Name: a new toolkit for evaluating gene functions using conditional cas9 stabilization
Uploaded: 2021-09-02T13:00:00
Description: Watch this Scientific Journal Video about A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization at JoVE.com

我们感谢我们的实验室前成员和科学家塞里夫·森图尔克以前的工作。我们感谢达尼洛·塞戈维亚批判性地阅读了这份手稿。这项研究是可能的，并得到了游泳横跨美国和国家癌症研究所癌症目标发现和发展中心计划的支持。

1.DD-Cas9向量
<ol><li>从Addgene（带填充序列的DD-Cas9和金星（EDCPV）、普拉斯米德90085获得DD-Cas9向量。 注：这是一个带 U6 促进器的扁豆 DD-Cas9 质粒，可驱动单导 RNA （sgRNA） 转录，而 EFS 发起人驱动 DD-Cas9 转录。DYKDDK序列（旗标）存在于Cas9的C终端，然后是2A自裂肽（P2A），它分离DD-Cas9和改性荧光蛋白金星（mVenus）。</li>
</ol>2. 小型导引RNA（sgRNA）设计
<ol><li>设计小型导引RNA（sgRNA...

<ol>
	<li>Jinek, 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=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337, 816-821 (2012).</li><li>Al-Attar, S., Westra, E. R., van der Oost, J., Brouns, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clustered+regularly+interspaced+short+palindromic+repeats+(CRISPRs):+the+hallmark+of+an+ingenious+antiviral+defense+mechanism+in+prokaryotes.">Clustered regularly interspaced short palindromic repeats (CRISPRs): the hallmark of an ingenious antiviral defense mechanism in prokaryotes.</a> Biological Chemistry. 392, 277-289 (2011).</li><li>Hsu, P. D., Lander, E. S., 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=Development+and+applications+of+CRISPR-Cas9+for+genome+engineering.">Development and applications of CRISPR-Cas9 for genome engineering.</a> Cell. 157, 1262-1278 (2014).</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, 823-826 (2013).</li><li>Barrangou, 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=Advances+in+CRISPR-Cas9+genome+engineering:+lessons+learned+from+RNA+interference.">Advances in CRISPR-Cas9 genome engineering: lessons learned from RNA interference.</a> Nucleic Acids Research. 43, 3407-3419 (2015).</li><li>Zhang, J., Chen, L., Zhang, J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+Inducible+CRISPR/Cas+Systems.">Drug Inducible CRISPR/Cas Systems.</a> Computational and Structural Biotechnology Journal. , (2019).</li><li>Wade, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Silencing+Using+the+CRISPR-Cas9+System:+A+Review+of+the+Benefits+and+Challenges.">High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges.</a> Journal of Biomolecular Screening. 20, 1027-1039 (2015).</li><li>Egeler, E. L., Urner, L. M., Rakhit, R., Liu, C. W., Wandless, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-switchable+substrates+for+a+ubiquitin-proteasome+system.">Ligand-switchable substrates for a ubiquitin-proteasome system.</a> Journal of Biological Chemistry. 286, 31328-31336 (2011).</li><li>Cao, 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=An+easy+and+efficient+inducible+CRISPR/Cas9+platform+with+improved+specificity+for+multiple+gene+targeting.">An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.</a> Nucleic Acids Research. 44, 149 (2016).</li><li>Oakes, B. 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=Profiling+of+engineering+hotspots+identifies+an+allosteric+CRISPR-Cas9+switch.">Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.</a> Nature Biotechnology. 34, 646-651 (2016).</li><li>Kamiyama, 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=Versatile+protein+tagging+in+cells+with+split+fluorescent+protein.">Versatile protein tagging in cells with split fluorescent protein.</a> Nature Communications. 7, 11046 (2016).</li><li>Roper, 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=In+vivo+genome+editing+and+organoid+transplantation+models+of+colorectal+cancer+and+metastasis.">In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.</a> Nature Biotechnology. 35, 569 (2017).</li><li>Chu, B. 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=Recent+progress+with+FKBP-derived+destabilizing+domains.">Recent progress with FKBP-derived destabilizing domains.</a> Bioorganic &amp; Medicinal Chemistry Letters. 18 (22), 5941-5944 (2008).</li><li>Banaszynski, L. A., Sellmyer, M. A., Contag, C. H., Wandless, T. J., Thorne, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+control+of+protein+stability+and+function+in+living+mice.">Chemical control of protein stability and function in living mice.</a> Nature Medicine. 14, 1123-1127 (2008).</li><li>Banaszynski, L. A., Chen, L. C., Maynard-Smith, L. A., Ooi, A. G., Wandless, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid,+reversible,+and+tunable+method+to+regulate+protein+function+in+living+cells+using+synthetic+small+molecules.">A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules.</a> Cell. 126, 995-1004 (2006).</li><li>Sentmanat, M. F., Peters, S. T., Florian, C. P., Connelly, J. P., Pruett-Miller, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Survey+of+Validation+Strategies+for+CRISPR-Cas9+Editing.">A Survey of Validation Strategies for CRISPR-Cas9 Editing.</a> Scientific Reports. 8, 888 (2018).</li><li>Hua, Y., Wang, C., Huang, J., Wang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+CRISPR/Cas9-induced+mutant+screening.">A simple and efficient method for CRISPR/Cas9-induced mutant screening.</a> Journal of Genetics and Genomics. 44 (4), 207-213 (2017).</li><li>Guschin, D. 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=A+rapid+and+general+assay+for+monitoring+endogenous+gene+modification.">A rapid and general assay for monitoring endogenous gene modification.</a> Methods in Molecular Biology. 649, 247-256 (2010).</li><li>Senturk, 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=Rapid+and+tunable+method+to+temporally+control+gene+editing+based+on+conditional+Cas9+stabilization.">Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.</a> Nature Communications. 8, 14370 (2017).</li><li>McJunkin, K., 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=Reversible+suppression+of+an+essential+gene+in+adult+mice+using+transgenic+RNA+interference.">Reversible suppression of an essential gene in adult mice using transgenic RNA interference.</a> Proceedings of the National Academy of Sciences. 108, 7113 (2011).</li><li>Davis, K. M., Pattanayak, V., Thompson, D. B., Zuris, J. A., Liu, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-triggered+Cas9+protein+with+improved+genome-editing+specificity.">Small molecule-triggered Cas9 protein with improved genome-editing specificity.</a> Nature Chemical Biology. 11, 316-318 (2015).</li><li>Dow, 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=Inducible+in+vivo+genome+editing+with+CRISPR-Cas9.">Inducible in vivo genome editing with CRISPR-Cas9.</a> Nature Biotechnology. 33, 390-394 (2015).</li><li>Wright, A. V., 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=Rational+design+of+a+split-Cas9+enzyme+complex.">Rational design of a split-Cas9 enzyme complex.</a> Proceedings of the National Academy of Sciences of the United States of America. 112, 2984-2989 (2015).</li><li>Albrecht, 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=Comparison+of+Lentiviral+Packaging+Mixes+and+Producer+Cell+Lines+for+RNAi+Applications.">Comparison of Lentiviral Packaging Mixes and Producer Cell Lines for RNAi Applications.</a> Molecular Biotechnology. 57, 499-505 (2015).</li><li>Froschauer, A., Kube, L., Kegler, A., Rieger, C., Gutzeit, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+Protein+Stabilization+In+Vivo+Mediated+by+Shield-1+in+Transgenic+Medaka.">Tunable Protein Stabilization In Vivo Mediated by Shield-1 in Transgenic Medaka.</a> PLOS One. , (2015).</li><li>Sellmyer, M. 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=Intracellular+context+affects+levels+of+a+chemically+dependent+destabilizing+domain.">Intracellular context affects levels of a chemically dependent destabilizing domain.</a> PLOS One. 7 (9), 43297 (2012).</li><li>Iwamoto, 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=A+general+chemical+method+to+regulate+protein+stability+in+the+mammalian+central+nervous+system.">A general chemical method to regulate protein stability in the mammalian central nervous system.</a> Chemistry &amp; Biology. 17 (9), 981-988 (2010).</li><li>Tai, K., 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=Destabilizing+domains+mediate+reversible+transgene+expression+in+the+brain.">Destabilizing domains mediate reversible transgene expression in the brain.</a> PLOS One. 7 (9), 46269 (2012).</li><li>Auffenberg, E., 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=Remote+and+reversible+inhibition+of+neurons+and+circuits+by+small+molecule+induced+potassium+channel+stabilization.">Remote and reversible inhibition of neurons and circuits by small molecule induced potassium channel stabilization.</a> Scientific Reports. 6, 19293 (2016).</li></ol>

CRISPR/Cas9技术彻底改变了功能性地询问基因组2的能力。然而，基因的灭活往往导致细胞致死性、功能缺陷和发展缺陷，限制了这种研究基因功能的方法的效用。此外，Cas9 的构成表达可能导致毒性和产生偏离目标的影响6。已开发出不同的方法，以时间控制CRISPR-Cas9为基础的基因组编辑技术6。这些系统要么基于Cas9和/或sgRNA的转...

