轨迹中删除增强剂。然而, 同时删除多个增强剂可能会导致基因组不稳定, 并且在单个细胞线中生成连续的强化剂删除是很耗时的。此外, 在修复后的删除站点上创建了新的基因组序列, 这一序列可能获得调节功能。Cas9 的另一种版本是专门用于调制基因表达的, 依赖于激活9、10或抑制11、12域到核酸酶不足形式 Cas9 的融合。(dCas9)。这些融合蛋白是理想的同时研究多个基因座, 因为它们不会物理改变 DNA 序列, 而是调整表观遗传学, 以询问一个调控区域。最广泛使用的压制融合是 KRAB, 招募 KAP1 联合阻遏复合体, 促进抑制相关组蛋白 H3 赖氨酸 9 trimethylation (H3K9me3)13的沉积。dCas9-KRAB, 也称为 CRISPR 干扰14, 已被用于目标和屏幕上的个体增强剂为他们的贡献基因表达15,16;但是, 它还没有针对多个区域同时进行优化。一个版本的复用 CRISPR 干扰促进剂, 马赛克序列17, 使用单细胞 RNA 序列作为读数, 但这种技术是昂贵的, 只适合研究高表达基因由于低灵敏度的单细胞RNA-
我们试图开发一种基于 CRISPR 干涉的方法来解剖组合增强函数在雌激素转录反应的背景下。大约一半的雌激素应答基因含有2或更多的增强剂, 受雌激素受体α (ER) 附近的18的约束, 这表明多重促愈剂可能参与雌激素的反应, 理解调控逻辑需要同时瞄准多个增强剂。在启动器中使用 CRISPR 干扰的初步研究表明, 并非所有的启动子都同样响应 KRAB 介导的镇压19, 我们推断, 增加一个独特的压制域, 以 dCas9 可以促进失活多样的促进剂。我们选择了 Mad1 (SID)20的 Sin3a 交互域, 因为它导致了组蛋白乙酰21的招募, 这将移除与转录活动相关的组织蛋白上的乙酰基团。重要的是, SID 域在将基因表达式融合到 dCas922和故事23时是有效的, Sin3a 在各种增强程序序列上下文24中被证明是一个强有力的压制性的共同因素。我们使用 SID4x-dCas9-KRAB (增强剂 i) 靶向10种不同的促进剂绑定的 er, 并确定 er 结合点 (ERBS), 是必要的雌激素转录应答的4基因18。我们还瞄准了促进剂的组合, 以确定在雌激素转录应答的生产中合作的地点。我们发现, 多达50个站点可能同时具有可检测的基因表达变化的目标。利用芯片序列和 RNA 序列, 我们证明了增强剂 i 是一种高度特异的技术, 同时研究多促能器。
在本协议中, 我们描述了执行增强器 i 的步骤, 这是一种灵活的技术, 能够同时在组织培养环境中同时进行多种促进剂的功能研究。增强器-i 与基因删除高度相关, 但提供了依赖于组蛋白乙酰 (HDACs) 的瞬态失活。通过通过瞬变转染提供导 rna, 而不是通过病毒载体稳定集成, 该协议避免了 H3K9me3 的沉积和潜在扩散。本协议详细介绍了通过吉布森组装进行 RNA 的设计和克隆, 利用 lipofection 对导 rna 进行转染, 并通过 qPCR 对由此产生的基因表达变化进行分析。我们还包括评估增强剂的特异性的方法-i 靶向基因组和转录的水平。该技术是在人类癌细胞系中应用 ER 结合促进剂研究基因调控的同时, 适用于任何哺乳动物增强剂的解剖。

