我们感谢达芙妮 Dambournet 为许多有见地的讨论和建议的基因编辑, 邵做插图, 安吉丽尼尔森的关键阅读手稿, 和安德鲁塔克生成 mEGFP 标签 Lamin B1 细胞线。我们希望感谢艾伦细胞科学研究所的干细胞和基因编辑和分析发展小组对基因编辑和质量控制过程的贡献。我们用来创建基因编辑细胞线的世贸中心线是由在旅行旅行学院和 UCSF 的布鲁斯 r. 康克林实验室提供的。我们感谢艾伦细胞科学研究所创始人保罗 g. 艾伦, 他的远见, 鼓励和支持。

1. crRNA 和供体模板质粒用于 FP 撞击的硅片设计
<ol><li>从 NCBI16或 UCSC 基因组浏览器17 (例如,基因库格式) 获取带注释的参考序列, 并将其导入到一个选择的生物信息学软件中。如果宿主基因组序列已知包含相对于引用的变体, 则现在通过调整生物信息学软件中的参考序列 (参见讨论) 来包含这些变量。</li>
	<li>找到所需的 FP 插入站点。对于 C 终端标记, FP 标记的序列将引入最后一个密码子的最后一个基元和停止密码子的第一个基元之间。对于 N 终端标记, FP 标记的序列通常会在起始密码子的最后一个基部和下一个密码子的第一个基之间引入。在某些情况下, 如起始密码子为单个密码子外显子, 或者在蛋白质总站附近存在信号序列, 则所需的 FP 插入站点可能位于更3ʹ的位置, 只要它继续处于帧中。</li>
	<li>在所需插入站点的每一侧使用 50 bp 作为任何公共可用 crRNA 设计工具的输入序列。在插入站点附近发现了 2-4 crRNA 目标后, 在生物信息学软件中对 crRNA 绑定站点和 protospacer 相邻的主题 (PAM) 序列 (NGG) 进行注释。这些 crRNAs 将被用来诱导双滞留断裂 (参见讨论更多的指导 crRNA 设计)。 注意: 自定义 crRNA 序列可以提交给一个商业供应商的合成 (推荐), 或序列可以作为一个出发点来设计克隆或体外综合战略, 这是超出本议定书的范围 (见讨论).</li>
	<li>要启动捐赠者模板质粒, 请使用所需插入站点上游的 1 kb 序列作为5ʹ同源臂 (这应该包括 N 终端插入的起始密码子), 并使用所需插入站点下游的 1 kb 序列作为3ʹ同源臂 (这应该包括 C 终端插入的停止密码子)。基地在二个同源胳膊之间通常不被省去。包括在同源武器中的细胞线特定变体将保留这些基因变体在结果编辑的细胞。</li>
	<li>在两个同源臂之间, 插入 FP (或其他敲入序列) 和链接器序列的序列 (有关连接器的更多指导, 请参见讨论)。对于 N 终端标签, 链接器序列应直接 3ʹ FP;对于 C 终端标记, 链接器序列应直接 5ʹ FP。</li>
	<li>中断捐赠者模板质粒中的 crRNA 绑定站点, 以防止 Cas9 切割捐赠者序列 (请参见讨论更改 crRNA 绑定站点时的注意事项)。如果可能, 将 PAM 中断到 NGG 或 NAG 以外的序列是首选。或者, 将点突变引入到 crRNA 种子区域中的三个碱基 (PAM 的10个碱基) 被预测足以扰乱 crRNA 的束缚。在捐赠者模板质粒中引入 FP 序列, 破坏了某些 crRNA 结合点;确保在这些情况下仍然不存在 PAM 或完整的绑定区域。 注:在硅供体模板质粒可以提交给基因合成的商业供应商, 或它可以作为一个出发点, 设计克隆战略, 这是超出了本议定书的范围。一个简单的骨干, 如 pUC19 或 pUC57 是足够的。</li>
</ol>2. Ribonucleoprotein (RNP) 转染 CRISPR/Cas9 介导的 hiPSCs 中的撞击
注: 在本协议中, 术语 "gRNA" 描述了合成 crRNA 和 tracrRNA 正确地重新悬浮、量化和预混合每个制造商的说明 (见材料表)。用1% 青霉素链霉素补充所有培养基。在艾伦细胞探险家18,19, 更详细地描述了世贸中心 hiPSC 线的一般培养指南。本协议采用了世台 hiPSCs, 但经过适当的转染优化, RNP 和供体模板质粒的电穿孔可以成功地适应其他细胞类型。
<ol><li>准备10µM gRNA 和野生型化脓Cas9 蛋白2,20的工作储存;保持冰块。准备1µg/µL 供体模板质粒的工作库存;保持室温 (RT)。对所有稀释使用 pH 值 8.0 TE 缓冲器。</li>
	<li>用5毫升新鲜生长培养基制备基质涂层的6井组织培养板, 并辅以10µM 岩石抑制剂 (Ri)。在孵化器的37°c 和 5% CO2保持板材与媒介, 直到准备板细胞在转染的做法以后 (最大 2 h)。 注: 本协议中使用的所有基体涂层板都是通过在冷 DMEM/F12 介质中添加一体积的冰冷胶稀释一点半, 根据艾伦细胞科学研究所的 hiPSC19号线培养协议。</li>
	<li>使用一个温和的单细胞离解试剂的建议, 在材料表中, 通道 hiPSCs 到单细胞悬浮和计数细胞使用自动细胞计数器, 或 hemocytometer。 注意: 这里使用的世贸 hiPSC 线的详细协议可以在艾伦细胞探险家19找到。简单地, 用 RT DPBS 冲洗细胞一次, 用离解试剂治疗3-5 分钟。然后将细胞 triturate 为单细胞悬浮, 由温和的吹打和颗粒离心。并用重悬生长培养基中的活细胞颗粒补充10µM Ri。<ol>
<li>准备一个整除 1.84 x 106细胞为每个实验条件将被转染在单独的1.5 毫升管。 注: 所有体积计算为4.5 µL 总反应量在100µL 电穿孔量, 乘以2.3 反应。这说明重复转染和多余的吹打错误。</li>
	</ol>
</li>
	<li>为每个实验条件准备 ribonucleoprotein (RNP) 复合管, 添加2.88 µL 10 µM gRNA 和2.88 µL 10 µM Cas9 到1.5 毫升管。在 RT 中孵育至少10分钟 (最大1小时)。</li>
