Name: introducing point mutations into human pluripotent stem cells using seamless genome editing
Uploaded: 2020-05-10T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing at JoVE.com

我们感谢Kosuke Yusa分享pMCS-AAT_PB-PGKpuroTK和pCMV-hyPBase质粒。我们感谢Jia-Yin Yang和Karamjit Singh-Dolt关于基因组编辑的有益讨论。D.C.H.实验室得到了首席科学家办公室（TC/16/37）和英国再生医学平台（MR/L022974/1）的奖项支持。Y.W.得到了中国国家留学基金委和爱丁堡大学资助的博士奖学金的支持。

1. CRISPR/Cas9n-sgRNA表达质粒的设计与构建
<ol><li>在要修改的位点附近任何5'-NGG的上游直接搜索20 bp引导序列。当使用来自 化脓性链球菌 的Cas9切口酶（Cas9n）时，需要一对单向导RNA（sgRNA）。这对sgRNA必须能够在切口时产生5'突出，偏移量为-4至20 bp，偏移量为6。</li>
	<li>订购必要的寡核苷酸和pSpCas9n-2A-puro载体。</li>
	<li>按照已发表的方案将 sgRNA 克隆到 pSpCas9n 载体中，以便与 Cas9n 共表达12.</li>
</ol>2. 设计和构建基于 piggyBac的靶向量
<ol><li>下载靶基因序列并搜索待修改位点附近的TTAA位点。 注意: TTAA 站点与要修改的站点之间的距离应尽可能小。如果在编码序列内且 100 bp 距离内不存在 TTAA 位点，则有必要通过进行同义核苷酸替换来引入一个位点。</li>
	<li>设计同源臂 （HA） 并在 HA 中加入所需的点突变。HA 的最佳长度应在 500 - 1200 bp 左右。 注意:建议引入同义核苷酸替换来突变PAM序列，以避免潜在的Cas9n重新切割。</li>
	<li>按照既定协议构建最终目标载体10. 注意:在这里使用的靶向载体中，PGK启动子驱动的puro-deltaTK选择盒两侧是转座子倒置重复。</li>
</ol>3. 人类多能干细胞的基因编辑
<ol><li>在 mTeSR1 培养基13 中的重组层粘连蛋白 521 包被表面 （5μg/mL） 上的加湿培养箱中保持人多能干细胞 （hPSC） 在 37 °C 和 5% CO2 上。细胞在 1:3 分流比后 72 小时后应达到 70-85% 汇合度。 注意:如果存在，请在每日培养基更换之前轻轻吸走自发分化的细胞。</li>
	<li>在转染当天，在37°C下用300μL层粘连蛋白521溶液（5μg/ mL）包覆24孔板的足够孔至少2小时。</li>
	<li>轻轻取出包衣溶液，向每个孔中加入 300 μL 补充有 10 μM ROCK 抑制剂的新鲜 mTeSR1 培养基，然后将板放回培养箱以接收细胞。 注意:去除层粘连蛋白涂层溶液后，不要让板在任何时候干燥。</li>
	<li>从培养箱中移出原液hPSC，除去用过的培养基，然后使用1 mL无菌1x DPBS洗涤细胞一次。</li>
	<li>向6孔板的每个孔中加入1mL温和的细胞解离试剂，并在37°C下孵育6-8分钟以解离细胞。 注意:轻轻敲击平板并检查细胞是否可以轻松分离，以确定消化时间是否足够长。</li>
	<li>使用 P1000 吸头轻轻上下移液，以去除所有 hPSC。</li>
	<li>通过将 2 mL 新鲜 mTeSR1 培养基加入补充有 10 μM ROCK 抑制剂 （Y-27632） 到细胞悬液中来终止解离。</li>
	<li>充分混合并将单细胞悬液转移到 50 mL 管中，并在室温下以 200 x g 离心 3 分钟。</li>
	<li>除去上清液，并将细胞重悬于补充有 10 μM ROCK 抑制剂的 2 mL 新鲜 mTeSR1 培养基中。</li>
	<li>使用血细胞计数器计数活细胞。使用台盼蓝染色并排除死细胞。</li>
	<li>将 8 x 10 5 至 1 x 106 活细胞转移到1.5 mL 管中以进行每次核转染反应，并以 200 x g 离心 3 分钟。然后小心地吸出上清液。</li>
	<li>将 3 μg 配对的 Cas9n-sgRNA 表达质粒和 5 μg 靶向载体质粒混合在人干细胞核转染试剂盒的 100 μL 混合核转染溶液/补充剂中。使用试剂盒中的GFP对照质粒，并制备GFP对照质粒混合物。 注意:重要的是对所有质粒进行最大程度的制备，以减少核转染前的内毒素污染。质粒的浓度应约为1μg/μL。</li>