<table><tbody><tr><td>ZR 96-well Quick-RNA Kit</td><td>Zymo Research</td><td>R1053</td><td></td></tr><tr><td>Power SYBR Green RNA-to-CT 1-Step</td><td>Applied Biosystems</td><td>4389986</td><td></td></tr><tr><td>AflII restriction enzyme</td><td>NEB</td><td>R0520S</td><td></td></tr><tr><td>Phusion High-Fidelity PCR Master Mix with HF Buffer</td><td>NEB</td><td>M0531L</td><td></td></tr><tr><td>NEBuilder HiFi DNA Assembly Master Mix</td><td>NEB</td><td>E2621L</td><td></td></tr><tr><td>FuGENE HD</td><td>Promega</td><td>E2312</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator Kit</td><td>Zymo Research</td><td>D4013</td><td></td></tr><tr><td>Buffer RLT Plus</td><td>Qiagen</td><td>1053393</td><td></td></tr><tr><td>b-estradiol</td><td>Sigma-Aldrich</td><td>E2758</td><td></td></tr><tr><td>Human: Ishikawa cells</td><td>ECACC</td><td>99040201</td><td></td></tr><tr><td>H3K27ac rabbit polyclonal</td><td>Active Motif&amp;nbsp;</td><td>39133</td><td></td></tr><tr><td>H3K9me3 rabbit polyclonal</td><td>Abcam&amp;nbsp;</td><td>ab8898</td><td></td></tr><tr><td>FLAG mouse monoclonal</td><td>Sigma-Aldrich&amp;nbsp;</td><td>F1804</td><td></td></tr><tr><td>ER alpha rabbit polyclonal</td><td>Santa Cruz</td><td>sc-544</td><td></td></tr><tr><td>pGL3-U6-PGK-Puro plasmid</td><td>Addgene&amp;nbsp;</td><td>51133</td><td>Shen et al., 2014</td></tr><tr><td>gRNA_cloningVector plasmid</td><td>Addgene&amp;nbsp;</td><td>41824</td><td>Mali et al., 2013</td></tr><tr><td>AflII U6 puromycin plasmid</td><td>Addgene&amp;nbsp;</td><td>106404</td><td>Carleton et al., 2017</td></tr><tr><td>SID4X-dCas9-KRAB plasmid</td><td>Addgene&amp;nbsp;</td><td>106399</td><td>Carleton et al., 2017</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250-10ML</td><td></td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>ThermoFisher Scientific</td><td>10977-023</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>KAPA Stranded mRNA-Seq Kit, with KAPA mRNA Capture Beads</td><td>Kapa Biosytems</td><td>KK8420</td><td></td></tr><tr><td>Pierce Protease and Phosphatase Inhibitor Mini Tablets</td><td>ThermoFisher Scientific</td><td>A32959</td><td></td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich</td><td>252549-25ML</td><td></td></tr><tr><td>Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)</td><td>ThermoFisher Scientific</td><td>10131035</td><td></td></tr><tr><td>LB Broth</td><td>ThermoFisher Scientific</td><td>10855001</td><td></td></tr><tr><td>Quick-DNA Miniprep Kit</td><td>Zymo Research</td><td>D3020</td><td></td></tr><tr><td>Quick-Load Purple 2-Log DNA Ladder</td><td>NEB</td><td>N0050S</td><td></td></tr></tbody></table>

dissection of enhancer function using multiplex crispr-based enhancer interference in cell lines

多促进剂经常调节一个给定的基因, 但对于大多数基因, 它仍然不清楚哪些促进剂是必要的基因表达, 以及这些增强剂如何结合产生转录反应。随着数以百万计的促进剂被确定, 需要高通量的工具来确定增强功能的基因组范围。目前研究增强功能的方法包括使用核酸酶熟练的 Cas9 进行基因缺失, 但要研究多种促发剂的组合效应, 还是很难的, 因为多重连续克隆细胞系必须生成。在这里, 我们提出了一种基于 CRISPR 干涉的方法, 它允许在其内源位置同时对多个增强剂进行功能性审问。增强器-i 利用两个压制域融合到核酸酶缺乏 Cas9, SID 和 KRAB, 以实现增强失活通过组蛋白脱乙酰在靶位点。该协议利用导 rna 的瞬态转染, 使靶区的瞬态灭活, 尤其有效地阻断诱导转录反应的刺激在组织培养设置。增强剂-i 是高度特异的, 无论是在其基因组定位和它对全球基因表达的影响。从本协议获得的结果有助于了解增强剂是否有助于基因表达、贡献的大小, 以及其他附近促进剂对其贡献的影响。