	<li>球团一细胞整除 (在步骤2.3.1 中制备) 在 211 x g 3 分钟的 RT. 吸入上清和并用重悬细胞颗粒220µL 制造商的电穿孔缓冲器。</li>
	<li>将步骤2.5 中的220µL 悬浮细胞添加到步骤2.4 中的1.5 毫升 RNP 复合管中。</li>
	<li>将4.60 µL 1 µg/µL 供体模板质粒添加到步骤2.4 中制备的1.5 毫升管中。</li>
	<li>使用 nucleofection 尖和吸管混合管的内容2-3 次, 然后转移100µL 的悬浮到准备的电穿孔装置。避免在提示中引入任何气泡。应用 1300 V, 1 脉30毫秒。</li>
	<li>将悬浮液从步骤2.2 中轻轻地转移到准备好的6井板中, 旋转运动。分散的细胞通过轻轻地移动板块侧对边和前后。</li>
	<li>使用新的 nucleofection 尖端, 重复步骤 2.8-2.9 与其余100µL 的悬浮和转移到第二井的准备6井板。<ol>
<li>对每个 gRNA 和捐赠者模板质粒组合重复步骤2.4 至 2.10, 包括非靶向 gRNA、仅供体模板质粒和仅限缓冲控制。注意更换吸管和 nucleofection 提示, 避免交叉污染。</li>
	</ol>
</li>
	<li>孵育转染细胞在37°c 和 5% CO2。将介质改变为常规生长介质 (no Ri) 在24小时, 并继续喂养 hiPSCs 每24小时为72-96 小时, 监测融合。当 hiPSCs 达到60-80% 汇合时, 继续步骤3。 注: 重细胞死亡 (> 70%, 估计) 是正常的24-48 小时后转染。</li>
</ol>3. 认定编辑 hiPSCs 的富集
注意: 在对干细胞进行分类时, 根据讨论中的建议, 调整仪器设置以促进细胞存活。简单地, 使用最大的喷嘴可能 (130 µm), 低流速 (≤24µL/分钟), 无防腐剂鞘液 (如生理盐水, 见材料表), 和低采样压力 (10 psi)。
<ol><li>在开始一项外地资产管制试验之前, 将培养基改成生长介质, 辅以10µM Ri 和孵化细胞37摄氏度和 5% CO2 2-4 h, 以促进生存后, 可在外地。</li>
	<li>使用一个温和的单细胞离解试剂的建议, 在材料表中, 通道 hiPSCs 到单细胞悬浮在生长培养基补充10µM Ri19。</li>
	<li>过滤 hiPSC 悬浮通过35µm 滤网过滤器成聚苯乙烯圆底管。</li>
	<li>使用正向散射和侧散射 (包括高度与宽度) 对单元格进行排序, 以排除碎片和双峰。使用实时、缓冲区仅控制单元格设置 FP 正浇口, 这样 < 0.1% 的缓冲细胞才会落到门内。</li>
	<li>将 FP 阳性细胞的整个种群分类为 1.5-15 毫升聚丙烯管, 其中含有 0.5-2 毫升的 RT 生长介质, 辅以10µM Ri。 注: 聚丙烯可降低细胞对塑料的黏附能力。</li>
	<li>离心机收集的细胞在 211 x g 3 分钟的 RT。</li>
	<li>仔细吸入上清和并用重悬细胞颗粒在200µL 生长培养基补充10µM Ri。将多达3000个已排序的单元转移到一个新的基体涂层的 96-井板19的单井。 注: 在适当的仪器设置下, 细胞也可以直接分类 (散装) 成一个单井的基质涂层的96井组织培养板, 其中包含200µL 生长培养基补充10µM Ri 在建议密度 1,000-3, 000 细胞每井为 hiPSC。</li>
	<li>孵育排列的细胞在37°c 和 5% CO2。将媒体改为增长媒体, 辅以5µM Ri 24 小时。在48小时开始喂养细胞定期生长培养基 (no Ri) 每24小时72-96 小时, 监测融合。如果在96井板的一个井中, 至少有500个细胞被播种, 则在其后的存活率估计大于50%。<ol>
<li>当 hiPSCs 达到60-80% 汇合并显示成熟的形态 (平滑, 包装好的菌落中心), 通道进入一个较大的格式板, 如24井板, 然后从24井板成6井板。</li>
		<li>当 hiPSCs 在一个6井板达到60-80% 汇合并且显示成熟形态学, 扩展到100毫米板材, 重新板材为成像, cryopreserve 或种子在克隆采摘密度 (步 4)19。</li>
	</ol>
</li>
</ol>4. 生成认定编辑的克隆 hiPSC 线
<ol><li>使用在材料表中推荐的温和单细胞离解试剂, 通道 hiPSCs 进入单细胞悬浮, 并确定每毫升19的细胞数量。</li>
	<li>将被编辑的 hiPSCs 的种子1万细胞放到一个新鲜的基质涂层的100毫米组织培养皿中, 使用生长培养基补充10µM Ri19。在播种后, 将培养基改成生长培养基而不需 Ri 24 小时, 每24小时将 hiPSCs 与新鲜生长培养基一起喂养5-7 天。</li>
	<li>当 hiPSCs 形成了肉眼可见的菌落 (大约500µm) 时, 它们的大小足以被隔离。用吸余基质制备基质涂层的96井板, 并添加100µL 的生长介质, 辅以10µM Ri, 每井19。</li>
	<li>在解剖显微镜上使用 P-200 吸管, 或类似的, 轻轻地刮, 并从板块表面吸入单个菌落。在步骤4.3 中制备的 96-井板的单个井的转移容积 (~ 20-100 µL)。<ol>
<li>在所有殖民地被转移了之后, 在37˚C 和 5% CO2在组织培养孵化器孵化板材。将介质改变为常规生长介质 (no Ri) 在24小时, 并继续喂养细胞每24小时72-96 小时, 直到菌落大约翻了几番 (约1500µm)。 注: 建议在转染中使用每 crRNA 24-96 个菌落。孤立克隆的生存率通常大于95%。</li>