	<li>使用DNA混合物（体积~111μL）重悬制备的细胞并将其转移到电穿孔比色皿（与核转试剂盒一起提供）中，避免任何气泡。</li>
	<li>通过选择人多能干细胞的优化条件，使用核转染装置对细胞进行电穿孔。 注意:如果首次使用人多能干细胞的核转检测试剂盒，则需要测试电穿孔程序并选择最有效的程序。</li>
	<li>立即将 500 μL 新鲜和温热的 mTeSR1 培养基加入 37 °C mTeSR1 培养基中，并补充有 10 μM ROCK 抑制剂到电穿孔细胞中。将混合物转移到从步骤3.2和3.3制备的24孔板的2孔中。</li>
	<li>快速将板放回37°C / 5%CO2 培养箱中，让细胞恢复。</li>
	<li>12-16小时后，更换细胞维持培养基。如果细胞建立了细胞间接触，请撤回ROCK抑制剂，如果没有，请继续用抑制剂补充培养基。</li>
	<li>在24-48小时之间，通过检查对照细胞中的GFP表达来检查核转染效率。GFP阳性细胞应至少为30%。</li>
	<li>核转染后48小时，通过向mTeSR1培养基补充1μg/ mL嘌呤霉素开始选择细胞。</li>
	<li>核转染后72小时，用0.5μg/ mL嘌呤霉素补充mTeSR1培养基。如果细胞汇合度低于30%，还要用10μM ROCK抑制剂补充培养基。</li>
	<li>核转染后4-6天，将嘌呤霉素耐药细胞传代至10-15 x 96孔板，浓度为0.8个细胞/孔。 注意:必须用 10 μM ROCK 抑制剂和 0.5 μg/mL 嘌呤霉素补充培养基。</li>
	<li>将这些细胞在37°C / 10%CO2 下维持10-12天以形成单细胞衍生的集落。播种后 7 天加满培养基一次。 注意:根据我们的经验，增加CO2 水平有助于单个hPSC形成菌落。</li>
	<li>标记含有单个菌落的孔，并用含有 0.5 μg/mL 嘌呤霉素但没有 ROCK 抑制剂的新鲜 mTeSR1 培养基替换培养基。 注意:从这一点开始，细胞将在37°C / 5%CO2下生长。</li>
	<li>两天后，更换含有未分化菌落的孔的培养基。用 10 μM ROCK 抑制剂和 0.5 μg/mL 嘌呤霉素补充培养基。 注意:在某些孔中，细胞可能已经分化，需要丢弃。</li>
	<li>使用 P2 移液器吸头轻轻刮掉菌落。将来自一个菌落的细胞悬液转移到单独的96孔板上的2个新孔中，并使用一个用于基因分型，一个用于维持。 注意:重要的是要小心地维持菌落并将自发分化水平保持在最低限度。</li>
	<li>一旦大多数孔中的汇合度达到50%或以上，就取出含有细胞的板进行基因分型。倾倒用过的培养基，然后用DPBS洗涤细胞一次。</li>
	<li>使用Bradley裂解缓冲液裂解孔中的细胞，并按照相关方案（https://mcmanuslab.ucsf.edu/protocol/dna-isolation-es-cells-96-well-plate）从板中的每个孔中分离基因组DNA。</li>
	<li>使用三引物连接PCR方法对左右同源臂进行独立基因分型10. 注意: 图2显示了PCR方法的方案。</li>
	<li>发送来自 4-5 个菌落的两个同源臂的连接 PCR 产物进行 Sanger 测序并获得序列。 注意:2个已建立菌落的测序结果如图 3A所示。</li>
	<li>保留具有正确基因型的菌落并丢弃其余的菌落。</li>
	<li>在连续嘌呤霉素选择下扩增正确的菌落，并在尽可能早的传代中冷冻它们。</li>
</ol>4. 从靶向人多能干细胞中去除转座子
<ol><li>将一个具有正确基因型的菌落维持在 0.5 μg/mL 嘌呤霉素选择下。</li>
	<li>如上所述，用5μg超活性转座酶（pCMV-hyPBase）对8 x 10 5至1 x 106个细胞进行核苷（第3节，步骤3.4-3.18）。并行执行GFP对照核转染。 注意:嘌呤霉素应在核化这些细胞后立即从mTeSR1培养基中去除。</li>
	<li>按照公布的程序10，用去除puro-deltaTK选择盒生长和筛选细胞。 注意:请务必仔细遵循原始程序。FIAU，或1-（2-脱氧-2-氟-β-d-阿拉伯呋喃糖基）-5-碘尿嘧啶）被用作基于单纯疱疹病毒衍生的胸苷激酶（HSV-tk）阴性选择的胸苷类似物。</li>
	<li>对修改区域进行排序以确认转座子的去除。 注意:3个已建立菌落的测序结果如图 3B所示。</li>
	<li>保持具有正确基因型的菌落并扩展以供进一步使用。</li>
	<li>在将其用于进一步分析之前，对所选菌落中的多能性标志物进行表征。 注意:两个已建立菌落的表征如图 4所示。</li>
</ol>

