Name: generation of genomic deletions in mammalian cell lines via crispr/cas9
Uploaded: 2015-01-03T14:00:00
Duration: 9 min 40 s
Description: Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

Thanks to Jason Wright for suggesting the Golden Gate Assembly cloning strategy and Katherine Helming and members of Orkin lab, particularly Jian Xu, Guoji Guo, Elenoe Smith, and Partha Das for helpful discussions. This work was supported by NIH R01HL032259 and P30DK049216 (Center of Excellence in Molecular Hematology) to S.H.O. and NIDDK K08DK093705 to D.E.B.

1. CRISPR设计<ol><li>手动或使用免费的在线工具7的设计sgRNAs。使用这些工具，以帮助识别引导顺序，最大限度地减少相同的基因组的比赛或接近的比赛，以减少离靶位点（脱靶效应）裂解的风险。确保引导序列，由20个碱基（“protospacer序列”）的“NGG”序列（“protospacer相邻的主题”或PAM）在基因组识别位点的上游。 注意： 图1描述了基因和非编码的元素可能删除策略。为了创建一个基因敲除，二因组外显子位于内会丰富甚至单等位基因缺失克隆的功能丧失。这是由于形成在非缺失等位基因12插入缺失，这很可能会导致移码突变，导致该基因转录物（ 图1B）的无义介导的衰减的高频率。 </li><li>使用例如导游为鼠标的预期删除PIM1的（ 小家鼠 ; 表1，图2A）。 注意：在这个例子中，因组-A的protospacer序列和PAM碰巧落在底部（克里克）链，而因组-B的protospacer序列和PAM落在顶部（沃森）链（ 图2A）。然而，DSB将发生独立protospacer序列/ PAM相的顶部或底部的链的取向。 </li><li>确定每个导引序列的反向互补（RC）。例如扭转PIM1因组的补体的序列。从表1中发现在表2中 。 </li><li>获得24或25聚体寡核苷酸的每个导向和其相关联的反向互补，包括额外的核苷酸用于克隆和表达目的。 注：在这里，我们讨论pSpCas9（BB）质粒（pX330）（Addgene质粒ID 42230）的使用。该质粒可以用于同时因组和SpCas9，但表达的不含标记物用于选择7。其它结构可用于，例如pX458（Addgene质粒编号48138）或pX459（Addgene质粒ID 48139），其包括GFP和嘌呤霉素作为选择标记物，分别或其中多个sgRNAs可以从单个质粒表达构建体。 <ol><li>导的反向互补用于克隆到用BBSI限制酶（ 见表3）的pX330矢量之前20聚体导向序列和“AAAC”之前加上“CACC”。 </li><li>后CACC序列和20聚体前添加一种G核苷酸，如果20聚体的第一位置是从pX330矢量的U6启动子不是G.因组表达被CACC序列之后列入A G核苷酸的增强。添加一个下以反向互补寡核苷酸的3&#39;末端（ 例如，因组-A的表4）。所得的寡核苷酸是25聚体寡聚物。 </li><li>怎么样以往，如果20个碱基（protospacer序列）的第一个位置为G，不添加其他G（ 如因组-B 表4），不添加C到反向互补寡核苷酸的最终位置。在这种情况下，所得到的寡核苷酸。将24-mer的寡核苷酸（ 表4）。 </li></ol></li></ol> 2.删除设计引物筛选<ol><li>设计一组引物内部的序列被删除（“非缺失带”）和另一组引物上游和的因组切割位点下游（“缺失带”; 图1 - 2）。在没有缺失时，“删除带”往往过大，无法高效地扩增。通常使用的引物的至少100碱基对，从所预测的切割位点，以确保检测不会由一个小INDEL在因组靶位受到影响。 <ol><li>设计额外的引物来分析疤痕（在因组Ç生产小型插入缺失leavage网站不打算删除）。使用一对正向和反向引物各侧翼因组目标部位（内150 - 350碱基对）以扩增因组靶位点以检查对疤痕。这可能是有用的，以单等位基因缺失克隆的非缺失等位基因表征。 </li></ol></li><li>对于小的缺失（称为“缺失带”可能仍然放大），解决在琼脂糖凝胶上的扩增子，以确定是否是大小与缺失的存在或不存在相一致。对于这种方法，在步骤2.1中所述的内部引物，也可以省略。 </li></ol> 3. CRISPR克隆<ol><li>退火和磷酸化寡核苷酸。 <ol><li>重悬寡聚在100μM在双蒸2 O的浓度</li><li>准备一个10微升反应混合物的每个指南，其反向互补：1.0微升因组24或25-mer的寡核苷酸（100μM;见步骤1.4），1.0微升因组24或25-mer的反补吨寡（100μM;参见步骤1.4），1.0微升10×T4连接缓冲液，6.5微升DDH 2 O的，以及0.5微升T4多核苷酸激酶（PNK）（10,000单位/毫升）。 注：磷酸寡核苷酸可以责令代替。对于这种方法，利用T4 PNK的被省略。 </li><li>退火在使用以下参数的热循环：37℃30分钟; 95℃下5分钟，然后减速至25℃以5℃/分。 </li><li>在DDH 2 O的稀释1:10的寡核苷酸（ 例如，1.0微升退火的寡聚+ 9.0微升DDH 2 O的产生的1μM的浓度）。 </li></ol></li><li>结扎寡核苷酸退火到使用金门组装克隆策略26 pX330。 <ol><li>准备一个50微升反应混合物：100毫微克的圆形pX330载体，1.0微升退火寡核苷酸（1μM;见步骤3.1.4），5.0微升限制酶缓冲液（10倍），4.0微升（20 U）BBSI限制性内切酶（5000 U /毫升），5.0μ升的ATP（10毫米），0.25微升（5微克）的BSA（20毫克/毫升），0.375微升（750 U）的T4 DNA连接酶（200万单位/毫升），和H 2 O至50μl的最终体积。这个反应可以按比例缩小到一个较小的最终体积，如果必要的。 </li><li>使用以下参数在热循环仪上运行样本：周期1-20（37℃5分钟，20℃5分钟）;周期21（80℃，20分钟）。这些循环条件允许消化和连接发生在一个反应（见步骤3.2）。 </li><li>将10微升DH5αE的大肠杆菌细胞与1微升反应（从3.2.1 - 3.2.2）。 </li><li>板到LB培养基（LB）琼脂板100微克/毫升氨苄青霉素和孵化O / N在37℃。 </li></ol></li><li>选择2 - 3个菌落，接种到小量制备文化。 <ol><li>对每个样品进行小量制备，并使用U6启动子的正向引物序列中的每个集落：CGTAACTTGAAAGTATTTCGATTTCTTGGC。这是ARepresentative测序引物;其它侧翼引物都可以使用。 </li></ol></li><li>选择一个序列验证菌落，接种到一个马克西准备文化。制备型尺寸可基于所要求的DNA的产率进行缩放。 </li><li>每个CRISPR / Cas9结构进行马克西准备。 </li></ol> 4.转染CRISPRs到感兴趣的细胞注：此协议涉及通过电27交付CRISPR / Cas9质粒。这个协议进行详​​细为鼠红白血病（MEL）细胞，悬浮细胞系进行说明。在所有步骤中的培养基由DMEM补充有2％青霉素/链霉素和1％L-谷氨酰胺，其用于MEL细胞。然而，CRISPR / Cas9质粒瞬时转染可以成功地使用，优选的培养条件和转染策略针对每个小区类型适于多种细胞类型。虽然MEL细胞是悬浮细胞，贴壁细胞ħ说明AVE也被包括在内。 <ol><li>确保有每CRISPR对2×10 6个细胞。悬浮2×10 6个细胞在100μl的电穿孔溶液，并加入到电穿孔杯中。 <ol><li>添加5微克每CRISPR / Cas9结构（10微克总）的。添加0.5 GFP表达构建体微克。 </li></ol></li><li>电穿孔细胞250伏5毫秒在2毫米比色皿使用电系统。 注：或者使用另一种转染方法，如阳离子脂质体为基础的转染。优化转染条件与记者构造每个细胞系，以确保在尝试基因组编辑之前强劲的质粒交付。 </li><li>立即从试管电后传输解决方案在1ml培养基。最小化电穿孔和转移溶液之间的时间成媒体，以提高细胞生存力。 </li><li>孵育在30 - 37℃，24 - 72小时。 30°C可增强基因组编辑效率，但37℃是可接受的。 </li></ol> 5.荧光激活细胞转染的细胞分选（FACS） <ol><li>通过一个50微米的过滤器过滤成在FACS管中制备的细胞用于FACS。 </li><li>流式细胞仪进行排序的GFP阳性细胞的顶〜3％，以充实该接收高水平CRISPR的细胞/ Cas9构造。 </li><li>板分选的细胞单独成用分拣机96孔圆底板或通过使用有限稀释以每96孔圆底板30细胞。优化镀在执行此步骤，以可靠地获得每96孔板大约30个细胞之前限制稀释使用的细胞类型。 </li><li>包括每个细胞培养基的孔100μl。 </li><li>对于其余的分选的细胞（“散装”）的未镀，冻结一半的细胞以供将来电镀。板的另一半用于筛选和引物验证（见步骤6）。 注：此Protocol是悬浮细胞。贴壁细胞可以使各单细胞衍生的克隆可以被拾取并移动到平底96孔板生长在96孔平底板中或在10cm皿在低浓度下作为单独的细胞。 </li><li>允许散装细胞孵育在37℃下进行3 - 7天，允许克隆孵育在37℃下7 - 14天。改变这些时间根据所使用的细胞系的倍增时间。 注：此孵化时间允许为预期删除通过PCR筛选基因组DNA（基因组DNA）足够的细胞增殖（见步骤6.1和7.1）。当浓度超过本体细胞已充分增殖〜100000个细胞/ ml或用于粘附细胞，该细胞已达到约80％汇合。该克隆已充分增殖以〜2毫米直径的一次肉眼可见。 </li></ol> 6.底漆验证和筛选CRISPR / Cas9介导的缺失<ol><li>通过重新悬浮亲和散装细胞沉淀于50μlDNA提取溶液分离的gDNA从亲和散装分选的细胞。 注意：通常〜100,000个细胞被用于DNA提取，虽然广泛的细胞数目是可以接受的。批量分选的细胞组成的多克隆群体暴露于因组A和因组-B的（见步骤5）。下述PCR的目的是验证的引物，并验证预期基因缺失的存在。 </li><li>运行样品在热循环仪，并运行下列程序：65℃6分钟，98℃2分钟，以提取的gDNA。测量DNA浓度。 注意：虽然步骤6.1和6.2的DNA提取建议一种有效的方法，任何方法对基因组DNA分离可利用，以便能够在步骤6.3进行PCR。 </li><li>组装20微升PCR以下组件：10微升2倍PCR组合，0.5微升正向引物（10 _6，M），0.5微升反向引物（10μM），50-100纳克的gDNA和H 2 O达20微升。使用设计的在上面的步骤2中的引物。进行PCR的“非删除带”和“删除带”在不同的反应。 注：许多聚合酶可用于步骤6.3。 <ol><li> 95℃15分钟，35个循环的（95℃，30秒，60℃1分钟，72℃1分钟）和72℃，10分钟：使用以下参数在热循环仪上运行样本。优化的PCR条件为每个引物对的基础上测试本体分选的细胞而设计的。 </li></ol></li><li>运行在2％琼脂糖凝胶的样品，在用1×Tris-醋酸-EDTA（TAE）缓冲液10伏/厘米。 </li><li>检查样本中存在/不存在非缺失和缺失带（ 图2）的。考虑多路复用“删除”和“非缺失”在一个单一的反应的PCR引物对。优化复在多克隆人群筛选单个克隆之前（ 即批量排序细胞）。至关重要的是，删除无删减扩增子可以轻松解决对复用琼脂糖凝胶。 </li></ol> 7.筛选CRISPR / Cas9克隆的缺失和克隆选择<ol><li>对于悬浮细胞，所有克隆转移到一个单一的96孔板已包含每孔50微升的细胞培养基为150微升的最终体积。这通过允许用于以下步骤在步骤7中的其余一个多道移液器便于筛选。 <ol><li>从各传送50μl的井用多道移液器（留在每个孔中加入100μl）至96孔PCR板。 </li><li>离心机PCR板在400×g离心5分钟，并通过轻弹PCR板通过水槽除去上清液。添加50微升每孔和重悬DNA提取溶液。继续加强7.3悬浮细胞。 </LI&gt; </ol></li><li>对于贴壁细胞，吸出介质。加入20微升的0.05％胰蛋白酶-EDTA至每孔用克隆本。 <ol><li>重悬的细胞在200μl的介质。吸管混合分离的细胞。 </li><li>板100微升每种成两个独立的96孔平底板。保持一个板以允许克隆生长，并使用另一个板来筛选各克隆为缺失。 </li><li>增加一个额外的100微升至每孔为200微升的总体积。等待24 - 72小时，使细胞生长。 </li><li>吸媒体。加入每孔，重悬和转让50微升DNA提取液为96孔PCR板。继续加强7.3。 </li></ol></li><li>从克隆提取基因组DNA中。运行样品在热循环：65℃，6分钟和98℃2分钟，以提取的gDNA。 </li><li>筛选使用相同的PCR引物和反应条件的优化上堆积的细胞的每个克隆（见步骤6）。 </li><li>选择克隆鉴定者tified与所需删除和移动，以较大的板或烧瓶中的生长。 </li></ol> 8.验证双等位基因缺失克隆<ol><li>为了获得克隆表征和验证成功敲除，评估在DNA克隆以及RNA和/或蛋白水平。 <ol><li>为了评估该DNA，扩增来自双等位基因缺失克隆缺失频带用校对聚合酶和克隆扩增（ 例如，用一个PCR克隆试剂盒）到质粒载体中。变换成质粒DH5αE.的大肠杆菌细胞和板到LB琼脂平板与相关抗生素。选择多个殖民地，小量制备各一台，并受各克隆Sanger测序每个缺失等位基因28表征- 30。重复PCR检测为缺失的初始画面后，确保了正确的克隆被选择和结果的可重复性。 </li><li>为了评估RNA，进行RT-qPCR的用于根相关基因30,31电子商务表达。 </li><li>为了评估蛋白质，执行使用抗体针对相关蛋白33的免疫印迹。 </li></ol></li></ol>