	</ol>
</li>
	<li>使用一个温和的单细胞离解试剂在材料表中建议, 通过 hiPSC 克隆成一个新的基质涂层96井板如下19。<ol>
<li>使用8通道吸引器, 从96井板的第一列中取出并丢弃介质。</li>
		<li>使用 P-200 多通道吸管, 添加〜200µL 的 DPBS 到第一列的96井板, 以洗涤细胞。使用8通道吸引器, 从96井板的第一列取出并丢弃 DPBS 洗涤。</li>
		<li>使用 P-200 多通道吸管, 将40µL 的离解试剂添加到96井板的第一柱上。</li>
		<li>重复步骤 4.5. 1-4. 5.3 为总共六列96井板, 改变提示, 确保不交叉污染水井。将该板块放置在37摄氏度孵化器中, 从分离试剂添加到第一列 (步骤 4.5.3) 的时间起3-5 分钟。 注: 建议每次只通过六列 (48 口井), 因为执行这些步骤所需的时间。第一次执行此协议时, 一次只传代一到两列。限制传代的列数, 确保细胞在离解试剂中不留太长时间, 这可能对 hiPSCs 有害。</li>
		<li>当板块第一列的单元格开始从底板上抬起时, 使用 P-200 多通道吸管将160µL 的 DPBS 添加到96井板的第一柱上, 并在 "12:00" 中轻轻 triturate 细胞。、"3:00"、"6:00" 和 "9:00" 位置的每一个井。将整个细胞悬浮量 (200 µL) 转移到 V 底96井板上。</li>
		<li>对具有离解试剂的细胞的剩余柱重复步骤 4.5.5;更改提示, 以不交叉污染水井。</li>
		<li>在 385 x g 的离心机上以3分钟的 RT 旋转 V 型底板。</li>
		<li>使用 P-200 多通道吸管, 轻轻地去除200µL 新鲜生长介质中的上清和并用重悬细胞, 补充10µM Ri 每井。对所有水井重复, 改变提示, 以免交叉污染。</li>
		<li>将所有的细胞悬浮液转移到一个新的基体涂层 96-井板19。在37摄氏度和 5% CO2的组织培养孵化器中孵化板块。将培养基改为正常生长培养基 (no Ri) 在24小时, 并继续喂养细胞每24小时72-96 小时, 直到大多数克隆达到60-80% 汇合。 注: 这篇文章有助于分散细胞, 并允许更多的增长超过96井板块的整个区域。</li>
	</ol>
</li>
	<li>观察克隆并为96井板 (例如, 1:10 或 1:8) 中的每个克隆确定适当的拆分比例。使用一个温和的单细胞离解试剂, 在材料表中建议, 通过 hiPSC 克隆成一个新的基质涂层96井板 (步骤 4.5. 1-4. 5.8) 转移适当的细胞悬浮率为每个克隆。在37˚C 和 5% CO2的组织培养孵化器中孵化板块。将培养基改为常规生长培养基 (no Ri) 24 小时, 并连续喂养细胞每24小时72-96 小时, 直到大多数克隆达到60-80% 汇合, 并显示成熟的形态学。 注意: 每个克隆可能有一个不同的分裂比率, 因为略有不同的增长率或生存从前一段, 所以这段经文有助于正常化的每一个克隆的细胞数量, 以冻结步骤遵循。由于生存率和生长速率的不同, 一些克隆在这些传代步骤中可能 overgrow 或无法生长。<ol>
<li>保存剩余的细胞悬浮物和颗粒为 gDNA 隔离由离心细胞在 V-底部96井板在 385 x g 为3分钟在 RT. 删除上清, 并进行 gDNA 隔离使用96井套件, 或储存板的颗粒细胞在20˚C 高达ee 周。</li>
	</ol>
</li>
</ol>5. 96 井板格式克隆细胞系的超低温保存
<ol><li>使用单细胞离解试剂, 通道 hiPSC 克隆如前所述 (步骤 4.5. 1-4. 5.7)。用 P-200 多通道吸管吸入上清液, 在60µL 的生长介质中重新悬浮, 辅以10µM Ri。对所有水井重复;更改提示, 以免交叉污染。</li>
	<li>将30µL 细胞悬浮液转移到非基质涂层的96井组织培养板上。然后迅速添加170µL 冻结缓冲器 (见材料表), 每井, 不混合。重复通过将剩余的30µL 的悬浮液转移到姊妹板中, 加入170µL 冷冻缓冲器。 注: 这一过程是在重复的姐妹板块, 以便在解冻后的细胞的一个单独的板块。将低温保存的细胞放入96井板中的每一个柱体, 可以加快解冻速度 (步骤 5.6)。</li>
	<li>包装板与 parafilm 和地方在 RT 发泡胶盒盖。把整个盒子放在-80 摄氏度的冰箱里。</li>
	<li>在 24 h 板以后可以从发泡胶盒转移并且存放在80˚C 至多四星期。 注: 虽然细胞暂时储存在-80 摄氏度, 基因质量控制化验可以执行的 gDNA 从细胞获得的步骤 4.6.1, 以确定克隆的解冻和传播, 如前发表的工作中讨论12. 简要地说, 拷贝数滴数字 PCR 法可用于鉴定含有 GFP 的一或两份拷贝的克隆, 而没有供体模板质粒的主干整合。最后点 PCR 检测和桑格测序的结合, 可以识别包含精确插入的克隆。</li>
	<li>解冻, 使整个板块到37°c 在一个组织培养孵化器, 仔细观察, 以冰丸融化。板块边缘的井往往先解冻。<ol>
<li>当所需克隆的冰粒融化时, 将整个200µL 轻轻地转移到15毫升圆锥管中, 其中含有3毫升的 RT 生长介质, 辅以10µM Ri 和离心机, 其 211 x g 为3分钟的 rt。</li>
		<li>在1毫升的 RT 生长培养基中吸出上清和并用重悬颗粒细胞, 辅以10µM Ri。转移到一个新的基质涂层24井板和孵化在37˚C 和 5% CO2。将介质改变为常规生长介质 (no Ri), 在24小时, 并继续喂养细胞每24小时72-96 小时, 直到克隆达到60-80% 融合, 并有成熟的形态学19。</li>
	</ol>
</li>
</ol>

