Name: selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells
Uploaded: 2017-06-16T15:00:00.000+00:00
Duration: 11 min 35 s
Description: Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

作者要感谢Gissell Sanchez，Megan Lee和Jason Estep的实验帮助，Weifeng Gu和Xuemei Chen分享试剂。

1.找出需要删除的同源区域注意:仅在使用基于选择的编辑时才需要。 <ol><li>在所需的缺失位点的任一侧选择两个最初为1.5-2 kb的区域，这将作为HDR模板中的同源臂（ 图1A ）。确定缺乏任何一条（GGTCTC）上的BsaI识别位点以促进克隆的区域。如果BsaI位点是不可避免的，则使用替代型IIs限制酶（BsmBI，SapI，BbsI）并修饰受体质粒（pUC19-BsaI），抗性标记供体质粒（pGolden-Neo或pGolden-Hygro）中的相应位点，同源臂PCR产物突出。 </li></ol> 2.产生Cas9-sgRNA表达质粒<ol><li>定义诱变的靶向位点。对于单切，无选择方法，目标5&#39;近端编码外显子增加非功能的概率功能突变对于双切法，选择两个跨越基因必需的位点。 注意:根据我们的经验，有效地创建高达53 kb的删除。 </li><li>输入200到300 bp的目标区域序列到crispr.mit.edu设计工具。对于基于选择的克隆，使用近似于缺失位点的200-300bp的鉴定的同源区（ 图1A ）。选择每个目标区域最高排名的sgRNA的2-3个，以解释其活动的差异，并分离不具有相同潜在的脱靶效应的独立克隆。 </li><li>为了设计具有适当突出端的用于插入表达质粒的双链体寡核苷酸，省略末端PAM序列（NGG），并将5-CACC突出端附着，然后附加G至顶链寡核苷酸。对于底部链寡核苷酸，将5-AAAC突出端附加到反向互补的靶序列，随后是3&#39;-C，如下面介绍: <img alt="序列1" src="/files/ftp_upload/55903/55903seq01.jpg" /></li><li>使用Zhang实验室方案29所述的金门克隆28 ，将对应于sgRNA的合成寡核苷酸插入pSpCas9（BB）质粒。 </li></ol>同源性修复模板质粒的生成注意:仅在使用基于选择的编辑时才需要。同源性修复模板质粒由两个与两个sgRNA靶位点之外的基因组互补的两个区域的耐药盒组成（ 图1B ）。 <ol><li>从步骤1.1中鉴定的更广泛的同源区域，选择距设计的Cas9切割位点不超过5-10bp的800-1,000bp 26 ，用作同源臂（ 图1A ）。确保不包括sgRNA目标位点及其PAM在同源臂中，这将导致CRISPR / Cas9切割模板质粒本身。如果同源性武器含有内部BsaI位点，则使用其他sgRNA位点。 </li><li> PCR使用高保真DNA聚合酶根据制造商的说明书使用具有引入BsaI位点（GGTCTC）的另外的5&#39;序列的正向引物和反向引物以及同源区两端的独特突出端扩增基因组DNA的同源性臂。突出部分必须包含适当的序列以产生正确的装配顺序和取向，如下所述（ 图1B ）: 左同源臂FP突出:5&#39;GGGTCTCA GGCC 左同源臂RP突出:5&#39;GGGTCTCT CACG 右侧同源臂FP突出:5&#39;GGGTCTCA GTCC 右侧同源臂RP突出:5&#39;GGGTCTCT CCAC </li><li>检查同源性臂适当的扩增在进行前进行凝胶电泳。 </li><li> 通过金门克隆28将具有电阻盒的右侧和左侧同源臂区域组装成HDR模板质粒。使用pGolden-Neo或pGolden-Hygro质粒30作为loxP-侧翼抗性盒（loxP-PGK-Neo-pA-loxP或loxP-PGK-Hyg-pA-loxP）的来源。使用pUC19-BsaI，在多克隆区域中具有BsaI位点的修饰的pUC19，并将其他位置的BsaI位点作为受体载体（可根据要求提供）消除。对于三个插入和矢量，使用1:1:1:1的比例。 <ol><li>准备以下反应混合物30 : <table><tbody><tr><td>右侧同源臂PCR产物</td><td> 0.06 pmol（30-40ng） </td></tr><tr><td>左同源臂PCR产物</td><td> 0.06 pmol（30-40ng） </td></tr><tr><td> pGolden-Neo质粒（100ng /μL） </td><td> 1＆＃181; l </td></tr><tr><td> pUC19-BsaI质粒（100ng /μL） </td><td> 1μL </td></tr><tr><td> 2x T7 DNA连接酶缓冲液</td><td> 5μL </td></tr><tr><td> BsaI（10 U /μL） </td><td> 0.75μL </td></tr><tr><td> T7 DNA连接酶（3000 U /μL） </td><td> 0.25μL </td></tr><tr><td>水</td><td>最多10μL </td></tr></tbody></table></li><li>使用以下热循环仪参数:37℃5分钟，20℃5分钟。