<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 (6096), 816-821 (2012).</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>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>Gaj, T., Gersbach, C. A., Barbas, C. F. Z. F. N., TALEN, <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-based+methods+for+genome+engineering.">CRISPR/Cas-based methods for genome engineering.</a> Trends in Biotechnology. 31 (7), 397-405 (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=CRISPR+provides+acquired+resistance+against+viruses+in+prokaryotes.">CRISPR provides acquired resistance against viruses in prokaryotes.</a> Science. 315, 1709-1712 (2007).</li><li>Ding, Q., 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=Enhanced+efficiency+of+human+pluripotent+stem+cell+genome+editing+through+replacing+TALENs+with+CRISPRs.">Enhanced efficiency of human pluripotent stem cell genome editing through replacing TALENs with CRISPRs.</a> Cell Stem Cell. 12 (4), 393-394 (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> Nature Protocols. 8 (11), 2281-2308 (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=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 (6), 1380-1389 (2013).</li><li>Larson, M. 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+interference+(CRISPRi)+for+sequence-specific+control+of+gene+expression.">CRISPR interference (CRISPRi) for sequence-specific control of gene expression.</a> Nature Protocols. 8 (11), 2180-2196 (2013).</li><li>Yang, 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=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Wang, 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=One-step+generation+of+mice+carrying+mutations+in+multiple+genes+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.</a> Cell. 153 (4), 910-918 (2013).</li><li>Canver, M. 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=Characterization+of+Genomic+Deletion+Efficiency+Mediated+by+CRISPR/Cas9+in+Mammalian+Cells.">Characterization of Genomic Deletion Efficiency Mediated by CRISPR/Cas9 in Mammalian Cells.</a> Journal of Biological Chemistry. 289 (31), 21312-21324 (2014).</li><li>Xiao, 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=Chromosomal+deletions+and+inversions+mediated+by+TALENs+and+CRISPR/Cas+in+zebrafish.">Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.</a> Nucleic Acids Research. 41 (14), e141 (2013).</li><li>Zhou, 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=Dual+sgRNAs+facilitate+CRISPR/Cas9+mediated+mouse+genome+targeting.">Dual sgRNAs facilitate CRISPR/Cas9 mediated mouse genome targeting.</a> The FEBS Journal. , (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=Genetic+screens+in+human+cells+using+the+CRISPR-Cas9+system.">Genetic screens in human cells using the CRISPR-Cas9 system.</a> Science. 343 (6166), 80-84 (2014).</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 (6166), 84-87 (2014).</li><li>Niu, 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=Generation+of+Gene-Modified+Cynomolgus+Monkey+via+Cas9/RNA-Mediated+Gene+Targeting+in+One-Cell+Embryos.">Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos.</a> Cell. 156 (4), 836-843 (2014).</li><li>Guo, X., 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=Efficient+RNA/Cas9-mediated+genome+editing+in+Xenopus+tropicalis.">Efficient RNA/Cas9-mediated genome editing in Xenopus tropicalis.</a> Development. 141 (3), 707-714 (2014).</li><li>Xie, K., Yang, Y. <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+genome+editing+in+plants+using+a+CRISPR-Cas+system.">RNA-guided genome editing in plants using a CRISPR-Cas system.</a> Molecular Plant. 6 (6), 1975-1983 (2013).</li><li>Waaijers, 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=CRISPR/Cas9-targeted+mutagenesis+in+Caenorhabditis+elegans.">CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans.</a> Genetics. 195 (3), 1187-1191 (2013).</li><li>Bassett, A. R., Liu, J. L. <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+and+Genome+Editing+in+Drosophila.">CRISPR/Cas9 and Genome Editing in Drosophila.</a> Journal of Genetics and Genomics. 41 (1), 7-19 (2014).</li><li>Schwank, 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=Functional+Repair+of+CFTR+by+CRISPR/Cas9+in+Intestinal+Stem+Cell+Organoids+of+Cystic+Fibrosis+Patients.">Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients.</a> Cell Stem Cell. 13 (6), 653-658 (2013).</li><li>Wu, 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=Correction+of+a+Genetic+Disease+in+Mouse+via+Use+of+CRISPR-Cas9.">Correction of a Genetic Disease in Mouse via Use of CRISPR-Cas9.</a> Cell Stem Cell. 13 (6), 659-662 (2013).</li><li>Lappalainen, T. 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=Transcriptome+and+genome+sequencing+uncovers+functional+variation+in+humans.">Transcriptome and genome sequencing uncovers functional variation in humans.</a> Nature. 501 (7468), 506-511 (2013).</li><li>Bauer, D. 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=An+Erythroid+Enhancer+of+BCL11A+Subject+to+Genetic+Variation+Determines+Fetal+Hemoglobin+Level.">An Erythroid Enhancer of BCL11A Subject to Genetic Variation Determines Fetal Hemoglobin Level.</a> Science. 342 (6155), 253-257 (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, e3647 (2008).</li><li>Gehl, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation:+theory+and+methods,+perspectives+for+drug+delivery,+gene+therapy+and+research.">Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research.</a> Acta Physiologica Scandinavica. 177, 437-447 (2003).</li><li>Zhang, S., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purifying+Plasmid+DNA+from+Bacterial+Colonies+Using+the+Qiagen+Miniprep+Kit.">Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit.</a> J. Vis. Exp. (6), e247 (2007).</li><li>Froger, A., Hall, 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=Transformation+of+Plasmid+DNA+into+E.+coli+Using+the+Heat+Shock+Method.">Transformation of Plasmid DNA into E. coli Using the Heat Shock Method.</a> J. Vis. Exp. (6), e253 (2007).</li><li>Finney, M., Nisson, P. E., Rashtchian, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+PCR+products.">Molecular cloning of PCR products.</a> Current Protocols in Molecular Biology. Chapter 15. (Unit 15.4), (2001).</li><li>Gordanpour, A., Nam, R. K., Sugar, L., Bacopulos, S., Seth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Detection+in+Prostate+Tumors+by+Quantitative+Real-time+PCR+(qPCR).">MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR).</a> J. Vis. Exp. (63), e3874 (2012).</li><li>Schularick, N. M., Clark, J. J., Hansen, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Culture+of+Human+Vestibular+Schwannomas.">Primary Culture of Human Vestibular Schwannomas.</a> J. Vis. Exp. (89), e51093 (2014).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting:+Sample+Preparation+to+Detection.">Western Blotting: Sample Preparation to Detection.</a> J. Vis. Exp. (44), e2359 (2010).</li><li>McVey, M., Lee, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMEJ+repair+of+double-strand+breaks+(director&#8217;s+cut):+deleted+sequences+and+alternative+endings.">MMEJ repair of double-strand breaks (director&#8217;s cut): deleted sequences and alternative endings.</a> Trends in Genetics. 24 (11), 529-538 (2008).</li><li>Symington, L. S., Gautier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-strand+break+end+resection+and+repair+pathway+choice.">Double-strand break end resection and repair pathway choice.</a> Annual Review of Genetics. 45, 247-271 (2011).</li><li>Fu, 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-frequency+off-target+mutagenesis+induced+by+CRISPR-Cas+nucleases+in+human+cells.">High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.</a> Nature Biotechnology. 31 (9), 822-826 (2013).</li><li>Wu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+binding+of+the+CRISPR+endonuclease+Cas9+in+mammalian+cells.">Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.</a> Nature Biotechnology. , (2014).</li><li>Hsu, P. 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=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (2013).</li><li>Fu, 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=Improving+CRISPR-Cas+nuclease+specificity+using+truncated+guide+RNAs.">Improving CRISPR-Cas nuclease specificity using truncated guide RNAs.</a> Nature Biotechnology. 32, 279-284 (2014).</li><li>Alem&#225;n, L. M., Doench, J., Sharp, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Comparison+of+siRNA-induced+off-target+RNA+and+protein+effects.">a Comparison of siRNA-induced off-target RNA and protein effects.</a> RNA. 13 (3), 385-395 (2007).</li><li>Anderson, E. 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=Experimental+validation+of+the+importance+of+seed+complement+frequency+to+siRNA+specificity+Experimental+validation+of+the+importance+of+seed+complement+frequency+to+siRNA+specificity.">Experimental validation of the importance of seed complement frequency to siRNA specificity Experimental validation of the importance of seed complement frequency to siRNA specificity.</a> RNA. 14, 853-861 (2008).</li><li>Marine, 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=Common+seed+analysis+to+identify+off-target+effects+in+siRNA+screens.">Common seed analysis to identify off-target effects in siRNA screens.</a> Journal of Biomolecular Screening. 17 (3), 370-378 (2012).</li></ol>