<ol>
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作者没有什么可透露的。

本文提出的在 hiPSCs 中产生内在调节荧光蛋白融合的方法是一种多功能而有力的方法, 用于生成基因编辑细胞系, 其应用范围从活细胞成像到各种功能研究, 以及 "疾病在菜 "模型使用患者衍生的 hiPSC 线13,14,15。虽然这种方法已经被用来引入大 FP 标记的 n-或 C 终点的内源蛋白, 它可能会被用来引入其他标记或小的基因改变模型或正确的疾病导致突变22,23.对于较小的插入, 可减小同源臂的大小, 但此方法中显示的一般编辑方法仍可能适用于24、25。虽然使用 hiPSCs 是强烈鼓励他们的巨大效用, 仔细优化, 这个协议可能会修改, 以编辑其他哺乳动物细胞线的基因组。
在确定一个对 FP 标记感兴趣的基因时, 成绩单丰度估计 (从微阵列或 RNA 序列数据) 是一个很好的起点, 用于评估是否表达了一个基因或异构体, 尽管成绩单水平并不总是与蛋白质水平。这里所描述的这种浓缩策略对于那些至少在细胞类型中表达良好的基因最有效。这个策略也成功地选择了融合, 显示有点和/或离散的本地化模式, 如 centrin, desmoplakin, 和桩蛋白的信号, 背景比是非常低的12,19。没有高度表达或仅在导数细胞类型中表达的基因可能需要额外的选择策略。
人类细胞系中使用的 crRNA 和供体模板质粒设计的起始点应该是人类参考基因组 (GRCh38)。因为不同细胞系的基因组在同一物种内可能不同, 而且由于 CRISPR/Cas9 是序列特异的, 所以识别细胞线特定的变体 (单核苷酸多态性或插入/删除 (indels)) 是非常有用的。这与参考基因组不同, 并将其纳入设计中。这确保 crRNAs 将与宿主基因组兼容, 捐赠者模板质粒同源臂将保留任何细胞线特定变种。建议的策略是在设计过程中将纯合变体纳入 crRNAs 和供体模板质粒同源臂。合并异型变体是可选的。下面讨论了用于大型撞击实验的特定试剂和此协议的其他关键考虑事项。
Cas9 蛋白
使用 Cas9 蛋白的主要好处是, 引入 Cas9 和 gRNA 作为 RNP 复合物, 表明核酸酶活动的持续时间与以质粒为基础的方法相比, Cas9 和 gRNA 的表达可能会持续数天并导致到更高的和非目标活动26,27。使用 Cas9 蛋白的另一个好处是它可以随时在细胞内进行切割。这与更传统的使用 Cas9 mRNA 或 Cas9/gRNA 质粒的方法形成对比, 需要转录, 翻译和蛋白质处理26,28。Wildtype 的化脓Cas9 蛋白现在可从许多商业来源获得。
指南 RNA
在主基因组29、30、31、32中, 有许多公开的工具可用于查找在所需的 FP 插入站点附近有零个或很少预测目标的 crRNA 目标。hdr 的效率和 hdr 结果的精确度在给定轨迹12中使用的 crRNA 目标之间有很大差异。因此, 建议每个轨迹测试几个 crRNAs (2-4 和最好在所需插入站点的 50 bp 内), 因为这可能会增加成功编辑实验的可能性。目前提供 gRNA 的可能性包括合成2部分 crRNA 和 tracrRNA, 合成单 gRNAs (sgRNAs),体外转录 sgRNAs, 或提供质粒的细胞表达 sgRNA 从 U6 启动子。该协议没有为高解裂活动进行优化。未修改的2部分 crRNA 和 tracrRNA (见材料表) 用于生成单等位的 FP 标记细胞线, 同时对细胞造成最小的潜在扰动。
供体模板质粒
由于捐助者模板质粒中提供的某些同源臂序列将在 hdr 事件期间纳入宿主基因组, 因此应引入 crRNA 识别点的突变, 以防止 Cas9 跟踪 hdr 后的进一步分裂。通常最简单的破坏性变化是改变 PAM 序列。由于某些非规范的 PAM 序列仍然可以被野生类型的化脓Cas9 识别, 所以最好避免使用 NGG、NAG 或33。当变异的同源臂, 避免非同义突变和引进稀有密码子。如果对 PAM 序列的同义变化是不可能的, 考虑做三同义点变异在种子区域 (10 bp 近到 pam) crRNA 结合站点。在5ʹ未翻译区域 (UTR) 进行这些更改时, 应格外小心, 因为这些区域可能包含重要的管理序列。咨询一个遗传保护数据库, 如 UCSC 基因组浏览器的比较基因组学轨道可以提供指导, 在这些情况下, 因为对非保守基地的变化可能比对高度保守的基础17的变化更好的耐受性。有时仅仅插入 FP 序列就足以扰乱 crRNA 绑定站点 (如图 1所示);但是, 应检查新追加的序列, 以 crRNA 绑定和 PAM 序列的持久性。
建议在 FP 和原生蛋白之间连接器氨基酸, 以保存融合蛋白34的功能。通常氨基酸链接器可以选择其特定的电荷或大小。如果一个与目标内源融合蛋白相似的设计的 cDNA 融合得到了很好的研究, 同样的链接器序列可以用于 CRISPR/Cas9 实验12,19。如果此类信息不可用, 则在12中也成功使用了诸如 GTSGGS 这样的短链接器。其他研究已经证明了成功与一个普通的小 3-氨基酸链接器序列的各种目标35。