重复30个周期。 </li><li>用制造商的说明书用外切核酸酶处理克隆产物，消化任何剩余的线性化DNA。 </li><li>根据提供给细胞的方案将反应混合物转化为感受态大肠杆菌菌株，并在含有氨苄青霉素的培养皿上培养。 </li></ol></li><li> 要使用正确的模板组合识别克隆，请执行细菌扩增组装的插入物的同源臂亚区域（因为整个插入物可能太大而不能可靠地扩增）。使用一个引物退火到侧翼质粒序列，另一个引物与抗性盒的边缘互补（ 图1B ，序列对Neo和Hygro插入物都是共同的），产生大约200bp的装配依赖性产物包含的同源臂: pUC19-Bsal-Left:5&#39;GGCTCGTATGTTGTGTGGAATTGTGAG 电阻左:5&#39;AAAAGCGCCTCCCCTACCC pUC19-BsaI-Right:5&#39;GCTATTACGCCAGCTGGCGAAA 抵抗权:5&#39;AAGACAATAGCAGGCATGCTGGG <ol><li>用无菌牙签挑取细菌菌落，并将细菌悬浮在20μL水中。在95℃下加热15分钟，并在桌面离心机中以最大速度旋转5分钟。立即放在冰上。 </li><li>准备以下PCR混合物31/ SUP&gt;: <table><tbody><tr><td>稀释菌落模板</td><td> 1.25μL </td></tr><tr><td> 10x Taq反应缓冲液</td><td> 1.25μL </td></tr><tr><td> 20mM dNTPs </td><td> 0.25μL </td></tr><tr><td> 10μM正向引物</td><td> 0.25μL </td></tr><tr><td> 10μM反向引物</td><td> 0.25μL </td></tr><tr><td> Taq聚合酶</td><td> 0.25μL </td></tr><tr><td>水</td><td> 9μL </td></tr></tbody></table></li><li>使用以下热循环仪参数:95℃30秒，（95℃30秒，61℃30秒，72℃1分钟/ kb），重复25个循环，72℃2分钟。 </li><li>通过琼脂糖凝胶电泳检查PCR产物的正确扩增子大小。 </li><li>任选地，来自具有正确插入片段大小和顺序的单个菌落的微量制备质粒DNA通过Sanger测序与上述扩增引物的同源臂区。 </li></ol></li></ol> 4.将CRISPR成分转染到培养细胞中<ol><li>转染前:在37℃和5％CO 2下补充有10％FBS的DMEM培养基中培养T-REx293细胞。 </li><li>将细胞板放置在6孔板上并生长至约70％汇合。包括一个未经转染的控件的井。 </li><li> 使用商业转染方案用2.5μg总质粒转染细胞。同时保持未转染的控制。 注意:转染方法和效率因细胞类型而异。在实验前确定系统的适当转染方法。 <ol><li>对于用选择的细胞进行转染，使用0.75μgCas9-sgRNA1（左切），0.75μgCas9-sgRNA2（右切）和1.0μg同源重组模板。 </li><li>用于转染在没有选择的细胞上，使用2.5μgCas9-sgRNA质粒。 </li></ol></li></ol> 5.药物选择<ol><li>转染后48 h，用合适的药物（新霉素/潮霉素）处理细胞。进行选择，直到未转染对照的所有细胞死亡（通常为Neo为3-5天，Hygro为7-14天）。 注意:对于新霉素和潮霉素，分别使用浓度为500μg/ mL和10μg/ mL的T-REx293细胞。对于其他细胞类型，可能有用的是在未转染的细胞上进行药物的先前滴定以确定有效浓度。 </li></ol> 6.克隆种群的分离<ol><li>选择后，在原始细胞中生长细胞至100％融合。将种子细胞以0.33个细胞/孔的密度进入96孔平板。 注意:播种三个96孔板是一个很好的起点，以确保分离多于一个正确的克隆，但是或多或少可能需要dep结束于sgRNA和HDR效率。 </li><li>在2-4周的时间内观察菌落，直到菌落对眼睛是可见的。用无菌移液器吸头挑取可见的菌落，并在新的孔中重新种植以促进单层生长。当使用悬浮细胞时，甚至可以使用更低的接种密度来确保更大比例的单菌落孔。 </li></ol> 7.候选人筛选<ol><li> 筛选候选人不使用选择:斑点印迹 <ol><li>将个体克隆培养至50-100％融合。通过在孔内移液来移走单层细胞。 </li><li>将100μL总体积的90μL等分成干净的微量离心管。