We have no conflicts of interest related to this report.

所述CRISPR / Cas9系统可以被用于产生一个尺寸范围的基因缺失。尽管我们已经观察到缺失的频率相对于预期缺失大小成反比变化，我们已经能够恢复的高达1 Mb缺失和缺失高达100kb的常规产率的多个等位基因缺失克隆。我们观察到在依次引入缺失的效率没有损失到的细胞系。这种策略可以用于创建组合删除许多基因和元件。获得双等位基因缺失克隆的方法可以通过估计所需的基础上删除尺寸，以获得克隆的期望数量与等位基因缺失12被筛选的克隆的最小数目可以加快。 获得单等位基因缺失的情况下等位基因缺失的概率分布的能力可能表明与功能完全丧失相关的细胞的杀伤力。低FRequency或不存在缺失可能反映了许多方案，包括转染较差，低效sgRNAs或低效PCR筛选引物（由于缺少一个阳性对照，以验证PCR引物来筛选缺失）。 GFP +细胞可以被用作一个替代转染效率（参见步骤5.2），因此，在GFP +细胞的降低可能反映转差和由此而来降低缺失效率。使用两种不同的因组对具有自主筛选引物可以帮助控制低效因组和筛选PCR引物，最大限度地获得等位基因缺失克隆的机会。细胞的GFP +细胞分选，丰富的缺失等位基因。虽然此步骤可以省略，遗漏将可能必要筛选更多的克隆，以确定那些具有单等位基因或等位基因缺失。到的程度的转染效率可被优化，我们希望被增强的基因组编辑效率。 图2B）的部位。通常，这些等位基因似乎是microhomology基于修34,35的结果。应当指出的是，我们描述了基于PCR的检测策略将无法确定更大或更复杂的插入，缺失，倒位，或重排。虽然这些事件是不常见的，我们观察到，其中既没有缺失，也没有非缺失的扩增子可被检测的克隆，并且在进一步的调查反映这些更复杂的结果。 我们已经观察到广泛CRISPR / Cas9介导的从单等位基因和非缺失克隆的非缺失等位基因“疤痕”（ 见图2B）。这些“疤痕”是由在因组切割位点不期望的缺失产生小插入缺失（ 即，缺失sgRNAs A和B之间的中间段）。这些伤痕常常中断了因组目标识别。因此，我们要敦促谨慎重新目标细胞等位基因此前曝光过使用相同的sgRNAs sgRNAs。一个更成功的重新定位策略将利用独特的序列因组从之前“伤痕累累”的识别位点不同的。在情况下，当一对sgRNAs的识别外显子序列（ 图1B，下图），移码等位基因可以甚至在没有缺失的产生。因此，单等位基因缺失克隆可以富集丢失的功能因移码突变在非缺失等位基因12的高频率。 一个关注与CRISPR / Cas9系统是脱靶效应， 即，基因组修饰在非预期位点36 - 38。 ř最近几个报告表明，较短的导的RNA用17 - 19个核苷酸可以减少CRISPR的频率/ Cas9基脱靶效应39。此外，使用每个目标部位2导板与一个切口酶双切口策略可以被用来创建双链断裂，同时尽量减少脱靶效应7。可替代地，类似于用于RNAi的策略，我们建议，不同对sgRNAs与非重叠protospacer序列可用于证明，所观察到的表型是在目标CRISPR / Cas9修改的结果，而不是潜在的脱靶效果。一种方便的方法是设计的至少两个相邻的，但不重叠的因组对，使得可用于多种因组对（ 图1A）一套单一的筛选的引物（参见步骤2）。此外，通过重新引入丢失的序列和/或破坏的基因互补的缺失的细胞系可以证实之间的因果关系一个给定的基因缺失和表型。 对于生物学家与细胞模型系统的工作，RNAi技术代表了一个强大的工具，功能基因组学。但是，这种方法的局限性已包括不完整的减少靶mRNA转录水平，靶向相同基因的独立的试剂的效果的异质性，和已知的脱靶效应，包括基于种子和非种子的影响40 - 42。基因组编辑的战略承诺，以解决许多这些关切，并代表了未来的遗传扰动8,36,37一个令人兴奋的，互补的办法。此外，基因组编辑允许的非编码的遗传元件在不通过RNAi可能的，并且通过常规的定位具有挑战性的方式接近25的研究。我们鼓励CRISPR / Cas9代基因缺失的作为一个强大的和具体的方法，以产生和表征丧失功能的等位基因。

