Name: using a fluorescent pcr-capillary gel electrophoresis technique to genotype crispr/cas9-mediated knockout mutants in a high-throughput format
Uploaded: 2017-04-08T14:00:00
Description: Watch this Scientific Journal Video about Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format at JoVE.com

笔者想感谢谭氏闵女士，海伦·Ong女士和越·Yi博士与毛细管电泳实验帮助。这项工作是由NMRC / IRG授予NMRC /二千零十一分之一千三百一十四和教育部ACRF二级基金赠款MOE2011-T2-1-051支持。

1.获取CRISPR / Cas9靶向单细胞克隆<ol><li>在6孔板种子HepG2细胞以每孔500,000个细胞在2mL不含抗生素的Dulbecco补充有10％胎牛血清（FBS）改良的Eagle培养基（DMEM）的。孵育在37℃下24小时和5％CO 2。 </li><li>转染细胞用质粒共表达使用适当的转染试剂，根据制造商的说明书和Cas9针对目标基因特异性因组。 注意：例如，因组可以克隆到pSpCas9（BB）-2A-GFP载体，如先前4所述。 </li...

<ol>
	<li>Chang, 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=CRISPR/cas9,+a+novel+genomic+tool+to+knock+down+microRNA+in+vitro+and+in+vivo.">CRISPR/cas9, a novel genomic tool to knock down microRNA in vitro and in vivo.</a> Sci. Rep. 6, 22312 (2016).</li><li>O'Connell, M. 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=Programmable+RNA+recognition+and+cleavage+by+CRISPR/Cas9.">Programmable RNA recognition and cleavage by CRISPR/Cas9.</a> Nature. 516, 263-266 (2014).</li><li>Ran, F. 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=Double+nicking+by+RNA-guided+CRISPR+Cas9+for+enhanced+genome+editing+specificity.">Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.</a> Cell. 154, 1380-1389 (2013).</li><li>Ran, F. 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=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nat. Protoc. 8 (11), 2281-2308 (2013).</li><li>Perez, E. 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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=Knockout+mice+created+by+TALEN-mediated+gene+targeting.">Knockout mice created by TALEN-mediated gene targeting.</a> Nat. Biotechnol. 31, 23-24 (2013).</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+knockout+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knockout screening in human cells.</a> Science. 343, 84-87 (2014).</li><li>Zhou, 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=High-throughput+screening+of+a+CRISPR/Cas9+library+for+functional+genomics+in+human+cells.">High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells.</a> Nature. 509, 487-491 (2014).</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 Mol. Biol. 649, 247-256 (2010).</li><li>Kim, H. J., Lee, H. J., Kim, H., Cho, S. W., Kim, J. 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+genome+editing+in+human+cells+with+zinc+finger+nucleases+constructed+via+modular+assembly.">Targeted genome editing in human cells with zinc finger nucleases constructed via modular assembly.</a> Genome Res. 19, 1279-1288 (2009).</li><li>Ramlee, M. K., Yan, T., Cheung, A. M., Chuah, C. T., Li, S. <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+genotyping+of+CRISPR/Cas9-mediated+mutants+using+fluorescent+PCR-capillary+gel+electrophoresis.">High-throughput genotyping of CRISPR/Cas9-mediated mutants using fluorescent PCR-capillary gel electrophoresis.</a> Sci. Rep. 5, 15587 (2015).</li><li>Liu, C. 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=Distinct+Responses+of+Stem+Cells+to+Telomere+Uncapping-A+Potential+Strategy+to+Improve+the+Safety+of+Cell+Therapy.">Distinct Responses of Stem Cells to Telomere Uncapping-A Potential Strategy to Improve the Safety of Cell Therapy.</a> Stem cells. 34, 2471-2484 (2016).</li><li>Doench, J. 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=Rational+design+of+highly+active+sgRNAs+for+CRISPR-Cas9-mediated+gene+inactivation.">Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation.</a> Nat. Biotechnol. 32, 1262-1267 (2014).</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=Identification+and+characterization+of+essential+genes+in+the+human+genome.">Identification and characterization of essential genes in the human genome.</a> Science. 350, 1096-1101 (2015).</li><li>Sanjana, N. E., Shalem, O., 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=Improved+vectors+and+genome-wide+libraries+for+CRISPR+screening.">Improved vectors and genome-wide libraries for CRISPR screening.</a> Nat. Methods. 11, 783-784 (2014).</li><li>Urnov, F. 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=Highly+efficient+endogenous+human+gene+correction+using+designed+zinc-finger+nucleases.">Highly efficient endogenous human gene correction using designed zinc-finger nucleases.</a> Nature. 435, 646-651 (2005).</li><li>Kim, H., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+genome+engineering+with+programmable+nucleases.">A guide to genome engineering with programmable nucleases.</a> Nat. Rev. Genet. 15, 321-334 (2014).</li><li>Dahlem, T. 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=Simple+methods+for+generating+and+detecting+locus-specific+mutations+induced+with+TALENs+in+the+zebrafish+genome.">Simple methods for generating and detecting locus-specific mutations induced with TALENs in the zebrafish genome.</a> PLoS Genet. 8, e1002861 (2012).</li><li>Thomas, H. R., Percival, S. M., Yoder, B. K., Parant, J. 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+genome+editing+and+phenotyping+facilitated+by+high+resolution+melting+curve+analysis.">High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.</a> PLoS One. 9, 114632 (2014).</li><li>Kim, J. M., Kim, D., Kim, S., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+with+CRISPR-Cas-derived+RNA-guided+endonucleases.">Genotyping with CRISPR-Cas-derived RNA-guided endonucleases.</a> Nat. Commun. 5, 3157 (2014).</li></ol>

中选择的模型细胞系敲除特定基因已成为阐明的作用，该基因在特定的细胞环境起着程序。事实上，一些全基因组的屏幕是目前可使用的CRISPR / Cas9系统基因组中的14，15，16靶向几乎所有已知的人类基因。随着这些大型屏幕（甚至小规模的利益个体基因靶向），它设计并利用sgRNAs针对同一基因的不同位点是非常重要的。整个使?...

反向遗传方法已使科学家能够阐明对细胞或整个生物体的基因组中特异性改变的影响。例如，特定基因的表达可通过基因敲减1，2（部分还原）或基因敲除3，4（完全消融），以便确定这对细胞的或对功能的影响减弱有机体的发展。 基因敲除实验已经成为自从引入序列特异性核酸酶的可编程，如锌指核酸酶（ZFN）和转录激活样效应核酸酶（TALEN的）的更容易。然而，比较近的聚集经常穿插短回文重复（CRISPR）表征/ Cas9系统使它非常容易为世界各地的任何实验室进行根基因敲除实验。在本质上，CRISPR / Cas9系统由两个基本组分-单个引导RNA（因组），其识别与通过碱基互补结合到基因组中的特定序列，并称为Cas9核酸内切酶。基因组DNA上的因组-Cas9复合物的特异性结合和行动的后果是DNA的双链切割。这，反过来，触发了细胞，这是通过非同源末端连接（NHEJ）或同源重组（HR）途径随后修复DNA损伤反应的机制。由于NHEJ修复机制（但不是HR机制）通常导致随机插入或缺失核苷酸在修复位点，从而导致插入/缺失（INDEL）突变，它可能会导致外显子的阅读框移位。然后这可能导致在该基因的敲除由于翻译和无义介导衰变5,6的过早终止，7。 尽管通过引入CRISPR / Cas9系统中敲除基因的所提供的便利，有针对性的细胞的克隆的基因分型仍然是一个瓶颈，尤其是在高通量设定8,9。现有技术或者遭受重大固有的局限性或在经济上昂贵。例如，调查或T7E1测定，这是一种酶测定法，其检测DNA的错配双链体10，是不能够野生型克隆，纯合突变体之间进行区分（克隆其等位基因相同的突变），因为这些克隆具有相同的等位基因，因此不会在其DNA序列11存在的错配。此外，采用Sanger测序，这被认为在基因分型突变体克隆，在高通量设置的金标准是不希望的，由于其成本较高。在这里，我们提出了一个DET荧光PCR-毛细管凝胶电泳技术，可以绕过的其他现有基因分型技术的局限性和是在执行核酸酶介导基因敲除克隆的高通量筛选中特别有用的ailed协议。这种方法在技术上简单执行并节省了时间和成本。