以6000rpm离心5分钟，除去培养基，并在10μL1x Lysis Buffer或1x SDS加载缓冲液（5x:250mM Tris-Cl pH 6.8,8％SDS，0.1％溴酚蓝，40％甘油，100mM DTT）。在其余的细胞中加入90μL新的培养基我继续传播。 </li><li>将1μL细胞裂解液移至干燥的硝酸纤维素膜上，形成一个点。在两个单独的膜上将每个样品两次打印，产生两个相同的样品样式。 </li><li>在室温下将TBST（Tris缓冲盐水+ 0.01％吐温20:8g NaCl，0.2g KCl，3g Tris碱，至多1L蒸馏水，pH 7.4） 31中的5％乳块中的膜封闭1小时摇摆，这里和整个程序）。 </li><li>在一个膜上使用针对靶蛋白的一抗进行印迹，另一个使用对照蛋白（微管蛋白，GAPDH或任何其他预期不会改变的蛋白质）的一抗。在TBST + 5％牛奶中使用推荐的一级抗体稀释度（对于ELAVL1为0.2μg/ mL，在这种情况下为Pum2为0.1μg/ mL）。在室温下孵育1小时。 </li><li>用TBST洗涤3次5分钟。 </li><li>在T中用合适的HRP缀合的二抗孵育每个膜BST + 5％牛奶在室温下1小时。 </li><li>用TBST洗涤3次5分钟。 </li><li>根据制造商的说明书应用化学发光底物溶液（参见材料表），并将数字化学发光成像仪上的印迹膜成像。 </li><li>使用适当的软件量化靶和对照蛋白的点强度。 </li><li>消除具有低控制蛋白信号的候选者，并计算目标的背景扣除比例以控制剩余候选物的蛋白质强度。选择最低通过率的候选物，并通过蛋白质印迹进一步验证。 </li></ol></li><li> 筛选候选人使用选择:菌落PCR <ol><li>使用DNA准备试剂盒收集细胞裂解物（参见材料表 ）。 <ol><li>在新的96孔中重复个体菌落，生长至100％融合。 </li><li>从一组克隆中删除介质，a并从试剂盒中将细胞重悬于30μL提取缓冲液中。转移到干净的1.5 mL微量离心管中。 </li><li>将溶液加热至96°C 15 min，冷却至室温。 </li><li>从试剂盒中加入30μL稳定缓冲液。混合好</li></ol></li><li>通过菌落PCR鉴定野生型和单等位基因/双等位基因/双等位突变体系。 <ol><li>设计两组单独的PCR引物（针对目标基因座的左侧和右侧），以基于电阻盒的成功或不成功的整合扩增同源臂周围的区域（ 图2C ）。在每一侧，使用在同源臂外退火的通用底漆（红色）。将其与与内源序列互补的相应配对引物（跨越同源区域（蓝色））进行测试，以测试野生型等位基因。使用与插入的电阻盒互补的另一配对引物（参见部分引物）2.3），以测试所需的突变。 </li><li> （推荐）使用原始选择的大块细胞群体的细胞裂解物验证引物组，因为它将含有WT和突变体等位基因的混合物（ 图2D ）。 </li><li>准备以下反应混合物: <table><tbody><tr><td>细胞裂解物</td><td> 0.5μL </td></tr><tr><td> 10倍KOD缓冲液</td><td> 1.25μL </td></tr><tr><td> 25mM MgSO 4 </td><td> 0.75μL </td></tr><tr><td> 2 mM dNTPs </td><td> 1.25μL </td></tr><tr><td> 10μM正向引物</td><td> 0.375μL </td></tr><tr><td> 10μM反向引物</td><td> 0.375μL </td></tr><tr><td> KOD聚合酶</td><td> 0.25μL </td></tr><tr><td>水</td><td> 7.75μL </td></tr></tbody></table></li><li>使用以下热循环仪条件:95℃2分钟，（95℃20s，Primer Tm 10s，70℃20s / kb）重复25个循环。 </li><li>通过琼脂糖凝胶电泳可视化PCR产物。 </li></ol></li><li>对预测整合的每一侧重复菌落PCR检测。选择并扩展显示存在整合的候选者，而不删除被删除区域的内源序列产物。 </li><li>通过蛋白质印迹和/或RT-qPCR验证阳性候选物。 </li></ol></li></ol> 8.通过测序验证基因组突变<ol><li>通过苯酚氯仿提取分离突变体细胞系的基因组DNA 31 。 </li><li> PCR扩增sgRNA靶区，使用5&#39;末端的引物，每端加入BsaI限制性位点。 注意:对于使用HDR编辑的克隆，其中两个等位基因可能相同，可以直接对PCR产物进行测序。 </li><li>通过金门克隆将扩增区克隆到pUC19-BsaI中。 </li><li>将反应混合物转化为感受态大肠杆菌菌株。 </li><li>来自6-10个单独菌落的Miniprep质粒DNA，并通过Sanger测序序列克隆的区域。 </li></ol>