Recent advances in genome engineering technology have allowed for unprecedented opportunities for site-specific modification of the genome. This technology may be utilized to investigate the function of genes and regulatory elements via prospective genetic perturbation. Zinc finger nucleases (ZFNs), transcription-activator like (TAL) effector nucleases (TALENs), and CRISPR/Cas9 RNA-guided nucleases each leverage customizable DNA specificity to localize a nuclease for the introduction of DSBs1&#8211;3. The resulting DSBs can be repaired by indel-forming NHEJ or by homology-directed repair (HDR) using a donor template4.
The CRISPR/Cas9 nuclease pathway, an adaptive immune system in prokaryotic cells5, has been recently adapted for mammalian genome engineering2,3. This tool has been demonstrated to be an inexpensive, efficient, and reliable genome engineering technique6. Briefly, a complex of Streptococcus pyogenes-derived Cas9 nuclease and a sgRNA achieve target recognition via Watson-Crick base-pairing with cognate genomic DNA sequences. sgRNAs include 20-mer sequences complementary to genomic sequences adjacent to an obligate protospacer adjacent motif (PAM) NGG. Cas9 induces a DSB at a predictable position within the target site. Additionally, variants of Cas9 with single-strand cleavage capacity or catalytic inactivity may be used to facilitate &#8220;nicking&#8221; or transcriptional regulation respectively7&#8211;9. CRISPR/Cas9 has been used for a wide range of applications including both knock-in and knockout10,11, large-scale genomic deletions12&#8211;14, pooled library screening for gene discovery15,16, genetic engineering of numerous model organisms10,11,17&#8211;21, as well as gene therapy22,23.
Here we describe a protocol for efficient deletion of desired genomic regions. The protocol includes CRISPR design, cloning, and delivery, as well as deletion, identification, and characterization. Genomic deletions can be generated by the introduction of two CRISPR sgRNAs with Cas9 to induce repair of the resultant two DSBs by NHEJ with deletion of the intervening segment. This strategy has been used to create deletions ranging from one kilobase to over one megabase12. Deletions can be informative for the study of genes and other genetic elements, either in isolation or in combination. There are several potential advantages of genomic deletions as compared to HDR or single-site small indel production. First, this method capitalizes on the high efficiency of NHEJ in many cellular contexts7. The high frequency of deletion limits the number of clones needed to be screened to identify informative clones. Deletion frequency is inversely related to deletion size. Biallelic deletion clones may be retrieved at frequencies at least as great as of probabilistic expectation12. Second, both monoallelic and biallelic deletions may be easily identified and distinguished by conventional PCR, simplifying the screening process. Strategies relying on small indels or point mutations may require RFLP, allele-specific PCR, T7EN1 cleavage assay, Sanger sequencing, RT-qPCR, or immunoblotting, which may be more laborious. Third, by removing a substantial portion of a gene or element of interest, a reliable loss-of-function allele may be obtained. In contrast, frameshift mutations in protein-coding sequences may not always induce nonsense-mediated decay, may produce a hypomorphic or neomorphic allele, or target an exon excluded from an alternate isoform24. Finally, deletions may be particularly revealing for the study of non-coding DNA such as regulatory elements since frameshift mutations as produced by single-site indels would not be relevant25.

<table border="0" cellpadding="0" cellspacing="0" width="842">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:388px;">Name of Material/ Equipment</td>
			<td style="width:168px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 Polynucleotide Kinase</td>
			<td>New England Biolabs</td>
			<td>M0201S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase (with associated ligation buffer)</td>
			<td>New England Biolabs</td>
			<td>M0202T</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#39;-Triphosphate (ATP)</td>
			<td>New England Biolabs</td>
			<td>P0756S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA, Molecular Biology Grade</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BbsI Restriction Enzyme (with associated NEB Buffer 2.1)</td>
			<td>New England Biolabs</td>
			<td>R0539S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pSpCas9(BB) (pX330)</td>
			<td>Addgene</td>
			<td>42230</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">S.O.C. Medium</td>
			<td>Life Technologies</td>
			<td>15544-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX ECM 830</td>
			<td>Harvard Apparatus</td>
			<td>45-0052</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BTX Solution and 2mm Cuvettes</td>
			<td>Harvard Apparatus</td>
			<td>45-0803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pmaxGFP Plasmid</td>
			<td>Lonza</td>
			<td>VPA-1003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuickExtract DNA Extraction Solution</td>
			<td>Epicentre</td>
			<td>QE09050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HotStarTaq PCR Master Mix Kit</td>
			<td>Qiagen</td>
			<td>203443</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zero Blunt PCR Cloning Kit</td>
			<td>Life Technologies</td>
			<td>K2700-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