<ol>
	<li>Rashidi, 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=3D+human+liver+tissue+from+pluripotent+stem+cells+displays+stable+phenotype+in+vitro+and+supports+compromised+liver+function+in+vivo.">3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.</a> Archives of Toxicology. 92 (10), 3117-3129 (2018).</li><li>Hockemeyer, 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=Efficient+targeting+of+expressed+and+silent+genes+in+human+ESCs+and+iPSCs+using+zinc-finger+nucleases.">Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases.</a> Nature Biotechnology. 27 (9), 851-857 (2009).</li><li>Porteus, M. H., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+nucleases+stimulate+gene+targeting+in+human+cells.">Chimeric nucleases stimulate gene targeting in human cells.</a> Science. 300 (5620), 763 (2003).</li><li>Watanabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nature Biotechnology. 25 (6), 681-686 (2007).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+programmable+dual-RNA-guided+DNA+endonuclease+in+adaptive+bacterial+immunity.">A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.</a> Science. 337, 816-822 (2012).</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>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), 819-824 (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>Yusa, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+correction+of+&#945;1-antitrypsin+deficiency+in+induced+pluripotent+stem+cells.">Targeted gene correction of &#945;1-antitrypsin deficiency in induced pluripotent stem cells.</a> Nature. 478 (7369), 391-394 (2011).</li><li>Yusa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seamless+genome+editing+in+human+pluripotent+stem+cells+using+custom+endonuclease-based+gene+targeting+and+the+piggyBac+transposon.">Seamless genome editing in human pluripotent stem cells using custom endonuclease-based gene targeting and the piggyBac transposon.</a> Nature Protocols. 8 (10), 2061-2078 (2013).</li><li>Wang, 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=Multiomics+Analyses+of+HNF4&#945;+Protein+Domain+Function+during+Human+Pluripotent+Stem+Cell+Differentiation.">Multiomics Analyses of HNF4&#945; Protein Domain Function during Human Pluripotent Stem Cell Differentiation.</a> iScience. 16, 206-217 (2019).</li><li>Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+engineering+using+the+CRISPR-Cas9+system.">Genome engineering using the CRISPR-Cas9 system.</a> Nature Protocols. 8 (11), 2281-2308 (2013).</li><li>Wang, 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=Defined+and+Scalable+Generation+of+Hepatocyte-like+Cells+from+Human+Pluripotent+Stem+Cells.">Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells.</a> Journal of Visualized Experiments. (121), 1-8 (2017).</li><li>Eggenschwiler, 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=Improved+bi-allelic+modification+of+a+transcriptionally+silent+locus+in+patient-derived+iPSC+by+Cas9+nickase.">Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase.</a> Scientific Reports. 6, 1-14 (2016).</li></ol>