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	<li>Carroll, D. <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+with+zinc-finger+nucleases.">Genome engineering with zinc-finger nucleases.</a> Genetics. 188 (4), 773-782 (2011).</li><li>Kim, Y. G., Cha, J., Chandrasegaran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+restriction+enzymes:+zinc+finger+fusions+to+Fok+I+cleavage+domain.">Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain.</a> Proc Natl Acad Sci U S A. 93 (3), 1156-1160 (1996).</li><li>Bibikova, 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=Stimulation+of+homologous+recombination+through+targeted+cleavage+by+chimeric+nucleases.">Stimulation of homologous recombination through targeted cleavage by chimeric nucleases.</a> Mol Cell Biol. 21 (1), 289-297 (2001).</li><li>Bibikova, M., Golic, M., Golic, K. G., Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+chromosomal+cleavage+and+mutagenesis+in+Drosophila+using+zinc-finger+nucleases.">Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases.</a> Genetics. 161 (3), 1169-1175 (2002).</li><li>Porteus, M. H., Cathomen, T., Weitzman, M. D., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+mediated+by+adeno-associated+virus+and+DNA+double-strand+breaks.">Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks.</a> Mol Cell Biol. 23 (10), 3558-3565 (2003).</li><li>Bogdanove, A. J., Voytas, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAL+effectors:+customizable+proteins+for+DNA+targeting.">TAL effectors: customizable proteins for DNA targeting.</a> Science. 333 (6051), 1843-1846 (2011).</li><li>Miller, J. 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=A+TALE+nuclease+architecture+for+efficient+genome+editing.">A TALE nuclease architecture for efficient genome editing.</a> Nat Biotechnol. 29 (2), 143-148 (2011).</li><li>Moscou, M. J., Bogdanove, A. 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+simple+cipher+governs+DNA+recognition+by+TAL+effectors.">A simple cipher governs DNA recognition by TAL effectors.</a> Science. 326 (5959), 1501 (2009).</li><li>Christian, 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=Targeting+DNA+double-strand+breaks+with+TAL+effector+nucleases.">Targeting DNA double-strand breaks with TAL effector nucleases.</a> Genetics. 186 (2), 757-761 (2010).</li><li>Jiang, W., Marraffini, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas:+New+Tools+for+Genetic+Manipulations+from+Bacterial+Immunity+Systems.">CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.</a> Annu Rev Microbiol. 69, 209-228 (2015).</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=CRISPR+provides+acquired+resistance+against+viruses+in+prokaryotes.">CRISPR provides acquired resistance against viruses in prokaryotes.</a> Science. 315 (5819), 1709-1712 (2007).</li><li>Karginov, F. V., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CRISPR+system:+small+RNA-guided+defense+in+bacteria+and+archaea.">The CRISPR system: small RNA-guided defense in bacteria and archaea.</a> Mol Cell. 37 (1), 7-19 (2010).</li><li>Wiedenheft, B., Sternberg, S. H., Doudna, J. A. <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+genetic+silencing+systems+in+bacteria+and+archaea.">RNA-guided genetic silencing systems in bacteria and archaea.</a> Nature. 482 (7385), 331-338 (2012).</li><li>Mojica, F. J., Diez-Villasenor, C., Garcia-Martinez, J., Soria, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervening+sequences+of+regularly+spaced+prokaryotic+repeats+derive+from+foreign+genetic+elements.">Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.</a> J Mol Evol. 60 (2), 174-182 (2005).</li><li>Pourcel, C., Salvignol, G., Vergnaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+elements+in+Yersinia+pestis+acquire+new+repeats+by+preferential+uptake+of+bacteriophage+DNA,+and+provide+additional+tools+for+evolutionary+studies.">CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies.</a> Microbiology. 151 (Pt 3), 653-663 (2005).</li><li>Brouns, S. 