该实验的目的是在MEL细胞缺失PIM1的。使用多个非重叠因组对（ 即，独立protospacer序列）可能有助于控制对脱靶效应（ 图1A）。一致的表型将更有可能从相对于由多个独立protospacer序列共有一个共同的脱靶效应的上靶效应的结果。每对会导致生产独特删除断点。如果并拢（ 即n小于〜150 bp）的，相同的筛选的引物可以用来检测由每组sgRNAs产生缺失。基因缺失可以通过使用在不同的位置相对于该基因（ 图1B）因组对扰乱的基因。例如，因组对可侧翼为缺失整个基因体的基因;一对可以位于两个外显子内，以创建移码插入缺失的电位即使一个或两个等位基因ES并没有被删除;或一对可侧翼的特定外显子，以允许特定同种型的中断。 用于PIM1删除策略是设计侧翼sgRNAs删除整个基因体，一个8 kb的缺失（ 图2）。这种策略被选择的部分，由于PIM1基因的相对小的尺寸。本实施例示出了一个PAM（绿色）在顶部（沃森）链和1的PAM在底部（克里克）链;然而，DSB是独立的PAM序列定位于顶部或底部链。因组对可以在底链兼得的PAM序列上的顶链，两者的，或每个中的一个。使用上述的协议，2因组设计，克隆到pX330表达载体中，并通过电穿孔连同GFP报告（ 图2A）递送至MEL细胞。 GFP +细胞的前3％的整理后两天，电，并在有限稀释镀克隆。屏幕荷兰国际集团引物被设计为在步骤2中描述的和如图2。PCR条件使用的gDNA从亲代的MEL细胞和来自“散装”分选的细胞分离最优化。 铺板后10天，基因组DNA是从所有克隆中分离并筛选通过PCR缺失，其中确定非缺失，单等位基因和双等位基因缺失克隆根据非缺失（ND）和缺失（D）的扩增子的图案（ 图3） 。非缺失克隆被确定为具有非缺失的扩增子的存在和不存在缺失扩增子。单等位基因的克隆被鉴定为具有两个非缺失和缺失的扩增子的存在。双等位基因的克隆被鉴定为具有不存在非缺失扩增子和缺失的扩增子的存在。对于这个缺失，400个克隆筛选其中确定126单等位基因缺失克隆和32个等位基因DELET<html>离子克隆（需要注意的是缺失的大小12删除频率变化是很重要的）。双等位基因缺失克隆进行选择并转移到烧瓶用8ml的介质。容许扩张后5天，每个克隆被重新测试通过的gDNA进行PCR以确认等位基因缺失和缺失扩增子进行Sanger测序，以确定精确的缺失（ 图2B）。删除扩增子内的异质性体现不完美的插入缺失形成NHEJ修复。测序在大多数情况下，发现了插入缺失monoallelelic缺失克隆的非缺失等位基因的，这表明即使是在非缺失等位基因经常编辑者CRISPR / Cas9，其中两个单等位基因和双等位基因缺失克隆需要其可以是对于应用是重要的。 RNA从双等位基因缺失克隆中分离并通过RT-qPCR的分析以确认PIM1表达的丧失（ 图4）。 方法“&gt; <img alt="图1" src="/files/ftp_upload/52118/52118fig1highres.jpg" /> 图1.原理可能删除策略。（A）两例如因组对的基因缺失（分别为黑色和橙色所示）。蓝色箭头表示引物，以检测所述非缺失的扩增子和红色箭头表示引物，以检测删除的扩增子。因组位置17和18的红色和蓝色的高亮因组站点-A 1 / A 2和紫色和橙色高亮显示，在因组站点-B 1 / B 2用红线指示位置17之间的预测Cas9乳沟和18（B）CRISPR-执导分裂显示为垂直的黑线。蓝色箭头表示引物检测非缺失扩增和红色箭头表示引物，检测缺失扩增。 <a href="https://www.jove.com/files/ftp_upload/52118/52118fig1large.jpg" target="_blank">请点击这里查看</a>一个更大的版本这个数字。 <img alt="图2" src="/files/ftp_upload/52118/52118fig2highres.jpg" /> 图2.策略以产生并检测缺失PIM1，包括测序缺失和非缺失的等位基因。基于PCR的筛选策略的（A）的示意图，以确定PIM1缺失克隆。一个引物对位于内部的缺失（蓝色箭头）和一个引物对被定位外部的缺失（红色箭头）。因组序列（protospacer序列）显示为紫色。垂直红色线表示的位置17和18因组序列的间预测Cas9裂解。（B）的 Sanger测序揭示INDEL形成在因组识别位点。因组序列示于绿色紫色和PAM序列。删除事件是SH通过自己几许标记和插入相同数量的蓝色高亮显示。垂直的红色线表示预计裂解位点，位置17和18因组之间。 <a href="https://www.jove.com/files/ftp_upload/52118/52118fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52118/52118fig3highres.jpg" /> 图3代表性凝胶鉴定非缺失，单等位基因，和双等位基因的克隆。对于每个克隆，左侧车道印记为非缺失扩增子（“ND”蓝色）和右车道表示缺失的扩增子（“D”，在红）。单个克隆用虚线分开。三个单等位基因缺失克隆是克隆5,6和2的三个等位基因缺失克隆是克隆15，17和13中所见的测序数据，则删除频带对C<html>孤13具有更小的尺寸，由于在删除结较大的缺失。 <img alt="图4" src="/files/ftp_upload/52118/52118fig4highres.jpg" /> 图4.双等位基因缺失克隆损失PIM1表达。PIM1表达计算两个等位基因缺失克隆通过RT-qPCR的。 ΔCT法-利用2数据被标准化为GAPDH。