D.C.H 是 Stemnovate Limited 的联合创始人、股东和董事。

本文描述的方案可用于将预定点突变引入hPSCs的内源性位点。基于 piggyBac的靶向载体和配对的CRISPR/Cas9n表达质粒的组合被证明是可靠和有效的11。在HNF4α基因编辑之后，通过连接PCR筛选确定的43个分析克隆中有12个被正确靶向。具体而言，双等位基因靶向效率约为21%（9/43），单等位基因靶向效率约为7%（3/43）。
在靶向实验之后，维持嘌呤霉素的选择至关重要，以便在第一轮筛选期间保持目标位点可及小 猪Bac 转座酶。这将最大限度地减少PGK启动子驱动的puro-deltaTK选择盒的沉默，并减少第二轮筛选10的背景。最近的一份报告显示，在hPSC14中，CAG启动子比PGK启动子更耐沉默。因此，更换选择盒的启动器可以在未来改进该系统。比较hyPBase和GFP转染细胞的耐药菌落数量也很重要。成功去除转座子后，hyPBase-的存活菌落应该比GFP转染的细胞更多。除了转座子切除的PCR分析外，还需要对修饰区域进行直接测序以确认切除保真度。
基于Yusa的基于piggyBac转座子的靶向载体策略9，10，上述程序侧重于提高hPSCs核转染和单细胞克隆效率。优化细胞接种密度以减少异质集落形成的可能性。我们还采用10%CO2下的10-12天培养来提高基因型筛选的菌落产量。根据我们的经验，每个96孔板可以获得大约20个菌落。尽管此步骤可能比其他方法花费更多时间，但它可靠且高效。
根据需要，可以设计靶向载体来引入或纠正点突变，创建报告细胞系以及敲除基因表达。总之，CRISPR/Cas9（n）系统和靶向载体的结合是递送不同类型转基因hPSC的有效方法。

人类多能干细胞（hPSCs）代表了用于研究或临床的无限体细胞来源1。hPSC中的基因靶向提供了一种强大的方法来研究细胞特异性过程中的基因功能和了解疾病的机制。尽管存在有效的基因组编辑方法，但修饰hPSC中的基因在技术上仍然具有挑战性。hPSCs中通过同源重组进行的标准基因靶向频率较低，甚至在某些基因2上检测不到，因此在这些细胞中需要DNA双链断裂（DSB）刺激的基因靶向（称为基因编辑）2，3。此外，hPSCs的转染和随后的单细胞克隆不是很有效，即使通过使用Rho相关蛋白激酶（ROCK）抑制剂4可以减少单细胞相关细胞凋亡。最后，目的基因上潜在的靶向和脱靶突变也可能是有问题的。因此，可靠的方案对于在hPSC中进行量身定制的遗传变化至关重要。
RNA引导的CRISPR（成簇规则间隔短回文重复序列）和CRISPR相关蛋白9（Cas9）技术现在是基因编辑的成熟工具。转录的CRISPR RNA（crRNA）和反式激活RNA（tracrRNA）形成单一向导RNA（sgRNA），其与Cas9蛋白复合，允许基因特异性切割5。CRISPR/Cas9系统是通过改变sgRNA的20 bp引导序列来编程的，这意味着几乎任何满足原间隔邻序（PAM）要求的位点都可以被编辑。然而，野生型Cas9可以容忍其引导序列和DNA靶标之间的一些不匹配，这可能会导致不必要的脱靶效应。为了提高其特异性，已经开发出一种切口酶突变体（Cas9n）。含有D10A或N863A突变的Cas9n仅具有一个功能性核酸酶结构域，因此只能在一条链上切开DNA。一对适当间隔和定向的Cas9n可以有效地诱导DNA DSB，并且脱靶效应显着降低6。为了确保有效的Cas9n修饰，已经表明，理想情况下，两个Cas9n-sgRNA应以-4至20 bp的偏移放置，并始终产生5'突出6。
Cas9（n）-sgRNA诱导的DSB可用于hPSC7，8的基因组编辑。它可以通过非同源末端连接修复产生基因敲除，或者如果存在供体DNA模板，则通过同源定向修复（HDR）引入基因修饰。这里描述的方案使用基于piggyBac转座子的供体质粒（或靶向载体）进行HDR，其中耐药性标记物两侧是转座子倒置重复序列。这种方法的优点包括有效的筛选和无缝的基因编辑，正如Yusa等人首先证明的那样9，10。药物选择盒允许用整合载体富集细胞，随后通过连接PCR筛选细胞以鉴定HDR衍生的细胞。此外，可以定位药物选择盒以替换Cas9（n）切割的目标序列，因此HDR后不会发生进一步的DNA断裂，从而消除了Cas9（n）重新切割引起的"on"靶突变。此外，通过利用piggyBac转座酶催化的精确切除，然后将选择标记从基因组中切除而不会留下疤痕。去除药物选择标记9后，仅保留用于piggyBac插入的原始内源性TTAA序列。即使必须通过引入替代来创建TTAA站点，与其他方法相比，干扰监管元素的机会也会减少10。
在这里，我们描述了在hPSC中实施无缝基因组编辑的详细程序。在两步遗传选择中结合基于 piggyBac的供体质粒和CRISPR-Cas9切口酶突变体，我们将两个预定点突变引入肝细胞核因子4α（HNF4α）基因11中。这种方法可靠且高效，并且可以深入分析HNF4α在人多能干细胞的内胚层和肝细胞规格中所起的重要作用11。