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=Small+CRISPR+RNAs+guide+antiviral+defense+in+prokaryotes.">Small CRISPR RNAs guide antiviral defense in prokaryotes.</a> Science. 321 (5891), 960-964 (2008).</li><li>Deltcheva, 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=CRISPR+RNA+maturation+by+trans-encoded+small+RNA+and+host+factor+RNase+III.">CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.</a> Nature. 471 (7340), 602-607 (2011).</li><li>Garneau, J. 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=The+CRISPR/Cas+bacterial+immune+system+cleaves+bacteriophage+and+plasmid+DNA.">The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA.</a> Nature. 468 (7320), 67-71 (2010).</li><li>Marraffini, L. A., Sontheimer, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+interference+limits+horizontal+gene+transfer+in+staphylococci+by+targeting+DNA.">CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.</a> Science. 322 (5909), 1843-1845 (2008).</li><li>Gasiunas, G., Barrangou, R., Horvath, P., Siksnys, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9-crRNA+ribonucleoprotein+complex+mediates+specific+DNA+cleavage+for+adaptive+immunity+in+bacteria.">Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.</a> Proc Natl Acad Sci U S A. 109 (39), E2579-E2586 (2012).</li><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 (6096), 816-821 (2012).</li><li>Davis, A. J., Chen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+double+strand+break+repair+via+non-homologous+end-joining.">DNA double strand break repair via non-homologous end-joining.</a> Transl Cancer Res. 2 (3), 130-143 (2013).</li><li>Guirouilh-Barbat, 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=Impact+of+the+KU80+pathway+on+NHEJ-induced+genome+rearrangements+in+mammalian+cells.">Impact of the KU80 pathway on NHEJ-induced genome rearrangements in mammalian cells.</a> Mol Cell. 14 (5), 611-623 (2004).</li><li>Moore, J. K., Haber, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+and+genetic+requirements+of+two+pathways+of+nonhomologous+end-joining+repair+of+double-strand+breaks+in+Saccharomyces+cerevisiae.">Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae.</a> Mol Cell Biol. 16 (5), 2164-2173 (1996).</li><li>Liang, F., Han, M., Romanienko, P. J., Jasin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homology-directed+repair+is+a+major+double-strand+break+repair+pathway+in+mammalian+cells.">Homology-directed repair is a major double-strand break repair pathway in mammalian cells.</a> Proc Natl Acad Sci U S A. 95 (9), 5172-5177 (1998).</li><li>Gratz, S. 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=Highly+specific+and+efficient+CRISPR/Cas9-catalyzed+homology-directed+repair+in+Drosophila.">Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila.</a> Genetics. 196 (4), 961-971 (2014).</li><li>Cong, 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=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Engler, C., Kandzia, R., Marillonnet, 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+one+pot,+one+step,+precision+cloning+method+with+high+throughput+capability.">A one pot, one step, precision cloning method with high throughput capability.</a> PLoS One. 3 (11), e3647 (2008).</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>Luo, Y., Lin, L., Bolund, L., Sorensen, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+construction+of+rAAV-based+gene+targeting+vectors+by+Golden+Gate+cloning.">Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.</a> Biotechniques. 56 (5), 263-268 (2014).</li><li>Green, M. R., Sambrook, J., Sambrook, 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> Molecular cloning: a laboratory manual. , (2012).</li><li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR/Cas9+delivery.">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> Elife. 3, e04766 (2014).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., 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=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Res. 24 (6), 1012-1019 (2014).</li></ol>