转染和富集
许多商业上可利用的转染试剂是为向细胞输送某些类型的分子而制定的, 而电穿孔系统可用于提供各种尺寸、电荷和成分的试剂。除了作为一种常见的转染方法, 如 hiPSCs 的硬到染细胞, 电穿孔也带来的好处, 提供所有三组件的 CRISPR/Cas9-mediated FP 敲, 如本方法所述。在开发此方法 (未显示数据) 的情况下, 与其他商用试剂相比, 电穿孔被发现能产生最佳效果, 其他产品也用于 RNP 交付26、28、36.
使用此协议编辑 hiPSCs 时, 应特别注意确保在基因编辑过程前后对细胞进行温和处理, 以获得最佳细胞存活率和最小自发分化。特别地, 应根据可能的最大喷嘴 (130 µm)、低流速 (≤24µL/分钟)、无防腐剂鞘液 (如生理盐水、见材料表) 和低样本来调整干细胞的分类方法。压力 (10 psi)。而不是单细胞排序, 结果在干细胞的最佳生存能力, hiPSCs 丰富的多项分类和扩大作为一个群体, 以优化细胞活力和干细胞完整性。但是, 单个单元格排序可能适合于不太敏感的单元格类型。为了促进细胞的存活, 细胞在收获后不超过一小时就被恢复到培养中, 在整个分拣过程中保持室温。对于某些细胞类型, 在整个排序过程中, 通过在冰 (4°c) 上孵化细胞来增强细胞存活率。
FP 阳性细胞的大量扩张为在生成克隆线之前的融合蛋白定位的影像学分析提供了一个评估人群的机会。虽然结果丰富的细胞数量可能是足够的一些研究, 这些人群经常显示的 FP 信号的强度不同。孤立的克隆线有一致的信号 (图 3), 使它们更适合于功能性实验12。
克隆细胞线生成
在整个编辑和克隆线生成过程中, 监测细胞形态学是很重要的。hiPSC 在无饲养条件下生长的殖民地应该展示光滑的边缘和均匀, 包装好的中心12,18,19。应在不到5% 的培养中观察分化细胞。挑选单个的殖民地时, 选择那些表现良好的形态。在96井板块传代事件中, 检查无性系的形态, 并停止那些杂草丛生的, 因为这可能导致分化或者是基因不稳定的征兆。
克隆细胞系的生成允许精确编辑的基因确认, 这是重要的, 因为 Cas9-induced 双链断裂在基因组经常被修复不精确地尽管在所需的轨迹中加入了标记。先前所描述的 PCR 检测结果显示, 十个独特的基因组位点 (45%) 在目标位点的捐赠质粒骨干整合或 (很少) 随机在基因组12中累积。此外, 23% 的 GFP 阳性克隆 (n=177) 横跨十个独特的基因座被发现在或接近预期的 crRNA 切割地点在未加标签的等位基因, 最有可能由于 NHEJ12。这种遗传分析的许多克隆线 (100 克隆/编辑) 强调了基因验证的重要性, 这是不可能在一个细胞的人口, 因为 FP 表达和预期融合蛋白定位不保证精确编辑12.此外, 这些基于 PCR 的化验方法不能对富集的细胞进行任何确定性的检测, 在有意义的分析完成之前, 需要克隆线的产生。对插入的 FP 标记的基因确认和验证未经编辑的等位基因的遗传完整性 (在单基因型的克隆) 是必要的, 以确保精确编辑在目标的轨迹以外的标记表达。
使用这种方法 (未公布的数据), exome 了低的双位基因编辑和缺乏非目标突变 (如由桑格和测序法测定).这与以前的研究描述的 CRISPR/Cas9 实验26,27的短期持久 RNP 的使用是一致的。缺乏双等位基因的克隆细胞系也可能是特定的轨迹, 或由于细胞无法容忍两个标记副本的基本蛋白质, 正如先前发表的实验所建议的, 假定双位基因编辑细胞是观察一个轨迹 (LMNB1), 但不是另一个 (TUBA1B)12。利用该方法对 ST6 β-半乳糖苷 alpha-2、6-sialyltransferase 1 (ST6GAL1) 和 RAB5A19RAB5A 成员 RAS 癌基因家族 (mEGFP) 进行标记, 生成了双位基因完全验证的克隆细胞系。
除了确认基因组中的编辑精确度外, 还有各种各样的质量控制化验, 可用于进一步表征克隆线, 并确定完成所有干细胞、基因组和细胞生物学标准的克隆, 以供今后研究使用。.细胞生物学和功能分析可以用来确认适当的表达, 定位和功能的融合蛋白12。与未编辑的家长控制的比较将有助于评估编辑过程对本地化、动态和功能的影响。其他的化验, 如生长分析和基因组稳定性测试也可以帮助确定标记蛋白是否扰动细胞。在该协议中使用 hiPSC 时, 对干细胞标记和分化电位的评估对于确定一个对下游研究有价值的克隆是至关重要的12。由于 hiPSC 的延伸培养导致遗传不稳定性, 监测克隆细胞系的生长速率和核型也很重要,12,37。然而, 最终打算使用的编辑的细胞将最终决定质量控制分析的水平和广度, 并将根据应用的不同。