<table><tbody><tr><td>Anti-Human SSEA4 PE</td><td>eBioscience</td><td>12-8843-42</td><td></td></tr><tr><td>Anti-Human TRA-1-60 PE</td><td>eBioscience</td><td>11-0159-42</td><td></td></tr><tr><td>DPBS</td><td>Thermo Fisher Scientific</td><td>14190144</td><td></td></tr><tr><td>DPBS with calcium and magnesium</td><td>Thermo Fisher Scientific</td><td>14040133</td><td></td></tr><tr><td>FIAU</td><td>Moravek</td><td>M251</td><td></td></tr><tr><td>Gentle cell dissociation reagent</td><td>Stem Cell Technologies</td><td>07174</td><td></td></tr><tr><td>H9 human embryonic stem cells</td><td>WiCell</td><td>WA09</td><td></td></tr><tr><td>Human NANOG antibody</td><td>R&amp;amp;D systems</td><td>AF1997</td><td></td></tr><tr><td>Human OCT4 antibody</td><td>Abcam</td><td>AB19857</td><td></td></tr><tr><td>Human stem cell Nucleofector kit 1</td><td>Lonza</td><td>VPH-5012</td><td></td></tr><tr><td>Mouse IgG3 isotype control PE</td><td>eBioscience</td><td>12-4742-41</td><td></td></tr><tr><td>mTeSR1 medium</td><td>Stem Cell Technologies</td><td>85850</td><td></td></tr><tr><td>Nucleofector 2b device</td><td>Lonza</td><td>AAB-1001</td><td></td></tr><tr><td>pCMV-hyPBase</td><td>Wellcome Trust Sanger Institute</td><td>N/A</td><td></td></tr><tr><td>pMCS-AAT_PBPGKpuroTK</td><td>Wellcome Trust Sanger Institute</td><td>N/A</td><td></td></tr><tr><td>pSpCas9n(BB)-2A-Puro (PX462)</td><td>Addgene</td><td>PX462</td><td></td></tr><tr><td>Puromycin dihydrochloride</td><td>Thermo Fisher Scientific</td><td>A11138-03</td><td></td></tr><tr><td>QIAprep spin maxiprep kit</td><td>Qiagen</td><td>12162</td><td></td></tr><tr><td>QIAprep spin miniprep kit</td><td>Qiagen</td><td>27106</td><td></td></tr><tr><td>Recombinant laminin 521</td><td>BioLamina</td><td>LN521</td><td></td></tr><tr><td>Trypan blue solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td></td></tr><tr><td>Y-27632(Dihydrochloride)</td><td>Millipore</td><td>SCM075</td><td></td></tr></tbody></table>

introducing point mutations into human pluripotent stem cells using seamless genome editing

定制设计的核酸内切酶，如RNA引导的簇状规则间隔短回文重复序列（CRISPR）-Cas9，能够在哺乳动物细胞中进行高效的基因组编辑。在这里，我们描述了无缝基因组编辑肝细胞核因子4α（HNF4α）位点的详细程序，作为人类多能干细胞的一个例子。在两步遗传选择中结合基于 piggyBac的供体质粒和CRISPR-Cas9切口酶突变体，我们证明了HNF4α位点的正确和有效靶向。

针对基于矢量的敲入策略 选择肝细胞核因子4α（HNF4α）基因进行靶向基因组编辑，将两点突变引入外显子8。在待修饰位点附近设计了一对具有11 bp偏移的Cas9n-sgRNA。基于 piggyBac的靶向载体作为同源定向修复模板，用于引入所需的点突变。将包含同义核苷酸取代或所需点突变的967 bp 5'-HA和1142 bp 3'-HA扩增并克隆到最终靶向载体中。 piggyBac 插入位点距离两个所需点突变分别为16 bp和22 bp。含有puro-deltaTK选择盒的菌落在第一轮中用嘌呤霉素选择。在第二轮通过转座酶切除去除选择盒后，仅将所需的点突变整合到基因中即可无缝修饰靶位点（图1）。
人类多能干细胞的基因编辑 为了筛选正确靶向的细胞，使用基于三引物的PCR方法进行基因分型（图2）。进行桑格测序以确认PCR结果（图3A）。取出选择盒后，再次对修饰区域进行测序以确认正确引入所需的点突变（图3B）。
编辑人多能干细胞的集落建立和表征 根据需要选择和扩增具有正确基因型的菌落。在用于进一步分析之前，需要对已建立的菌落进行表征。编辑的细胞具有与亲本细胞相同的形态（图4A）。它们还表达具有代表性的人类多能干细胞标志物，包括转录因子NANOG和OCT4（图4 B），以及细胞表面标志物SSEA4和TRA-1-60（图4C）。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61152/61152fig1.jpg"> 图 1:针对基于矢量的敲入策略11. 使用一对Cas9n-sgRNA表达质粒诱导HNF4α基因外显子8的DNA双链断裂。使用带有选择盒的靶向载体引入位于16 bp和22 bp之外的预定点突变。该选择盒包含在 piggyBac 转座子中，由由组成型活性启动子（PGK）驱动的正阴性选择标记（puro-deltaTK）组成。通过同源定向修复途径，靶向细胞掺入选择盒。由转座酶介导的转座子切除导致仅存在点突变的无缝修饰。红叉表示所需点突变的位置。HA = 同源臂;PB = 小猪巴克;PM = 点突变。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61152/61152fig1large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61152/61152fig2.jpg"> 图 2:三种基于引物的 PCR 方法。 为了筛选基因靶向细胞，使用了基于PCR的基因分型。三个引物LA-F1，-R1和-R2用于扩增左同源臂区域。独立地，RA-F1，-F2和-R1用于扩增右同源臂区域。根据凝胶电泳结果，将非靶向细胞与杂合子和纯合子细胞区分开来。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61152/61152fig2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61152/61152fig3.jpg"> 图 3:转基因细胞的测序结果11. 对来自两个克隆的PCR产物进行测序，并确认选择盒在目标位点（A）上正确插入。5'和 3'piggyBac 倒置终端重复（ITR）两侧是TTAA直接重复。转座子切除后对3个克隆进行测序（B）。一对具有11 bp偏移的Cas9n-sgRNA用于引入DNA双链断裂。两个预定的点突变（A到G和A到C）被引入基因中。引入一个同义突变来突变原间隔相邻基序（PAM），另一个突变用于创建 piggyBac 切除所需的TTAA位点。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61152/61152fig3large.jpg" target="_blank">请点击此处查看此图的大图。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61152/61152fig4.jpg"> 图4:编辑的人多能干细胞的表征。 两个编辑的细胞系和亲本细胞的形态（A），比例尺= 100μm。通过免疫染色（B，比例尺= 50μm）检查多能干细胞标志物NANOG和OCT4的表达，以及通过流式细胞术（C）在两个修饰细胞系和亲本细胞中的SSEA4和TRA-1-60。IgG被用作阴性对照。DAPI用于染色细胞核。百分比计算为三个独立实验的平均值。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61152/61152fig4large.jpg" target="_blank">请点击此处查看此图的大图。</a>

Watch this Scientific Journal Video about Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing at JoVE.com

Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

在这里，我们描述了一种使用基于 piggyBac的供体质粒和Cas9切口酶突变体在人类多能干细胞中进行无缝基因编辑的详细方法。将两点突变引入人胚胎干细胞（hESCs）肝细胞核因子4α（HNF4α）位点的外显子8。

使用无缝基因组编辑将点突变引入人类多能干细胞

Custom designed endonucleases, such as RNA-guided Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9, enable efficient genome editing ...