图 1显示了协议中描述的工作流示意图。为了确定在雌激素调控基因MMP17附近的 ER 增强增强剂的贡献, 它有3个结合点在附近由芯片序列 (图 2A) 定义, 指南 rna 为每个区域被设计了。为了设计导 rna, 选择了一个 600-900 bp 围绕每个 ER 结合点的序列的窗口, 并将其放入向导 RNA 设计程序中。结果引导 RNA 序列与0-2 被预言的目标站点被排列了对人类基因组使用 BLAT。为确定目标 (图 2B), 选择了四个跨越了芯片序列和 DNaseI 超敏的区域的非重叠导 rna。添加了附加序列 (表 1), 以方便下游克隆, 并订购了所产生的59核苷酸片段。到达后, 引导 rna 被稀释和聚集的站点, 并进行了短期 PCR 添加同源区域之前, 吉布森组装。图 2C在使用 "U6_internal" 引物 (表 1) 的短 PCR 后显示预期的指南 rna 产品, 它将在 59 basepair 指南 RNA 片段的每一端添加 20 basepairs 序列, 从而产生 100 basepair 序列。在吉布森组装后, 这些导 RNA 池被转化为细菌, 并在第二天制备质粒 minipreps。图 2D显示了增强器解剖实验的结果, 在该试验中, 在MMP17附近的多个增强剂是单独使用的, 并且是用促进剂 i 进行组合的。以增强器为目标的站点-我用黑色六边形表示。引导 RNA 质粒靶向表明的地点被转染成雌激素剥夺的石川细胞系稳定表达 SID4X-dCas9-KRAB。两天后, 媒体被改变, 嘌呤霉素被添加, 以丰富的转染细胞。第二天, 这些细胞是在8小时10纳米雌二醇治疗后收获的。RNA 被分离, 并且一步 qPCR 被执行了。在此示例中, 站点1和2对于MMP17的完全雌激素响应是必需的, 而站点3在这些条件下不作出贡献 (图 2D, 第二条车道)。当只有2或3的站点处于活动状态 (vi 和 vii) 时, 雌激素的反应类似于没有活动的站点 (viii), 这表明这些站点不能独立贡献。站点1可以自行贡献一些表达式 (v), 但当站点1和2处于活动状态 (iv) 时, 就会看到最大的活动。
为了同时操作近4种不同基因的10促进剂 (图 3A), 生成了包含42个增强器指南和16个启动器参考线的导 rna 复合池。指南 rna 寡核苷酸在最初的指南 rna 引伸 PCR (步骤 3.3) 之前汇集了, 并且结果 PCR 产品被纯化了并且结合了空嘌呤霉素 U6 克隆载体用吉布森汇编。在吉布森装配之后, 进行了多次独立变换并进行了电镀。板块被刮成磅, 允许在 maxiprep 之前长出 2-4 小时。图 3B显示, 当这些指南 RNA 池被转染成雌激素剥夺的石川细胞系稳定表达 SID4X-dCas9-KRAB 和治疗如上所述 (图 2D) 时, qPCR 基因表达的代表性减少。增强剂的减少-我是类似于那些通过靶向启动者的假定目标基因。图 3C显示了在使用增强剂 i 的情况下, 导 rna 稀释对降低雌激素反应的影响。1:50 稀释的指南 RNA 池靶向增强剂附近的G0S2仍然产生显著的减少基因表达, 表明增强剂-我可以被用于目标多达50个网站一次。但是, 停用可以被稀释, 表明数以百计的站点不能同时被瞄准, 除非使用更灵敏的检测方法。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57883/57883fig1.jpg"> 图1。使用增强器的多路增强剂解剖协议示意图指南 rna (红色和蓝色) 是设计使用电子脆和选择使用 UCSC 基因组浏览器。四引导 rna 被选择横跨感兴趣的区域 (转录因子结合站点由芯片序列定义)。指南 RNA 寡核苷酸已汇集的利益区 (红和蓝) 进行 PCR, 添加同源区域 (橙色) 之前, 吉布森组装和转换。结果质粒池通过 lipofection 转染成细胞系, 与 SID4X-dCas9-KRAB 质粒结合, 稳定地表达 SID4X-dCas9-KRAB 或与野生型细胞。引导 RNA 质粒池可以单独转染, 一次瞄准一个站点, 或者组合在一起, 同时瞄准多个站点。转染细胞用抗生素治疗, 以丰富含有导 rna 的细胞。在72小时后转染, 细胞被收获。核酸可以提取 qPCR, RNA 序列, 或芯片-后<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57883/57883fig1large.jpg" target="_blank">向请单击此处查看此图的更大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57883/57883fig2.jpg"> 图2。指南 RNA 设计和增强器解剖为MMP17.(A)基因组浏览器在 MMP17 附近瞄准的 ER α增强增强剂 (灰色) 的截图.此数字已从卡尔顿 ( et .) 中进行了修改。18. (B)指南 RNA 设计为3个绑定站点18。由芯片序列定义的 ER 的绑定站点是目标, 4 导 rna 在这个区域内平铺。DNaseI 灵敏度信号, 它跨越绑定站点, 也可以用来定义目标序列的指导 RNA 设计。从 10 nM 雌二醇处理的石川细胞中提取了两种芯片序列和 DNaseI 的数据, 1 h. (C)代表指南 RNA 顺序, 为吉布森装配准备好, 经过短 PCR 添加同源区域。(D) MMP17的相对表达式, 通过 qPCR 对特定区域的靶向增强 i 和 8-h10 nM 雌二醇治疗后测量。表达式相对于CTCF和MMP17在未用雌二醇处理的单元格中的表达式级别。控制指南 rna 瞄准 IL1RN 的启动子.所有误差线都代表 SEM, 双星号指示p < 0.01 和单星号指示p < 0.05 在配对 t 测试。此数字已从卡尔顿 ( et al) 中进行了修改。18.<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57883/57883fig2large.jpg" target="_blank">请单击此处查看此图的更大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57883/57883fig3.jpg"> 图3。同时用联合增强剂 i. 在不同基因附近的多个促进剂的靶向性.(A) "绑定站点" 和 "启动器" 的示意图, 其目标是汇集增强器-i。(B)在 E2 治疗后, 用增强型质粒池 (绿色)、启动子 i. 质粒池 (蓝色) 或控制 gRNAs (白色)18转染的石川细胞对表达的影响 qPCR。用增强剂 i 观察所有基因的显著减少。此数字是从卡尔顿 ( et al) 中修改的。18. (C)在 E2 治疗后, 以不同数量的导 rna 为靶向G0S2转染的石川细胞对G0S2表达水平的影响。即使有少量的导 RNA (1:50 稀释), 也可以看到显著的减少, 这表明多达50个站点可能同时被瞄准。所有误差线都代表 SEM, 双星号指示p < 0.01 和单星号指示p < 0.05 在配对 t 测试。<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57883/57883fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>名字</td>
			<td>序列</td>
		</tr>
<tr>
<td>U6_internal_F</td>
			<td>TTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG</td>
		</tr>
<tr>
<td>U6_internal_R</td>
			<td>GACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC</td>
		</tr>
<tr>
<td>U6_PCR_F</td>
			<td>CCAATTCAGTCGACTGGATCCGGTA</td>
		</tr>
<tr>
<td>U6_PCR_R</td>
			<td>AAAAAAAGCACCGACTCGGTGCCA</td>
		</tr>
<tr>
<td>gRNA_qPCR_F</td>
			<td>GCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG</td>
		</tr>
<tr>
<td>gRNA_qPCR_R</td>
			<td>AAAAAGCACCGACTCGGTGCC</td>
		</tr>
<tr>
<td>dCas9_qPCR_F</td>
			<td>GTGACCGAGGGAATGAGAAA</td>
		</tr>
<tr>
<td>dCas9_qPCR_R</td>
			<td>AGCTGCTTCACGGTCACTTT</td>
		</tr>
<tr>
<td>pAC95_PCR_F</td>
			<td>AGAAGAGAAAGGTGGAGGCC</td>
		</tr>
<tr>
<td>pAC95_PCR_R</td>
			<td>CGTCACCGCATGTTAGAAGG</td>
		</tr>
<tr>
<td>SID4X_PCR_F</td>
			<td>CAATAGAAACTGGGCTTGTCG</td>
		</tr>
<tr>
<td>SID4X_PCR_R</td>
			<td>TCGTGCTTCTTATCCTCTTCC</td>
		</tr>
</tbody></table>表1。引物用于引导 RNA 的延伸和排序, qPCR 和检测的融合蛋白。