使用基因组编辑策略, 特别是 CRISPR/Cas9, 研究细胞过程越来越容易接近和有价值的1,2,3,4,5,6,7. CRISPR/Cas9 的许多应用之一是引入 (通过同源定向修复 (HDR)) 的大型外源序列, 如 GFP 成特定基因组的基因座, 然后作为记者的活动的基因或蛋白质产品8.该技术可用于将荧光蛋白序列加入到内源开放阅读框架中, 由此产生的内在调节融合蛋白可用于可视化蛋白的亚细胞定位和动力学5 ,6,9,10,11。虽然内在标记的蛋白质提供了许多好处相比过度表达系统, 插入大序列到人类基因组是一个效率低下的过程, 通常要求选择或浓缩战略, 以获得细胞的数量, 可以容易地被学习5,12。
该协议描述了将荧光蛋白 (FP) 编码为理想基因组轨迹的 DNA 序列的插入。该协议包括设计和交付供体模板质粒, 和 ribonucleoprotein (RNP) 复合物 (野生型S 化脓Cas9 蛋白结合合成 CRISPR RNA (crRNA) 和反激活 crRNA (tracrRNA))。还描述了通过荧光活化细胞分选 (认定) 和克隆细胞线生成过程丰富的编辑细胞。到目前为止, 该方法已用于生成 hiPSC 线与 monoallelic 或 (很少) 双等位的绿色荧光蛋白 (GFP) 标签标签二十五蛋白代表主要细胞结构。这些努力所产生的经过编辑的细胞已被证实有预期的基因插入, 表达正确的本地化融合蛋白, 并保持干细胞和稳定的核型12 (和未发表的数据)。这种方法也被用来产生多个其他单一和双 (两个不同的蛋白质标记在同一细胞) 编辑的人口 hiPSCs (未发表的数据)。
人类 iPSCs 从一个健康的捐赠者被选择为这些基因组编辑的努力, 因为, 不同于许多常规细胞系, 他们是双, karyotypically 稳定, 不转化, 和增生。这些特性为研究基础细胞生物学和疾病建模提供了一个有吸引力的模型。此外, hiPSCs 的分化潜力提供了机会, 研究多个不同的血统和细胞类型平行的发展阶段, 使用等基因系细胞, 包括 organoids, 组织和 "疾病在一碟" 模型13 ,14,15。虽然这项协议是为 hiPSCs (世贸中心线) 开发的, 它可能是信息, 以发展的协议使用其他哺乳动物细胞线。

<table border="0" cellpadding="0" cellspacing="0" style="width:1169px;" width="1169">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Geneious R9</td>
			<td style="width:231px;">Biomatters, or similar</td>
			<td style="width:119px;"></td>
			<td style="width:515px;">bioinformatics software for in silico donor plasmid design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">TE Buffer pH 8.0</td>
			<td style="width:231px;">IDT, or similar</td>
			<td style="width:119px;">11-01-02-05</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:305px;">HERAcell VIOS 160i CO2 incubator, or similar</td>
			<td style="width:231px;">ThermoFisher Scientific, or similar</td>
			<td style="width:119px;">51030408</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Pipettes (1000 &#181;L, 200 &#181;L, 20 &#181;L, 10 &#181;L, 2 &#181;L)</td>
			<td style="width:231px;">Rainin, or similar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Pipette tips (1000 &#181;L, 200 &#181;L, 20 &#181;L)</td>
			<td style="width:231px;">Rainin, or similar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Multi-channel Pipette (200 &#181;L)</td>
			<td style="width:231px;">Rainin, or similar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Serological pipetes (25 mL, 10 mL, 5 mL)</td>
			<td style="width:231px;">Costar, or similar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">BRAND 8-Channel Manifold for Quiksip, Autoclavable</td>
			<td style="width:231px;">Millipore Sigma, or similar</td>
			<td style="width:119px;">BR704526-1EA</td>
			<td style="width:515px;">use with non-filtered pipet tips, such as Molecular BioProducts Low Retention Pipet Tips, Pure 10, below</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Molecular BioProducts Low Retention Pipet Tips, Pure 10</td>
			<td style="width:231px;">Thermo Fisher, or similar</td>
			<td style="width:119px;">3501-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">XP2 Pipette Controller</td>
			<td style="width:231px;">Drummond, or similar</td>
			<td style="width:119px;">4-000-501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Disposable Pasteur Pipets</td>
			<td style="width:231px;">VWR, or similar</td>
			<td style="width:119px;">53300-567</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Class II, Type A2 Biological Safety Cabinet</td>
			<td style="width:231px;">CELLGARD, or similar</td>
			<td style="width:119px;">NU-481</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Matrigel Matrix, Growth Factor Reduced</td>
			<td style="width:231px;">Corning</td>
			<td style="width:119px;">354230</td>
			<td style="width:515px;">lot tested before use with hiPSC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">DMEM/F12 (-phenol red)</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:119px;">11039-021</td>
			<td>cold, for diluting Matrigel 1:30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">mTeSR1 Complete Media</td>
			<td style="width:231px;">StemCell Technologies</td>
			<td style="width:119px;">85850</td>
			<td>recommended growth media for WTC hiPSC line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Penicillin-streptomycin</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:119px;">15070-063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">WTC hiPSC line</td>
			<td style="width:231px;">Coriell</td>
			<td style="width:119px;">GM25256</td>
			<td>the hiPSC line used in this protocol is available through Coriell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Tissue culture dish 100 mm</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:119px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">6-well Cell Culture Plate</td>
			<td style="width:231px;">CELLSTAR</td>
			<td style="width:119px;">657160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">StemPro Accutase</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:119px;">A11105-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Dulbecco&#39;s Phosphate Buffered Saline (DPBS) no calcium, no magnesium</td>
			<td style="width:231px;">Gibco</td>
			<td style="width:119px;">14190144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">15 mL polystyrene conical</td>
			<td style="width:231px;">Sarstedt</td>
			<td style="width:119px;">62.554.100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Y-27632 (ROCK Inhibitor)</td>
			<td style="width:231px;">StemCell Technologies</td>
			<td style="width:119px;">72308</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Edit-R CRISPR-Cas9 Synthetic crRNA, unmodified (custom sequence)</td>
			<td style="width:231px;">Dharmacon</td>
			<td style="width:119px;">Custom0247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Edit-R CRISPR-Cas9 Synthetic tracrRNA</td>
			<td style="width:231px;">Dharmacon</td>
			<td style="width:119px;">U-002005-05</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Recombinant wild-type Streptococcus pyogenes Cas9-NLS purified protein, 40 &#181;M</td>
			<td style="width:231px;">University of California-Berkeley QB3 Macrolab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">Custom donor plasmid (PriorityGENE)</td>
			<td style="width:231px;">Genewiz</td>
			<td style="width:119px;"></td>
			<td style="width:515px;">donor insert design was synthesized and cloned into pUC57 backbone by Genewiz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">DNA LoBind Tube 1.5 mL</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:119px;">22431021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">NucleoBond Xtra Maxi EF</td>
			<td style="width:231px;">Clontech</td>
			<td style="width:119px;">740424.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Neon Transfetion System</td>
			<td style="width:231px;">ThermoFisher Scientific</td>
			<td style="width:119px;">MPK5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Neon Transfection System, 100 &#181;L kit</td>
			<td style="width:231px;">ThermoFisher Scientific</td>
			<td style="width:119px;">MPK10096</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">5 mL Polystyrene Round-bottom Tube with Cell Strainer Cap</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:119px;">352235</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:305px;">15 mL High Clarity Polyproylene Conical Tube</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">FACSAriaIII Fusion</td>
			<td style="width:231px;">BD Biosciences</td>
			<td style="width:119px;">656700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">FACSDiva software</td>
			<td style="width:231px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">FlowJo version 10.2</td>
			<td style="width:231px;">TreeStar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">NERL Blood Bank Saline</td>
			<td style="width:231px;">ThermoFisher Scientific</td>
			<td style="width:119px;">8504</td>
			<td>used as preservative-free FACS Buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Olympus SZX7 Stereo Microscope, or similar</td>
			<td style="width:231px;">Olympus, or similar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Tissue culture plate, 96-well</td>
			<td style="width:231px;">Falcon</td>
			<td style="width:119px;">353072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">96 well Cell Culture Plate, V-Bottom</td>
			<td style="width:231px;">CELLSTAR</td>
			<td style="width:119px;">351180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">CryoStor CS10</td>
			<td style="width:231px;">Sigma</td>
			<td style="width:119px;">C2874-100ML</td>
			<td>used as cryopreservation buffer for cells in 96-well plate format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Parafilm</td>
			<td style="width:231px;">Bemis</td>
			<td style="width:119px;">PM-996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">24 well Cell Culture Plate</td>
			<td style="width:231px;">CELLSTAR</td>
			<td style="width:119px;">662160</td>
			<td></td>
		</tr>
	</tbody>
</table>