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td> Protospacer序列</td></tr><tr><td>因组-A </td><td> 5&#39;-TAGGAAAATGACTCGTCACT-3&#39; </td></tr><tr><td>因组-B </td><td> 5&#39;-GTCTATCCTATCTCGATAAA-3&#39; </td></tr></tbody></table> 表2因组的删除PIM1的1. 20聚体protospacer序列。 543“&gt; <tbody><tr height="23"><td height="23" style="height:23px;width:101px;"></td><td style="width:443px;"> Protospacer序列逆补</td></tr><tr height="23"><td height="23" style="height:23px;">因组-A-RC </td><td> 5&#39;-AGTGACGAGTCATTTTCCTA-3&#39; </td></tr><tr height="23"><td height="23" style="height:23px;">因组-B-RC </td><td> 5&#39;-TTTATCGAGATAGGATAGAC-3&#39; </td></tr></tbody></table> 两个因组从表1中的protospacer序列表2反向互补。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>序列</td></tr><tr><td>因组-A </td><td> 5&#39;-CACCTAGGAAAATGACTCGTCACT-3&#39; </td></tr><tr><td>因组-A-RC </td><td> 5&#39;-AAACAGTGACGAGTCATTTTCCTA-3&#39; </td></tr><tr><td>因组-B </td><td> 5&#39;-CACCGTCTATCCTATCTCGATAAA-3&#39; </td></tr><tr><td> sgRNA-B-RC </td><td> 5&#39;-AAACTTTATCGAGATAGGATAGAC-3&#39; </td></tr></tbody></table> 表3. Protospacer序列及其反向互补包含“CACC”和“AAAC”添加用于克隆到用BBSI限制性内切酶的pX330向量。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>序列</td></tr><tr><td>因组-A </td><td> 5&#39;CACCGTAGGAAAATGACTCGTCACT-3&#39; </td></tr><tr><td>因组-A-RC </td><td> 5&#39;AAACAGTGACGAGTCATTTTCCTAC-3&#39; </td></tr><tr><td>因组-B </td><td> 5&#39;CACCGTCTATCCTATCTCGATAAA-3&#39; </td></tr><tr><td>因组-B-RC </td><td> 5&#39;AAACTTTATCGAGATAGGATAGAC-3&#39; </td></tr></tbody></table> 从pX330矢量的U6启动子表4因组表达通过后CACC序列和前加入一种G核苷酸的增强20聚体。加入额外ģ核苷酸的要求在反向互补寡核苷酸的3&#39;末端（ 例如，因组-A）中的加成一个C核苷酸。然而，如果20聚体（protospacer序列）的第一位置是已经A G核苷酸，没有必要添加其他G（ 例如，因组-B）和无需添加C到反向互补的最终位置寡。

Watch this Scientific Journal Video about Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9 at JoVE.com

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

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 ...

generation-of-genomic-deletions-in-mammalian-cell-lines-via-crisprcas9

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Biology

Unit 2

Analyzing Gene Expression and Function

SCIENTISTS IN ACTION

MISSION esiRNA for RNAi Screening in Mammalian Cells

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein1,2,3. RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs.
Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown10.
In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.