CRISPR-Cas9代表"定期间歇性短回流相关蛋白质9"，作为细菌自适应免疫1，2研究的一部分首次被发现。今天，CRISPR/Cas9已成为可编程基因编辑的最知名的工具，系统中不同的迭代已被开发，允许转录和表观遗传调制3。这项技术使DNA4的几乎任何序列都能够进行高度精确的基因操作。
任何CRISPR基因编辑的基本组成部分是一个可定制的指南RNA序列和Cas9核素5。RNA指南与DNA中的目标互补序列结合，指示Cas9核素在基因组3、4的特定点进行双链断裂。由此产生的部位然后修复由非同源端连接（ NHEJ ）或同源定向修复（ HDR ），随之而来的是目标 DNA 序列5的变化。
CRISPR/Cas9基于基因编辑是易于使用的，相对便宜相比，以前的基因编辑技术，它已被证明既有效又强大的多重系统2，4，5。然而，这个系统存在一些局限性。Cas9的构成表达经常被证明会导致偏离目标的数量增加和高细胞毒性4，6，7，8。此外，Cas9对基本和细胞生存基因的构成定位剥夺了其进行某些类型的功能研究的能力，如细胞死亡的动能研究。
已开发了不同的诱导或有条件控制的CRISPR-Cas9工具，以解决这些问题6，如泰特-ON和泰特-关闭9：特定地点重组10：化学诱导的接近11：因汀依赖拼接3：和4-羟基甲基雄激素受体（ER）为基础的核定位系统12。一般来说，这些程序（因汀拼接和化学诱导的接近分割系统）大多不提供可逆的控制，对药物治疗（Tet-On/Off系统）的动能反应非常缓慢，或不适合高通量操纵6。
为了解决这些限制，我们开发了一个新的工具包，不仅提供快速和强大的时间控制基因编辑，而且还确保可追溯性，可调和适应高吞吐量基因操作。这种新技术可用于细胞系、器官和动物模型。我们的系统基于一个工程领域，当融合到Cas9时，它会导致其快速退化。然而，它可以迅速稳定与高度选择性，无毒，细胞渗透的小分子。更具体地说，我们设计了人类FKBP12突变的"破坏稳定领域"（DD）到Cas9，标志着Cas9通过无处不在的蛋白体系统快速和构成退化时，在哺乳动物细胞13表达。DD合成配体，盾-1可以稳定DD构象，从而防止蛋白质的降解融合到DD（如Cas9）在一个非常有效的方式，并与快速动能反应14，15。值得注意的是，Shield-1与突变的FKBP12的强度比其野生型对应物14的强度要紧三个数量级。
DD-Cas9/Shield-1对可用于研究培养细胞和动物模型中基本基因的系统识别和表征，正如我们以前通过有条件地瞄准CypD基因所表明的那样，CypD基因在线粒体的新陈代谢中起着重要作用：EGFR，在致癌转化的关键角色;和Tp53，DNA损伤反应中的核心基因。除了时间和条件控制的基因编辑，该方法的另一个优点是DD-Cas9的稳定独立于其转录。此功能可在同一促进器下共同表达可追溯标记以及重组，如雌激素受体依赖重组酶、CREER。在这项研究中，我们展示了我们的方法如何在体外成功使用，以有条件地瞄准DNA复制基因，RPA3。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100mM DTT</td>
			<td>Thermosfisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X FastDigest buffer</td>
			<td>Thermosfisher</td>
			<td>B64</td>
			<td></td>
		</tr>
		<tr>
			<td>10X T4 Ligation Buffer</td>
			<td>NEB</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>colorimetric BCA kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, glutaMax</td>
			<td>Thermo Fisher</td>
			<td>10566024</td>
			<td></td>
		</tr>
		<tr>
			<td>FastAP</td>
			<td>Thermosfisher</td>
			<td>EF0654</td>
			<td></td>
		</tr>
		<tr>
			<td>FastDigest BsmBI</td>
			<td>Thermosfisher</td>
			<td>FD0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Flag [M2] mouse mAb</td>
			<td>Sigma</td>
			<td>F1804-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Genomic DNA extraction kit</td>
			<td>Macherey Nagel</td>
			<td>740952.1</td>
			<td></td>
		</tr>
		<tr>
			<td>lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td></td>
		</tr>
		<tr>
			<td>oligonucleotides</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma Aldrich</td>
			<td>TR-1003-G</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR &#38; Gel Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28506</td>
			<td></td>
		</tr>
		<tr>
			<td>secondary antibodies</td>
			<td>LICOR</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shield-1</td>
			<td>Cheminpharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stbl3 competent bacterial cells</td>
			<td>Thermofisher</td>
			<td>C737303</td>
			<td></td>
		</tr>
		<tr>
			<td>SURVEYOR Mutation Detection Kit</td>
			<td>Transgenomic/IDT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 PNK</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>NEB</td>
			<td>M0273S</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-tubulin [DM1A] mouse mAb</td>
			<td>Millipore</td>
			<td>CP06-100UG</td>
			<td></td>
		</tr>
	</tbody>
</table>

a new toolkit for evaluating gene functions using conditional cas9 stabilization

聚类定期间歇性短回流（CRISPR-）相关蛋白质9（CRISPR/Cas9）技术已成为一种流行的实验室工具，在基因组中引入准确和有针对性的修饰。其巨大的受欢迎程度和快速的传播归因于其易于使用和准确性相比，其前身。然而，该系统的构成激活应用有限。在本文中，我们描述了一种新方法，允许基于Cas9蛋白质有条件稳定对CRISPR/Cas9活性进行时间控制。将拉帕霉素结合蛋白FKBP12的工程突变物与Cas9（DD-Cas9）融合，使Cas9迅速降解，而这种降解又可以通过存在FKBP12合成配体（盾牌-1）来稳定。与其他诱导方法不同，该系统可以很容易地适应生成双系统，以与另一个感兴趣的基因共同表达DD-Cas9，而无需对第二个基因进行有条件的调节。这种方法能够生成可追溯系统，以及Cas9核酸酶靶向的等位基因的平行、独立操作。该方法的平台可用于对基本基因进行系统识别和定性，并研究基因在体外和体内的功能相互作用。

为了实现Cas9的有条件表达，我们开发了一个双扁豆载体结构，包括一个U6驱动的促进器，构成表达sgRNA，和一个EF-1+核心促进器，以驱动DD-Cas9融合蛋白（图1A）19的表达。作为说明系统的稳健性和效率的范例，我们用扁豆结构转导了肺致病性A549细胞系。Cas9 在配体盾-1 存在或不存在的情况下的水平是通过反转录聚合酶链反应 （RT-PCR）...

Watch this Scientific Journal Video about A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization at JoVE.com

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

在这里，我们描述了一个基因组编辑工具，基于时间和条件稳定聚类定期间歇性短腹膜重复（CRISPR-）相关蛋白质9（Cas9）下的小分子，盾牌-1。该方法可用于培养细胞和动物模型。

使用有条件Cas9稳定评估基因功能的新工具包

The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 (CRISPR/Cas9) technology has become a prevalent laboratory ...

这种方法提供快速，温度控制的CRISPR-Cas9基因编辑下的小分子，盾牌-1。在将其与Cas9的构成激活进行比较时，它还提供了更少的脱靶效应、更低的细胞毒性，以及更高的内部控制基因编辑。这种粒子的主要优点是，它可用于基本基因的表征，以及肿瘤存活基因。破坏DD-Cas9的稳定与其表达无关，这允许在同一促进者下共同表达感兴趣的基因。这种方法可以很容易地应用于体内和体外研究。盾牌-1也可以穿透血脑屏障，这使得研究参与大脑发育的基因也变得容易。均匀地播种健康的 HEK293T 细胞，密度为每 10 厘米组织培养板 10 到 200 万细胞，使用 10 毫升葡萄糖 DMEM 和 10% 过滤 FBS。在组织培养孵化器中将细胞在5%的二氧化碳和37摄氏度下孵化20小时。第二天，确认细胞通过亮场显微镜达到70%的汇合度，并且它们均匀地分散在板块中。在转染前一小时更换介质，最终体积为 10 毫升。准备两个管与500微升的温暖介质。加入25微升转染试剂管一，并在室温下孵育5分钟。在另一根管子上，加入质粒的混合物。将一管一与管二混合，形成转染混合物，并在室温下孵育20分钟。将转染混合物滴入 HEK293T 细胞，并在组织培养培养器中孵化板。18 小时后，小心地将介质换成 10 毫升新鲜 DMEM，并配以 10% FBS 以去除转染试剂。在接下来的48小时内孵育细胞。48 小时后，用 10 毫升注射器收集超高纳坦，并通过 0.45 微米过滤器将其传递。阿利奎特病毒超高，并储存在零下80摄氏度。为了确定病毒滴答机，将每口井的500万个HED293T细胞种子成两个六井板。在37摄氏度和5%的二氧化碳照时将两个板块孵化过夜，达到50%至60%的汇流。第二天，使用六井板块中的一个来计算六口井中的细胞。解冻用于执行序列稀释的病毒，每毫升聚乙烯试剂添加 8 微克，用 10% FBS 制备 DMEM 以连续稀释病毒，并添加聚乙烯试剂。在含有介质的聚乙烯中，准备 0.1 到 0.0001 的 10 倍系列稀释的两毫升。从六井板中取出介质，在井中加入一毫升病毒稀释剂，留下一口井，单独将介质作为负控制，一口井与 100% 病毒一起。孵育细胞24小时。第二天，从六井板中取出带有病毒的介质，并将其换成两毫升的新鲜 DMEM，并配以 10% FBS。孵育细胞48至72小时，每天用荧光显微镜观察GFP。48 至 72 小时后，分离细胞，并在最大缓冲区重新悬浮。使用流细胞计确定 GFP 表达的百分比，并使用文本手稿中给出的公式计算病毒滴答声。将细胞系在10厘米的板中镀上，在一夜之间孵育，达到50%至60%的汇合，然后在10毫升的培养介质中感染500至2000微升病毒颗粒的细胞。孵育病毒介质24小时。第二天，将病毒介质更改为培养介质，并使用流细胞学确定 GFP 阳性细胞的百分比。使用 BD FACSARIA II 进行细胞分拣，选择 GFP 阳性细胞。扩大积极选择的细胞并冻结库存。在感染后24小时内，将积极选择的细胞和未转化的细胞分别镀在12井板中。等到细胞连接并改变介质到含有200纳米摩尔盾-1的细胞培养介质。用转导和未转化的细胞替换板中的介质，每个板材留下两口井，并作为负控。在将盾-1加入井中后，在零、二、六、十二、二十四、四十八和七十二小时内从每口井中孵育和提取蛋白质。然后从其他油井中取出带有 Shield-1 的介质，并将其更改为常规细胞介质。在改变介质后2小时、6小时和12小时从这些井中收集蛋白质。Cas9蛋白的诱导被证实其表达水平在转导细胞和车辆之间相似，尽管存在或不存在盾-1。使用 Shield-1 治疗两小时后，在转导的 A549 细胞系中充分感应了 Cas9 表达，在盾牌-1 退出后 6 到 12 小时内，该表达可以忽略不计的。Shield-1 治疗的转导 A549 细胞在 48 小时后数量减少，而不影响 Rinella 样本，该样本通过使用抗体对抗耗尽的 RPA3 蛋白进行免疫膨胀验证。基因编辑反转或内德尔突变通过测量师核酸酶检测得到确认。所设计的伦蒂病毒载体结构是一个双生体系统，在同一EF-1a促进器下具有另一个感兴趣的基因。一种改良的荧光蛋白P2A被添加到追踪受感染的细胞中。在车辆和 A549 转导细胞中观察到 mVenus 蛋白质的表达，独立于 DD-Cas9 表达和盾牌-1 治疗。使用 DD-Cas9 矢量有效导电细胞线是限制速率的步骤之一，因为具有健康的 HEK293T 细胞并使用其低通量是至关重要的。DD-Cas9 质粒、包装质粒和包络质粒之间的比例优化对于最佳转化非常重要。但除此之外，稳定DD-Cas9目标细胞所需的盾-1数量也是您需要小心的事情。DD-Cas9 与 Cre 质粒也可用，它可用于研究基于 Cre-lox 系统的活体研究中的遗传相互作用。

Research

JoVE Journal

Genetics

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

科研

遗传学

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

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. ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

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 ...