作者宣称他们没有竞争的经济利益。

CRISPR / Cas9系统允许有效地产生稳定的基因组修饰，这提供了与其他瞬时操作方法更一致的替代方案。在这里，我们提出了两种在哺乳动物细胞系中快速鉴定CRISPR / Cas9基因敲除的方法。这两种方法都需要很少的细胞材料，因此可以在克隆培养的早期阶段进行测试，节省时间和试剂。为了提高两种方法的效率，我们建议测试多种sgRNA，因为效率因序列和基因组位置而异。此外，使用多个sgRNA可用于通...

<html>稳定的遗传改变比细胞扰动的瞬时方法提供了一个优点，这可以在其效率和持续时间上变化。由于目标特异性核酸酶的发展，如锌指核酸酶1,2,3,4,5，转录激活物样效应核酸酶（TALEN） 6,7,8,9 ，近年来，基因组编辑越来越普及和衍生自聚集的，定期交织的短回文重复（CRISPR）系统的RNA引导的核酸酶10 。 CRISPR / Cas9编辑机器改编自细菌和古菌用于防御病毒感染的免疫系统ass ="xref"&gt; 11，12，13。在这个过程中，入侵病毒序列的短的20-30nt片段被引入到基因组基因座中，作为侧重于重复单元14,15的 "间隔物"。随后的转录和RNA加工产生小的CRISPR相关RNA 16 （crRNA），其与反式激活的crRNA17（tracrRNA）一起与效应物Cas9核酸内切酶组装。因此，crRNA提供对Cas9靶向的特异性，指导复合物切割互补的病毒DNA序列并防止进一步的感染18,19 。目标DNA中的任何"原始孢子"序列都可以作为crRNA的来源，只要它直接与短原型相邻基序（PAM）5&#39;相连，在化脓性链球菌 Cas9 21中的单个引导RNA（sgRNA）组分中。 在真核细胞中Cas9和sgRNA表达后，Cas9蛋白在目标基因座处切割两条DNA链。在没有合适的同源序列区域的情况下，细胞通过非同源末端连接（NHEJ） 22,23,24修复了该断裂，其通常引入小的缺失或很少插入。定位开放时阅读框，修复可能导致产生非功能蛋白质产物的翻译移码。相比之下，当提供具有大的同源区域的外源模板时，细胞可以通过同源性定向修复25,26固定双链断裂。该途径允许在基因组中进行更大的精确的缺失，置换或插入，以及引入可切除的选择标记27 。 在这里，我们提出了通过这两种CRISPR / Cas9方法产生敲除细胞系的方案（ 图1A ）。 NHEJ方法使用单个sgRNA切割位点和选择无关筛选，因此需要很少的前期准备。当使用这种方法时，必须设计指导与最有可能产生敲除的转录物5&#39;末端附近的外显子互补的RNA。自从修改在这种情况下，到基因组的离子量很小，筛选克隆克隆基于斑点印迹，其中以高通量方式评估蛋白质产物。我们使用ELAV样1蛋白（ELAVL1）敲除生产线的生成作为例子。第二种方法依赖于同源性定向修复（HDR），并使用跨越感兴趣的基因或区域的两个sgRNA切割位点，允许几十kb的缺失。在切割位点侧面具有两个具有同源性区域的质粒提供替代模板（ 图1B ），引入可选择的抗性标记，其提高敲除产生效率。该方法也可适用于引入具有适当设计的同源臂的基因修饰。在这种情况下，新的DNA片段的整合允许基于PCR的筛选（ 图1C ）。在这里，我们使用Pumilio RNA结合家族成员2（PUM2）敲除生产线的生成作为例子。