introducing-point-mutations-into-human-pluripotent-stem-cells-using

Universidad San Sebastian

Research

JoVE Journal

Genetics

4.0K 次观看.  University of Edinburgh. 在这里，我们描述了一种使用基于 piggyBac的供体质粒和Cas9切口酶突变体在人类多能干细胞中进行无缝基因编辑的详细方法。将两点突变引入人胚胎干细胞（hESCs）肝细胞核因子4α（HNF4α）位点的外显子8。

视频: Introducing Point Mutations into Human Pluripotent Stem Cells Using Seamless Genome Editing

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

科研

发育生物学

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic &#946;-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Protocols to generate hPSC mutant lines using the iCRISPR platform and to differentiate hPSCs into glucose-responsive &#946;-like cells are described. Combining genome editing technology with hPSC-directed differentiation provides a powerful platform for the systematic analysis of the role of lineage determinants in human development and disease progression.

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding ...

遗传学

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human development and investigate the pathophysiology of disease. By employing site-specific nucleases (SSNs) for genome editing, the rapid derivation of new hPSC lines harboring specific genetic alterations in an otherwise isogenic setting becomes possible. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are the most commonly used SSNs. All of these nucleases function by introducing a double stranded DNA break at a specified site, thereby promoting precise gene editing at a genomic locus. SSN-meditated genome editing exploits two of the cell&#39;s endogenous DNA repair mechanisms, non-homologous end joining (NHEJ) and homology directed repair (HDR), to either introduce insertion/deletion mutations or alter the genome using a homologous repair template at the site of the double stranded break. Electroporation of hPSCs is an efficient means of transfecting SSNs and repair templates that incorporate transgenes such as fluorescent reporters and antibiotic resistance cassettes. After electroporation, it is possible to isolate only those hPSCs that incorporated the repair construct by selecting for antibiotic resistance. Mechanically separating hPSC colonies and confirming proper integration at the target site through genotyping allows for the isolation of correctly targeted and genetically homogeneous cell lines. The validity of this protocol is demonstrated here by using all three SSN platforms to incorporate EGFP and a puromycin resistance construct into the AAVS1 safe harbor locus in human pluripotent&#160;stem cells.

Genome editing of human pluripotent stem cells (hPSCs) can be done quickly and efficiently. Presented here is a robust experimental procedure to genetically engineer hPSCs as exemplified by editing the AAVS1 safe harbor locus to express EGFP and introduce antibiotic resistance.

建立基因组编辑的人多能干细胞系:从定位到隔离

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

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 ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

生物工程

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成<em&gt; AAVS1</em&gt;轨迹

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

医学

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

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

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

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

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

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

生物学

Zinc Finger Nuclease-Based Genome Editing: A Technique for Modifying Genome in Human Pluripotent Stem Cells by Double-Stranded Homology Dependant Repair Mechanism

Source: Blair, J. D., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/53583">Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation.</a> J. Vis. Exp. (2016).
This video demonstrates a technique of zinc-finger nuclease-based genome editing in human pluripotent stem cells (hPSCs). As hPSCs can potentially be differentiated into various cell types, this technology is helpful for in vitro pathological and pharmacological studies in a patient-specific manner.

基于锌指核酸酶的基因组编辑:一种通过双链同源依赖性修复机制修饰人类多能干细胞基因组的技术

Source: Blair, J. D., et al. Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation. J. Vis. Exp. (2016).
This ...

使用无缝基因组编辑将点突变引入人类多能干细胞

摘要

探索更多视频

此视频中的章节

转染，选择和人类诱导多能干细胞集落采摘TALEN靶向用GFP基因进入AAVS1安全港

基因组编辑和hPSCs定向分化为胰腺癌发展讯问谱系行列式

建立基因组编辑的人多能干细胞系:从定位到隔离

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

利用 CRISPR-Cas9 系统高效生成和编辑人胰细胞无饲养干细胞

CRISPR/Cas9介导的胚胎干细胞中的高效基因靶向，用于开发基因操纵的小鼠模型

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

CRISPR-Cas9 介导在无饲养层条件下培养的人多能干细胞中的基因缺失

基于锌指核酸酶的基因组编辑:一种通过双链同源依赖性修复机制修饰人类多能干细胞基因组的技术