Here we use a human esiRNA library in a high-throughput screen for genes involved in cell division. We demonstrate how to set up and conduct an esiRNA screens, as well as how to analyze and validate the results.

在哺乳动物细胞的RNAi筛选使命esiRNA

RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as ...

科研

生物学

Generation of RNA/DNA Hybrids in Genomic DNA by Transformation using RNA-containing Oligonucleotides

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be produced to contain any desired DNA or RNA sequence and can be prepared to include a wide variety of base and sugar modifications. Moreover, oligos can be designed to mimic specific nucleic acid alterations and thus, can serve as important tools to investigate effects of DNA damage and mechanisms of repair. We found that Thermo Scientific Dharmacon RNA-containing oligos with a length between 50 and 80 nucleotides can be particularly suitable to study, in vivo, functions and consequences of chromosomal RNA/DNA hybrids and of ribonucleotides embedded into DNA. RNA/DNA hybrids can readily form during DNA replication, repair and transcription, however, very little is known about the stability of RNA/DNA hybrids in cells and to which extent these hybrids can affect the genetic integrity of cells. RNA-containing oligos, therefore, represent a perfect vector to introduce ribonucleotides into chromosomal DNA and generate RNA/DNA hybrids of chosen length and base composition. Here we present the protocol for the incorporation of ribonucleotides into the genome of the eukaryotic model system yeast /Saccharomyces cerevisiae/. Yet, our lab has utilized Thermo Scientific Dharmacon RNA-containing oligos to generate RNA/DNA hybrids at the chromosomal level in different cell systems, from bacteria to human cells.

This work shows how to form an RNA/DNA hybrid at the chromosomal level and reveal transfer of genetic information from RNA to genomic DNA in yeast cells.

改造使用含RNA寡核苷酸基因组DNA的RNA / DNA杂交一代

Synthetic short nucleic acid polymers, oligonucleotides (oligos), are the most functional and widespread tools of molecular biology. Oligos can be ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

绿色怪物承载多基因缺失的酵母菌株的产生过程

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

（TET-ON）与四环素诱导shRNA或cDNA表达生成稳定的人类细胞系

A major approach in  the field of mammalian cell biology is the manipulation of the expression of  genes of interest in selected cell lines, with the aim ...

Anaerobic Growth and Maintenance of Mammalian Cell Lines

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that the incubation in normoxic (5% CO2 in air) or hypoxic (low oxygen levels) conditions followed by an anoxic incubation (an absence of free oxygen) results in limited viability (2&#8211;3 days). A novel methodology was developed that enables an anoxic cell cultivation (for at least 17 days; the maximum time tested). The most critical aspect of this methodology is to ensure that no oxygen is introduced into the system. This is obtained by the degassing of media, and by flushing tubes, dishes, flasks, and pipettes with an anaerobic gas mixture (H2, CO2, N2) followed by permitting the materials to equilibrate to the anoxic (non-oxygen) environment prior to usage.&#160;Additional care must be exercised when acquiring photomicrographs to ensure that the micrographs obtained do not contain artifacts. In the absence of oxygen, cell morphology is significantly altered. Two distinct morphotypes are noted for all anaerobically-grown cells. The ability to grow and maintain mammalian cells in the absence of oxygen can be applied to the analysis of cell physiology, polymicrobial interactions, and the characterization of biosynthetic pathways for novel cancer drug development.

Here, we describe a new method that enables the anaerobic long-term cultivation of established cell lines. The maximum survival time that was tested is 17 days. This method is suitable for the testing of cytotoxic agents and exploring the physiology of anoxically replicating cells.

哺乳动物细胞系的厌氧生长与维持

Most mucosal surfaces along with the midpoints in tumors and stem cell niches are geographic areas of the body that are anoxic. Previous studies show that ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

沉默的火花：CRISPR/Cas9基因组编辑在弱电鱼

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

Genome Engineering of Primary Human B Cells Using CRISPR/Cas9

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make attractive candidates for cell-based therapies because of their ease of isolation from peripheral blood, their ability to expand in vitro, and their longevity in vivo. Additionally, their normal biological function&#8212;to produce large amounts of antibodies&#8212;can be utilized to express very large amounts of a therapeutic protein, such as a recombinant antibody to fight infection, or an enzyme for the treatment of enzymopathies. Here, we provide detailed methods for isolating primary human B cells from peripheral blood mononuclear cells (PBMCs) and activating/expanding isolated B cells in vitro. We then demonstrate the steps involved in using the CRISPR/Cas9 system for site-specific KO of endogenous genes in B cells. This method allows for efficient KO of various genes, which can be used to study the biological functions of genes of interest. We then demonstrate the steps for using the CRISPR/Cas9 system together with a recombinant, adeno-associated, viral (rAAV) vector for efficient site-specific integration of a transgene expression cassette in B cells. Together, this protocol provides a step-by-step engineering platform that can be used in primary human B cells to study biological functions of genes as well as for the development of B-cell therapeutics.

Here we provide a detailed, step-by-step protocol for CRISPR/Cas9-based genome engineering of primary human B cells for gene knockout (KO) and knock-in (KI) to study biological functions of genes in B cells and the development of B-cell therapeutics.

使用CRISPR/Cas9的人类B细胞基因组工程

B cells are lymphocytes derived from hematopoietic stem cells and are a key component of the humoral arm of the adaptive immune system. They make ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

TGF-β介质到中微过渡（末端MT）和使用CRISPR/Cas9基因编辑对末端效应器的功能评估

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.