<table border="0" cellpadding="0" cellspacing="0" width="1548">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:401px;">HEPG2 cells</td>
			<td style="width:196px;">ATCC</td>
			<td style="width:135px;">HB-8065</td>
			<td style="width:816px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Dulbecco&#39;s Modified Eagles Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>SH30022.01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>SV30160.03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pSpCas9(BB)-2A-GFP plasmid</td>
			<td>Addgene</td>
			<td>PX458</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.5%), no phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HyClone Water, Molecular Biology Grade</td>
			<td>GE Healthcare</td>
			<td>SH30538.02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CRISPR sgRNA insert oligonucleotide (sense)</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CACCGCTAACCTTTCAGCCTGCCTA-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CRISPR sgRNA insert oligonucleotide (anti-sense)</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-AAACTAGGCAGGCTGAAAGGTTAGC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unlabeled PCR amplification forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CACTAACTCCAATGCTTCAGTTTC-3&#39;; this primer is also used to sequence PCR amplified alleles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-FAM-labeled fluorescent PCR forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-6-FAM-CACTAACTCCAATGCTTCAGTTTC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEX-labeled fluorescent PCR forward primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-HEX-CACTAACTCCAATGCTTCAGTTTC-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Unlabeled PCR reverse primer</td>
			<td>AITbiotech</td>
			<td>None</td>
			<td>Sequence: 5&#39;-CCTCTTCCAAGTCTGCTTATGT-3&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Taq PCR Core Kit</td>
			<td>QIAGEN</td>
			<td>201223</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hi-Di Formamide</td>
			<td>Thermo Fisher Scientific</td>
			<td>4311320</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GeneScan 500 LIZ Dye Size Standard</td>
			<td>Thermo Fisher Scientific</td>
			<td>4322682</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Optical 96-Well Reaction Plate</td>
			<td>Thermo Fisher Scientific</td>
			<td>4306737</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3500xL Genetic Analyzer</td>
			<td>Thermo Fisher Scientific</td>
			<td>4405633</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3500 Series 2 program</td>
			<td>Thermo Fisher Scientific</td>
			<td>4476988</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gene Mapper 5 program</td>
			<td>Thermo Fisher Scientific</td>
			<td>4475073</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gentra Puregene Cell Kit</td>
			<td>QIAGEN</td>
			<td>1045696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Wizard SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NAP1L1 Antibody (N-term)</td>
			<td>Abgent</td>
			<td>AP1920b</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclear Matrix Protein p84 antibody [5E10]</td>
			<td>GeneTex</td>
			<td>GTX70220</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase AffiniPure Goat Anti-Rabbit IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-035-144</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase AffiniPure Sheep Anti-Mouse IgG</td>
			<td>Jackson ImmunoResearch</td>
			<td>515-035-003</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a fluorescent pcr-capillary gel electrophoresis technique to genotype crispr/cas9-mediated knockout mutants in a high-throughput format

可编程基因组编辑工具的发展促进了利用反向遗传学的理解角色的特定基因组序列在细胞和整个生物体的功能发挥。该事业由近期出台的CRISPR的/ Cas9系统一个多功能的工具，使研究人员能够以操纵基因组和转录组除其他事项外，敲，敲下来，或有针对性的基因敲了极大的帮助方式。用于敲除基因的目的，CRISPR / Cas9介导的双链断裂招收非同源末端连接的DNA修复途径在断裂位点上引入核苷酸的移码 - 导致插入或缺失。然而，一个单独的导向RNA可以导致不期望的脱靶效应，并排除这些出来，使用多个引导RNA的是必要的。的目标这种多重性也意味着克隆的高容量筛选是必需的，这又引出了一个使用的EF的够的高通量技术基因型的敲除克隆。目前的基因分型技术无论是从内在的局限性遭受或招致成本高，因此使得它们不适合高通量的目的。在这里，我们详细协议用于使用荧光PCR，其使用基因组DNA从粗细胞裂解物作为模板，然后通过解析毛细管凝胶电泳的PCR片段。这种技术是不够准确区分片段之间的一个碱基对差异，因此是在指示移码的在目标基因的编码序列的存在或缺乏足够的。这种精确的知识有效地排除了一个确定的顺序步骤的需求，并允许用户以节省时间和成本的过程。此外，该技术已被证明是通过基因分型对许多基因的RNA指导有针对性的各种组织器官的各种哺乳动物细胞多才多艺，因为这里和其他地方所示。

这里描述的荧光PCR-毛细管凝胶电泳技术被预期在几乎任何细胞系是适于外源DNA递送到适用于基因组中的任何靶向区域。之前我们已经通过在结肠癌细胞株12靶向三个基因显示出它的应用。在这里，我们将展示其基因分型肝癌细胞系HepG2，针对性与有效性CRISPR / Cas9构建针对核小体装配蛋白1样1（NAP1L1）基因。事实上，我们已经成功地利用荧光PCR-毛细管凝胶?...

Watch this Scientific Journal Video about Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format at JoVE.com

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

这里所描述的基因分型技术，荧光聚合酶链式反应（PCR），以毛细管凝胶电泳，其耦合，允许核酸酶介导敲除克隆的高通量基因分型。它规避所面临的其他基因分型技术的限制，并且比测序方法更具成本效益。

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 ...

Research

JoVE Journal

Genetics

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

肠易激综合征的循环miRNA的扰动检测使用复用高通量基因表达平台

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

科研

遗传学

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

High-Throughput Robotically Assisted Isolation of Temperature-sensitive Lethal Mutants in Chlamydomonas reinhardtii

温度敏感致死突变体的高通量机器人辅助隔离<em&gt;莱茵衣藻</em

Systematic identification and characterization of genetic perturbations have proven useful to decipher gene function and cellular pathways. However, the ...

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 ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

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 ...

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 ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

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 ...