endogenous protein tagging in human induced pluripotent stem cells using crispr/cas9

提出了一种用于生成人类诱导的多能干细胞 (hiPSCs) 的协议, 该方法将内源蛋白融合到帧内 n-或 C 末端荧光标记中。原核 CRISPR/Cas9 系统 (聚簇定期 interspaced 短回文重复/CRISPR 相关 9) 可用于通过同源定向修复 (HDR) 将大型外源序列引入基因组基因座。为了达到预期的效果, 本协议采用了基于 ribonucleoprotein (RNP) 的方法, 其中野生型链球菌化脓Cas9 蛋白, 合成2部分导 RNA (gRNA), 捐赠者模板质粒通过穿孔。认定编辑细胞表达荧光标记蛋白是丰富的荧光活化细胞排序 (外地资产管制署)。然后生成克隆线, 并可对精确编辑结果进行分析。通过在兴趣基因基因组轨迹上引入荧光标记, 可以在内源调节控制下研究融合蛋白的亚细胞定位和动力学, 这是传统过度表达的关键改进系统。利用 hiPSCs 作为基因标记模型系统, 为研究 nontransformed 细胞中标记蛋白提供了机会。由于 hiPSCs 可以被区分成多种细胞类型, 这种方法提供了在各种等基因系细胞环境中创建和研究标记蛋白的机会。