Watch this Scientific Journal Video about Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines at JoVE.com

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

本协议描述了设计和执行多路化增强剂的步骤, 该方法与停用融合蛋白 SID4X-dCas9-KRAB (即增强器干扰) 相结合。该协议能够识别出调节基因表达的增强剂, 并促进调节共同靶基因的促促剂之间的关系的解剖。

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 ...

dissection-enhancer-function-using-multiplex-crispr-based-enhancer

Universidad San Sebastian

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Genetics

9.2K 次观看.  University of Utah. 本协议描述了设计和执行多路化增强剂的步骤, 该方法与停用融合蛋白 SID4X-dCas9-KRAB (即增强器干扰) 相结合。该协议能够识别出调节基因表达的增强剂, 并促进调节共同靶基因的促促剂之间的关系的解剖。

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

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

科研

发育生物学

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

遗传学

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

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

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence has shown that enhancers not only provide DNA binding motifs for transcription factors (TFs) but also generate non-coding RNAs that are referred to as eRNAs. Studies have demonstrated that eRNA transcripts can play significant roles in gene regulation in both physiology and disease. Commonly used methods to investigate the function of eRNAs are constrained to &#8220;loss-of-function&#8221; approaches by knockdown of eRNAs, or by chemical inhibition of the enhancer transcription. There has not been a robust method to conduct &#8220;gain-of-function&#8221; studies of eRNAs to mimic specific disease conditions such as human cancer, where eRNAs are often overexpressed. Here, we introduce a method for precisely and robustly activating eRNAs for functional interrogation of their roles by applying the dCas9 mediated Synergistic Activation Mediators (SAM) system. We present the entire workflow of eRNA activation, from the selection of eRNAs, the design of gRNAs to the validation of eRNA activation by RT-qPCR. This method represents a unique approach to study the roles of a particular eRNA in gene regulation and disease development. In addition, this system can be employed for unbiased CRISPR screening to identify phenotype-driving eRNA targets in the context of a specific disease.

