Name: generation of genomic deletions in mammalian cell lines via crispr/cas9
Uploaded: 2015-01-03T14:00:00.000+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>

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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><tbody><tr><td>T4 polynucleotide kinase</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>T4 DNA ligase (with associated ligation buffer)</td><td>New England Biolabs</td><td>M0202T</td><td></td></tr><tr><td>Adenosine 5&#039;-triphosphate (ATP)</td><td>New England Biolabs</td><td>P0756S</td><td></td></tr><tr><td>BSA, molecular biology grade</td><td>New England Biolabs</td><td>B9000S</td><td></td></tr><tr><td>BbsI restriction enzyme (with associated NEB Buffer 2.1)</td><td>New England Biolabs</td><td>R0539S</td><td></td></tr><tr><td>pSpCas9(BB) (pX330)</td><td>Addgene</td><td>42230</td><td></td></tr><tr><td>S.O.C. medium</td><td>Life Technologies</td><td>15544-034</td><td></td></tr><tr><td>BTX ECM 830</td><td>Harvard Apparatus</td><td>45-0052</td><td></td></tr><tr><td>BTX solution and 2 mm cuvettes</td><td>Harvard Apparatus</td><td>45-0803</td><td></td></tr><tr><td>pmaxGFP plasmid</td><td>Lonza</td><td>VPA-1003</td><td></td></tr><tr><td>QuickExtract DNA Extraction Solution</td><td>Epicentre</td><td>QE09050</td><td></td></tr><tr><td>HotStarTaq PCR Master Mix Kit</td><td>Qiagen</td><td>203443</td><td></td></tr><tr><td>Zero Blunt PCR Cloning Kit</td><td>Life Technologies</td><td>K2700-20</td><td></td></tr><tr><td>Plasmid</td><td><a href="http://www.addgene.org/42230/" target="_blank"><img src="/img/new_window.png" /> Addgene</a></td><td>42230</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://jove.unicartagenaproxy.elogim.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://jove.unicartagenaproxy.elogim.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

Universidad de Cartagena UDEC

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Unit 2

Analyzing Gene Expression and Function

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95.2K 次观看.  Harvard Medical School. 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.

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

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

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

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

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 ...

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

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

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

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

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

发育生物学

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 ...

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

用于微RNA簇网络分析的CRISPR基因编辑工具

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...

生物学

使用 CRISPR/Cas9 系统进行着丝粒相关 Protein-E 基因编辑

Generation of Centromere-Associated Protein-E CENP-E-/- Knockout Cell Lines using the CRISPR/Cas9 System

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing in a variety of organisms. Centromere-associated protein-E (CENP-E) is a plus-end-directed kinesin required for kinetochore-microtubule capture, chromosome alignment, and spindle assembly checkpoint. Although cellular functions of the CENP-E proteins have been well studied, it has been difficult to study the direct functions of CENP-E proteins using traditional protocols because CENP-E ablation usually leads to spindle assembly checkpoint activation, cell cycle arrest, and cell death. In this study, we have completely knocked out the CENP-E gene in human HeLa cells and successfully generated the CENP-E-/- HeLa cells using the CRISPR/Cas9 system.
Three optimized phenotype-based screening strategies were established, including cell colony screening, chromosome alignment phenotypes, and the fluorescent intensities of CENP-E proteins, which effectively improve the screening efficiency and experimental success rate of the CENP-E knockout cells. Importantly, CENP-E deletion results in chromosome misalignment, the abnormal location of the BUB1 mitotic checkpoint serine/threonine kinase B (BubR1) proteins, and mitotic defects. Furthermore, we have utilized the CENP-E knockout HeLa cell model to develop an identification method for CENP-E-specific inhibitors.
In this study, a useful approach to validate the specificity and toxicity of CENP-E inhibitors has been established. Moreover, this paper presents the protocols of CENP-E gene editing using the CRISPR/Cas9 system, which could be a powerful tool to investigate the mechanisms of CENP-E in cell division. Moreover, the CENP-E&#160;knockout cell line would contribute to the discovery and validation of CENP-E inhibitors, which have important implications for antitumor drug development, studies of cell division mechanisms in cell biology, and clinical applications.

作者聚焦:建立 CENP-E 敲除 HeLa 细胞 – 一种研究驱动蛋白 7 CENP-E 生物学及其抑制剂的新方法

This article reports the construction of centromere-associated protein-E (CENP-E) knockout cells using the CRISPR/Cas9 system and three phenotype-based screening strategies. We have utilized the CENP-E&#160;knockout cell line to establish a novel approach to validate the specificity and toxicity of the CENP-E inhibitors, which is useful for drug development and biological research.

使用 CRISPR/Cas9 系统生成着丝粒相关蛋白- E CENP-E-/- 敲除细胞系

The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system has emerged as a powerful tool for precise and efficient gene editing ...