本实验的目的是将 mEGFP (单体增强 GFP) 与核 lamin B1 蛋白相融合, 将 mEGFP 序列引入5ʹ基因的 LMNB1 端 (蛋白质的 N 终点)。根据 SGLRSRAQAS 荧光蛋白收集21的先前 cDNA 构造, 选择了一个链接器 (氨基酸序列)。由于每个候选 crRNA 的供体模板质粒中的 crRNA 结合区在 mEGFP 和链接器序列的硅插入后被打乱, 因此不需要进行点突变来破坏潜在的 crRNA 识别和裂解捐赠者序列的 Cas9 (图 1)。捐赠者序列包含 1 kb 同源武器侧翼两端的 mEGFP 链接器序列。结果 2734 DNA 的 bp 被克隆成一个 pUC57 骨干, 序列验证, 并得到的捐赠模板质粒被纯化使用无内毒素的最大的准备。对供体模板质粒和 RNP 复合物进行转染, 认定编辑细胞丰富, mEGFP 核 lamin B1 融合蛋白的定位由荧光显微镜 (图 2) 确定。只有 crRNA1 转染的结果在这里描述, 虽然两个 crRNA 序列产生认定编辑的人口12。
与不含 gRNA、Cas9 蛋白或供体模板质粒的负控制相比, 电穿孔反应 (仅限缓冲器), LMNB1 crRNA1 转染细胞包含 0.95% mEGFP 阳性细胞, 代表认定编辑mEGFP 核 lamin B1 人口 (图 3a)。这一结果是在许多基因组基因座上观察到的撞击效率范围内使用这种方法, 如前所报告的12。
mEGFP 阳性细胞被观察者隔离, 并通过活体显微成像来确认 mEGFP 核 lamin B1 融合蛋白对核包络的预期定位。在 LMNB1 crRNA1 种群中, 约有90% 的分类细胞被 mEGFP 阳性 (如显微镜所确定), 提示某些 mEGFP 阴性细胞在分拣过程中与 GFP 阳性细胞共纯化。这是一个可接受的浓缩水平, 允许采摘96克隆, 然后可以进行基因筛选, 以成功编辑。通常, 成功浓缩的截止是 50% GFP 阳性。
多数被排序的细胞人口显示荧光在核信封 (核边缘) 在 nondividing 细胞和在细胞质之内的一个延长的核叶片在有丝分裂期间提供信心正确基因组编辑在 LMNB1轨迹。丰富的人口包含有明亮或微弱信号的细胞。这种信号强度的差异可能反映正确和不正确的编辑结果的组合, 并强调了生成基因验证的克隆线用于进一步研究的效用 (见讨论) (图 3b)12。克隆线生成后, 经基因验证的细胞在显微实验中显示出均匀的 GFP 强度 (图 3c)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58130/58130fig1.jpg"> 图 1.LMNB1 基因 n-终点 GFP 标记的设计策略.GFP 标签是专为 N 终端插入5ʹ的第一个外显子的 LMNB1 位于5号染色体。5ʹ和3ʹ同源武器分别为 1 kb, 在起始密码子 (ATG) 和第二个密码子 (同源臂仅部分代表图) 之间相遇。两个候选 crRNAs 的设计, 以指导 Cas9, 以尽可能接近预期的插入点, 而仍然是唯一的基因组。序列为 mEGFP 和氨基酸链接器入了仅3ʹ开始密码子 ( mEGFP 和链接器序列不缩放 ) 。<a href="https://www.jove.com/files/ftp_upload/58130/58130fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58130/58130fig2.jpg"> 图 2.生产内在标记 hiPSC 克隆线的工作流.转染成分, 包括 Cas9/crRNA/tracrRNA RNP 络合物 (如红色 Cas9 蛋白与金 crRNA 和紫色 tracrRNA), 捐赠模板质粒包含同源武器 (如金), 和 FP + 链接器序列 (显示为绿色), 是转.4天后, FP 阳性认定编辑细胞通过将所有已排序的细胞播种到一个96井板 (1000 细胞) 的单个井中, 并在文化中扩展到数以百万计的工作人口, 从而丰富了它的数量。细胞可以被鉴定为 "丰富的人口" (参见协议步骤 3.8)。由于 HDR12的变化率, FP 阳性细胞的产量因实验的不同而异;成功的浓缩通常包括 300-5, 000 细胞在转染大约 1.6 x 106臀部细胞之后。初步的影像学研究证实了融合蛋白在富集人群中的信号和定位。菌落被手动挑选成一个96井板, 用于扩张和冷冻保存。进一步的基因组质量控制筛选使用液滴数字 pcr (ddPCR) 和其他 PCR 的化验, 然后用于确定正确编辑克隆, 如前所述12。<a href="https://www.jove.com/files/ftp_upload/58130/58130fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58130/58130fig3.jpg"> 图 3.认定编辑细胞群的富集.(A)转染后四天 LMNB1 编辑细胞的流式细胞仪图。y 轴显示 GFP 强度, x 轴显示正向散射。排序门是基于仅限缓冲区的控件设置的。由于 hiPSCs 是敏感的摄动, 活/死污点被省略和一个非常保守的 FSC/SSC 门使用。(B)富集后, LMNB1 Cr1 编辑细胞的数量显示, 90% 的细胞与 GFP 定位到核信封 (预期 LMNB1 本地化)。种群中含有不同的 gfp 强度的细胞以及一些 gfp 阴性细胞。刻度条是10微米。(C)在克隆线生成后, 细胞显示出均匀的 GFP 强度, 其中某些细胞周期依赖差异。刻度条是20微米。

Watch this Scientific Journal Video about Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9 at JoVE.com

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

这里描述的是一个协议, 用于标记内在表达的蛋白与荧光标记在人类诱导多潜能干细胞使用 CRISPR/Cas9。认定编辑细胞通过荧光活化细胞分选, 生成克隆细胞系。

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

endogenous-protein-tagging-human-induced-pluripotent-stem-cells-using

Nanyang Technological University

Research

JoVE Journal

Genetics

26.0K 次观看.  Allen Institute for Cell Science. 这里描述的是一个协议, 用于标记内在表达的蛋白与荧光标记在人类诱导多潜能干细胞使用 CRISPR/Cas9。认定编辑细胞通过荧光活化细胞分选, 生成克隆细胞系。

视频: Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using 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 ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

CRISPR/Cas9-mediated 目标集成在体内使用基于同源介导的端接连接策略

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

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. ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical regions, is caused by infection with Plasmodium parasites. The development of more effective drugs to combat malaria can be accelerated by improving our understanding of the biology of this complex parasite. Genetic manipulation of these parasites is key to understanding their biology; however, historically the genome of P. falciparum has been difficult to manipulate. Recently, CRISPR/Cas9 genome editing has been utilized in malaria parasites, allowing for easier protein tagging, generation of conditional protein knockdowns, and deletion of genes. CRISPR/Cas9 genome editing has proven to be a powerful tool for advancing the field of malaria research. Here, we describe a CRISPR/Cas9 method for generating glmS-based conditional knockdown mutants in P. falciparum. This method is highly adaptable to other types of genetic manipulations, including protein tagging and gene knockouts.

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

We describe a method for generating glmS-based conditional knockdown mutants in Plasmodium falciparum using CRISPR/Cas9 genome editing.

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

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 ...