Enhancer RNAs (eRNAs) are non-coding RNAs produced from active enhancers. An optimal approach to study eRNA functions is to manipulate their levels in the native chromatin regions. Here we introduce a robust system for eRNA studies by using CRISPR-dCas9-fused transcriptional activators to induce the expression of eRNAs of interest.

Enhancers are pivotal genomic elements scattered through the mammalian genome and dictate tissue-specific gene expression programs. Increasing evidence ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

免疫与感染

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

生物工程

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific eukaryotic genome engineering. CRISPR/Cas9 is an inexpensive, facile, and efficient genome editing tool that allows genetic perturbation of genes and genetic elements. Here we present a simple methodology for CRISPR design, cloning, and delivery for the production of genomic deletions. In addition, we describe techniques for deletion, identification, and characterization. This strategy relies on cellular delivery of a pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order to delete the intervening DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared to single-site small indels given the efficiency of biallelic modification, ease of rapid identification by PCR, predictability of loss-of-function, and utility for the study of non-coding elements. This approach can be used for efficient loss-of-function studies of genes and genetic elements in mammalian cell lines.

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using CRISPR/Cas9.

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

The prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be re-purposed for site-specific ...

生物学

Electroporation-Based CRISPR-Cas9-Mediated Gene Knockout in THP-1 Cells and Single-Cell Clone Isolation

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including their interplay with significant human pathogens such as the human immunodeficiency virus (HIV). Compared to other immortalized cell lines of myeloid origin, THP-1 cells retain many intact inflammatory signaling pathways and display phenotypic characteristics that more closely resemble those of primary monocytes, including the ability to differentiate into macrophages when treated with phorbol-12-myristate 13-acetate (PMA). The use of CRISPR-Cas9 technology to engineer THP-1 cells through targeted gene knockout (KO) provides a powerful approach to better characterize immune-related mechanisms, including virus-host interactions. This article describes a protocol for efficient CRISPR-Cas9-based engineering using electroporation to deliver pre-assembled Cas9:sgRNA ribonucleoproteins into the cell nucleus. Using multiple sgRNAs targeting the same locus at slightly different positions results in the deletion of large DNA fragments, thereby increasing editing efficiency, as assessed by the T7 endonuclease I assay. CRISPR-Cas9-mediated editing at the genetic level was validated by Sanger sequencing followed by Inference of CRISPR Edits (ICE) analysis. Protein depletion was confirmed by immunoblotting coupled with a functional assay. Using this protocol, up to 100% indels in the targeted locus and a decrease of over 95% in protein expression were achieved. The high editing efficiency makes it convenient to isolate single-cell clones by limiting dilution.

The THP-1 cell line is widely used as a model to investigate the functions of human monocytes/macrophages across various biology-related research areas. This article describes a protocol for efficient CRISPR-Cas9-based engineering and single-cell clone isolation, enabling the production of robust and reproducible phenotypic data.

THP-1 细胞中基于电穿孔的 CRISPR-Cas9 介导的基因敲除和单细胞克隆分离

The human acute monocytic leukemia (AML) THP-1 cell line is widely used as a model to study the functions of human monocyte-derived macrophages, including ...

基于多重 CRISPR 增强器的细胞系干扰增强功能解剖

摘要

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此视频中的章节

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

THP-1 细胞中基于电穿孔的 CRISPR-Cas9 介导的基因敲除和单细胞克隆分离

基于多重 CRISPR 增强器的细胞系干扰增强功能解剖

摘要

探索更多视频

此视频中的章节

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

Enforced Activation of Enhancer RNAs In Situ through the dCas9 Synergistic Activation Mediator System

Lentiviral CRISPR/Cas9介导基因组编辑，用于疾病模型中造血细胞的研究

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

基因缺失在通过CRISPR / Cas9哺乳动物细胞系生成

THP-1 细胞中基于电穿孔的 CRISPR-Cas9 介导的基因敲除和单细胞克隆分离

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择