<table><tbody><tr><td>Competent &lt;em&gt;E. coli&lt;/em&gt; cells</td><td></td><td></td><td></td></tr><tr><td>plasmid prep kit</td><td></td><td></td><td></td></tr><tr><td>pSpCas9(BB) plasmid</td><td>Addgene</td><td>42230</td><td>Ran &lt;em&gt;et al.&lt;/em&gt; 2013, cloning sgRNAs</td></tr><tr><td>BbsI enzyme</td><td>ThermoFisher</td><td>FD1014</td><td>Ran &lt;em&gt;et al.&lt;/em&gt; 2013, cloning sgRNAs</td></tr><tr><td>T7 DNA ligase</td><td>NEB</td><td>M0318L</td><td>Ran &lt;em&gt;et al.&lt;/em&gt; 2013, cloning sgRNAs</td></tr><tr><td>Tango Buffer</td><td>ThermoFisher</td><td>BY5</td><td>Ran &lt;em&gt;et al.&lt;/em&gt; 2013, cloning sgRNAs</td></tr><tr><td>PlasmidSafe exonuclease</td><td>Epicentre</td><td>E3105K</td><td>Ran &lt;em&gt;et al.&lt;/em&gt; 2013, cloning sgRNAs</td></tr><tr><td>Q5 hot start high fidelity polymerase</td><td>NEB</td><td>M0494A</td><td>HA amplification</td></tr><tr><td>pUC19-BsaI</td><td></td><td></td><td>Modified pUC19 plasmid, mutated existing BsaI site and inserted two outward facing BsaI sites after BamHI/EcoRI digestion</td></tr><tr><td>pGolden-Neo</td><td>Addgene</td><td>51422</td><td>Resistance cassette</td></tr><tr><td>pGolden-Hygro</td><td>Addgene</td><td>51423</td><td>Resistance cassette</td></tr><tr><td>BsaI enzyme</td><td>NEB</td><td>R3535S</td><td>Homology arm contruction</td></tr><tr><td>T7 DNA ligase</td><td>NEB</td><td>M0318L</td><td></td></tr><tr><td>PlasmidSafe exonuclease</td><td>Epicentre</td><td>E3105K</td><td></td></tr><tr><td>Toothpicks</td><td></td><td></td><td>bacterial PCR</td></tr><tr><td>Taq polymerase</td><td></td><td></td><td>bacterial colony PCR</td></tr><tr><td>HEK293 human cells</td><td></td><td></td><td></td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td></td></tr><tr><td>FBS</td><td>Corning</td><td>MT35010CV</td><td></td></tr><tr><td>Penicillin-Strep (opt.)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>6 well plates</td><td>BioLite</td><td>12556004</td><td></td></tr><tr><td>TransIT-LTI Transfection Reagent</td><td>Mirus</td><td>MIR2300</td><td>for lipofection only</td></tr><tr><td>Opti-MEM</td><td>ThermoFisher</td><td>31985062</td><td>for lipofection only</td></tr><tr><td>G418 (Neomycin)</td><td>Sigma Aldrich</td><td>A1720-5G</td><td></td></tr><tr><td>Hygromycin</td><td>Sigma Aldrich</td><td>H3274-250MG</td><td></td></tr><tr><td>96 well plates</td><td>ThermoFisher</td><td>12556008</td><td></td></tr><tr><td>Passive Lysis Buffer, 5x</td><td>Promega</td><td></td><td></td></tr><tr><td>1x SDS loading buffer</td><td></td><td></td><td>recipe decribed in protocol</td></tr><tr><td>Nitrocellulose Membrane</td><td>Bio-Rad</td><td>162-0115</td><td></td></tr><tr><td>TBST</td><td></td><td></td><td>recipe decribed in protocol</td></tr><tr><td>Dehydrated milk</td><td></td><td></td><td></td></tr><tr><td>SuperSignal West Dura Extended Duration Substrate</td><td>ThermoFisher</td><td>34075</td><td>for HRP-conjugated secondary antibodies</td></tr><tr><td>Extracta DNA prep for PCR</td><td>Quantabio</td><td>95091-025</td><td></td></tr><tr><td>KOD polymerase</td><td>Novagen</td><td>71316</td><td></td></tr></tbody></table>

selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells

CRISPR / Cas9基因组工程系统通过允许精确的基因组编辑，很少的努力，彻底改变了生物学。在引导特异性的单指导RNA（sgRNA）的指导下，Cas9蛋白在靶位点切割两条DNA链。 DNA断裂可以触发非同源末端连接（NHEJ）或同源性定向修复（HDR）。 NHEJ可以引入导致帧转移突变的小缺失或插入，而HDR允许更大和更精确的扰动。在这里，我们提出了通过将建立的CRISPR / Cas9方法与下游选择/筛选的两个选项相结合来产生敲除细胞系的方案。 NHEJ方法使用单个sgRNA切割位点和选择无关筛选，其中以高通量方式通过点免疫印迹评价蛋白质产生。 HDR方法使用跨越感兴趣基因的两个sgRNA切割位点。与提供的HDR模板一起，该方法可以实现删除几十kb，由插入的可选电阻标记辅助。讨论每种方法的适用应用和优点。

为了产生ELAVL1敲除生产线，可以使用强大的抗体，因此使用单个sgRNA进行编辑（ 图1A ，左侧），然后进行点免疫印迹。独立地转染三种sgRNA以比较效率并排除所得克隆中的靶效应。在将克隆群体的细胞裂解物收集并印迹到两个硝酸纤维素膜上之后，对ELAVL1和PUM2（作为归一化对照）检测印迹（ 图2A ）。 ELAVL1与PUM2信号的量化比例?...

Watch this Scientific Journal Video about Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells at JoVE.com

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

遗传操纵体细胞系的能力的最新进展对于基础和应用研究具有巨大的潜力。在这里，我们提出了CRISPR / Cas9在哺乳动物细胞系中产生敲除生产和筛选的两种方法，使用和不使用选择标记。

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 ...

selection-dependent-independent-generation-crisprcas9-mediated-gene

Research

JoVE Journal

Genetics

12.4K 次观看.  University of California, Riverside. 遗传操纵体细胞系的能力的最新进展对于基础和应用研究具有巨大的潜力。在这里，我们提出了CRISPR / Cas9在哺乳动物细胞系中产生敲除生产和筛选的两种方法，使用和不使用选择标记。 

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

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

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

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

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

科研

遗传学

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

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

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

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

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

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

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. ...

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 ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

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 cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

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 ...

A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.

This protocol describes the steps for cloning multiple single guide RNAs into one guide RNA concatemer vector, which is of particular use in creating multi-gene knockouts using CRISPR/Cas9 technology. The generation of double knockouts in intestinal organoids is shown as a possible application of this method.

使用CRISPR并发剂的小鼠小肠组织中多基因敲除的方案

CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In ...

生物工程

Construction of CRISPR Plasmids and Detection of Knockout Efficiency in Mammalian Cells through a Dual Luciferase Reporter System

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).

Here, we present a protocol describing a streamlined method for the efficient generation of plasmids expressing both the CRISPR enzyme and associated single guide RNA (sgRNAs). Co-transfection of mammalian cells with this sgRNA/CRISPR vector and a dual luciferase reporter vector that examines double-strand break repair allows evaluation of knockout efficiency.

通过双路西法酶检测系统构建CRISPR质粒和检测哺乳动物细胞的挖空效率

Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. ...

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 ...

免疫与感染

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system. The sgRNAs targeting exon 1 of the L2HGDH gene were chemically synthesized and cloned into the lentiCRISPR v2-puro vector, which combines the constitutive expression of sgRNAs with Cas9 in a highly efficient single-vector system to achieve higher lentiviral titers for hESC infection and stable selection using puromycin. Puromycin-selected cells were further expanded, and single-cell clones were obtained using the limited dilution method. The single clones were expanded, and several homozygous knockout clones for the L2HGDH gene were obtained, as confirmed by a 100% reduction in L2HGDH expression using Western blot analysis. Furthermore, using MSBSP-PCR, the CRISPR mutation site was mapped upstream of the PAM recognition sequence of Cas9 in the selected homozygous clones. Sanger sequencing was performed to analyze the exact insertions/deletions, and functional characterization of the clones was conducted. This method produced a significantly higher percentage of homozygous deletions compared to previously reported non-viral gene delivery methods. Although this report focuses on the L2HGDH gene, this robust and cost-effective approach can be used to create homozygous knockouts for other genes in pluripotent stem cells for gene function studies.

<meta charset="utf8"></head><body>CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery system.

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical ...