这项工作得到得克萨斯州癌症预防和研究所 (RP160283、RP140001 和 R1201) 的支持, 加布里埃尔的癌症研究天使基金会, 爱德华. 埃文斯基金会和 NIH (DK092883, CA183252, CA125123, P50CA126752,CA193235 和 DK107413)。M.C.G. 是由贝勒研究倡导者支持的学生科学家。流式细胞术由 NIH (国家研究资源赠款 S10RR024574、国家过敏和传染性疾病研究所 AI036211 和国立癌症研究所 P30CA125123) 部分支持贝勒医学院细胞分选核心。

该议定书遵循贝勒医学院人类伦理委员会的指导方针。所有实验程序都是由贝勒医学院的动物护理和使用委员会批准的。
1. sgRNA 前进设计
<ol><li>导航到 http://www.crisprscan.org/？page=track8以开始设计感兴趣的 sgRNAs。</li>
	<li>根据感兴趣的单元格类型, 单击 "鼠标" 或 "人" 按钮。</li>
	<li>在 UCSC 搜索框中输入感兴趣的基因, 然后按 "去"。</li>
	<li>放大并移动到该基因的区域 (i. e), 这是一个目标感兴趣的特定外显子。 注意:为了实现有效的基因破坏, 建议针对特定细胞类型中所有转录亚型中最早出现的外显子。</li>
	<li>确保潜在的 sgRNA 目标序列在 "基因 (CDS) 的 CRISPRscan 预测" 标题下列出。</li>
	<li>查找并单击一个目标序列, 其得分相对较高, 并且没有目标 (亮绿色突出显示)。复制显示在 "寡聚序列" 部分下的完整序列, 并将其粘贴到文本文档中。将 "ATAGC" 添加到序列的 3 ' 末尾。完成后, 请为全长序列放置顺序。 注意:对于每个目标序列, CRISPRscan8提供了一个 "集成的" 前向 sgRNA 底漆, 包括 T7 启动子、protospacer (目标) 序列和通用脚手架重叠序列 (请参见图 1A)。全长序列由56或57核苷酸组成, 这取决于 protospacer 的长度。</li>
</ol>2. sgRNA DNA 模板合成
<ol><li>溶解和稀释 sgRNA 和通用的转速底漆 (见表1为完整的序列)。为了获得10µM 最终浓度, 按照制造商的指示, 如何稀释底漆到100µM, 然后做1:10 稀释与核酸酶无水。</li>
	<li>要执行重叠 PCR (请参见图 1B), 在 PCR 条管中混合以下材料: 2 µL 前 sgRNA 底漆 (10 µM), 2 µL 通用转速脚手架底漆 (HPLC 纯化) (10 µM), 10 µL 高保真 DNA 聚合酶主混合 (2x) 和6µL 核酸酶-免费水。</li>
	<li>运行 PCR 以以下节目:95 °c 为 3 min,98 °c 为 5 s, 60 °c 为 5 s, 72 °c 为十年代, 去2为6个周期, 72 °c 为1分钟, 4 °c 永远。</li>
	<li>用 DNA 纯化柱纯化 PCR 产物 (见材料表)。按照制造商的指示和洗脱11.5 µL 洗脱缓冲器。</li>
	<li>在分光光度计上测量 PCR 产物的浓度。用洗脱缓冲器将仪器留空。 注意:DNA 浓度应介于50至120µL 之间。</li>
</ol>3.体外转录 sgRNA
<ol><li>将以下成分混合在 PCR 条管中 (试剂在 RNA 合成试剂盒中提供): 4 µL 洗脱 DNA, 4 µL dNTPs, 1 µL 10x 反应缓冲器, 1 µL RNA 聚合酶组合。</li>
	<li>将样品孵育37摄氏度, 至少4小时。</li>
	<li>应用 RNase 清洗剂从戴手套的手上取下 RNase。</li>
	<li>使每个 rna 样品多达50µL 的总容量与核酸酶水 (RNA 纯化的第一步跟随制造商指示)。</li>
	<li>进行 RNA 纯化后, 制造商的指示和洗脱在50µL 套件提供的核酸酶免费水。</li>
	<li>测量洗脱 sgRNA 在分光光度计上的浓度。用无核酸酶的水将仪器清空。 注意:纯化后的预期产量为 RNA 的 50-80 µg (即浓度为 1.0-1.5 µg/µL)。</li>
	<li>使用纯化的 sgRNA 立即或存储在整除数 2-4 µL 在-80 摄氏度长期。</li>
</ol>4. HSPC 隔离和文化
<ol><li>
小鼠 HSPCs 分离与培养 注意:在 2-6 月的时间内, 通过与变风量地球复兴社区 (JAX008610) 交叉的雄性和雌性 JAX004353 和 Rosa26-LSL-tdTomato (JAX007914), 获得如下结果。<ol>
<li>弄死麻醉小鼠颈椎脱位。 注意:两名训练有素的人应该通过注意到呼吸不足和心跳至少5分钟来独立验证成功的安乐死. 从动物身上取出皮肤。</li>
		<li>解剖胫骨, 股骨和髂嵴的小鼠, 并删除所有肌肉和结缔组织周围的解剖骨。在冰上放置完整的骨骼成组织培养皿与 HBSS 补充与2% 血清 (HBSS +)。当铝骨被清洗并转移到组织培养皿中时, 立即移动到层流罩。</li>
		<li>将被清洗的骨骼转移到无菌砂浆中, 含2毫升冰冷 HBSS + 每3块骨头。用杵把骨头粉碎成骨头碎片, 从内部释放骨髓。继续烦人, 直到骨头停止在杵下开裂。</li>
		<li>用40µm 细胞过滤器从砂浆中收集上清, 过滤成50毫升锥形管。用相同的40µm 过滤器冲洗剩下的骨头碎片, 用5毫升冰冷 HBSS + 和过滤成相同的50毫升圆锥管。</li>
		<li>用冰冷 HBSS + 和离心在 400 x g 的7分钟内, 将细胞洗净两次, 然后将其去掉。</li>
		<li>继第二次洗涤并用重悬细胞颗粒在20毫升冰冷 PBS。用台盼蓝排除9计算可行单元格。</li>
		<li>孵育 1 x 108可存活细胞每1毫升冰冷 HBSS + 与生物素共轭 c-试剂盒抗体在1:100 稀释为45分钟在冰上。</li>
		<li>用冰冷 HBSS + 离心在 400 x g 处清洗细胞, 并丢弃上清液。</li>
		<li>在制造商的指导下, 用抗生物素磁性珠子孵化细胞。</li>
		<li>使用磁性柱或磁性活化细胞分拣的 c 套件阳性细胞, 按照制造商的指示隔离。收集15毫升圆锥管中的 c 套件阳性细胞。</li>
		<li>用 PBS 冲洗细胞两次, 15 毫升圆锥管和离心 400 x g 7 分钟。在两个洗涤之间计算的活细胞总数由台盼蓝排除9。</li>
		<li>并用重悬 c 套件 + 细胞浓度高达10 6 viablecells 每100µL 培养基为小鼠 HSPCs 补充2% 血清, 50 ng/毫升小鼠干细胞因子 (超临界), 50 ng/毫升小鼠血小板生成素 (TPO), 10 ng/毫升小鼠 IL-3, 10 ng/毫升小鼠 IL-6。</li>
		<li>将细胞在37摄氏度, 5% CO2上孵育至少1小时, 在电穿孔前的最大值为 24 h。</li>
	</ol>
</li>
	<li>
人类 HSPCs 的分离与培养 注意:这些步骤必须在层流罩中执行。<ol>
<li>通过40µm 细胞过滤器筛选骨髓/脐带血细胞。</li>
		<li>稀释1:2 过滤骨髓/脐血细胞与 PBS 补充1毫米 edta (PBS/edta)。</li>
		<li>将15毫升的密度梯度介质分配到50毫升圆锥管中。在密度梯度介质表面轻轻倒入稀释的骨髓/脐血30毫升。如果细胞悬浮量大于30毫升, 则准备多管。 注意:确保保持血液和密度梯度介质之间的界面尽可能尖锐。</li>
		<li>旋转管在 750 x g 30 分钟, 不减速。</li>
		<li>收集介质界面可检测到的单个核细胞层, 并将其转移到清洁的50毫升圆锥管。用 PBS/EDTA 填充管, 将细胞旋转280克, 15 分钟。</li>
		<li>在 PBS/EDTA 旋转 400 x g 7 分钟, 并丢弃上清, 两次清洗细胞。</li>
		<li>在制造商的指导下, 用 anti-CD34 磁性珠子孵化单个核细胞。</li>
		<li>使用磁性柱或磁性活化细胞分拣, 根据制造商的指示隔离 CD34+ 细胞。在15毫升锥形管中收集 CD34+ 细胞。</li>
		<li>清洗收集的 CD34+ 细胞两次通过顶部与 PBS 和纺纱 400 x g 7 分钟, 并丢弃上清。在两个洗涤之间计算的活细胞总数由台盼蓝排除9。</li>
		<li>并用重悬细胞在培养基中补充100的人类超临界, 100 ng/毫升人类 FLT3 配体 (FLT3L), 100 ng/毫升人类 TPO 浓度为 2.5 x 105可行细胞/毫升. 培养分离的 CD34+ 细胞在37°c, 5% CO2 为 24 h 到最大 48 h 在先去电穿孔。 注意:用流式细胞仪对富 CD34+ 种群进行电穿孔检查纯度。</li>
	</ol>
</li>
</ol>5. 电穿孔
注意:下面的协议是优化10µL 电穿孔提示。所有步骤都应在层流罩中进行。
<ol><li>用台盼蓝排除9计数单元格。每复制收集 2 x 105可行单元格 (例如, 为 10 electroporations 收集 6 x 53单元格)。</li>
	<li>用 PBS 冲洗两次细胞, 在 300 x g 处旋转7分钟, 小心地取出上清液。</li>
	<li>同时, 通过在 RT 1.5 µg Cas9 上孵化 20-30 分钟的 PCR 管 Cas9-sgRNA RNP 配合物, 除µg 唯一的控制外, 每种复制的总 sgRNA 1 Cas9。 注意:Cas9 蛋白提供10µg/µl 浓度。为确保准确的吹打, 稀释1:10 的 Cas9 蛋白所需的核酸酶免费水 (e. g 10 electroporations 采取µl 蛋白 1 Cas9 和稀释它与9µl 无核酸酶水和孵化1µl 稀释 Cas9 每复制。根据 sgRNA 的浓度, Cas9 和 sgRNA 的总体积应由每次复制的1.7 和2µl 组成。如果使用两个 sgRNAs 孵化每个 sgRNA 每复制 500 ng;如果使用 3 sgRNAs 孵化330每 sgRNA 每个复制, 等等。</li>
	<li>计算10µL 乘以所需的总复制数 (例如, 3 复制的30µL) 所需的泥沙缓冲 T 的总体积。并用重悬颗粒细胞与计算量的缓冲吨确保最后浓度的细胞悬浮是 2 x10 7 细胞/毫升。</li>
	<li>整除10µL 的悬浮细胞/复制到每个 pcr 管包含预复合 sgRNA/Cas9 RNP (例如, 一三个整除 30 ul 的细胞悬浮进入 PCR 管含有预复杂的 Cas9-sgRNA)。</li>
	<li>建立电穿孔系统, 打开仪器, 将3毫升的缓冲 E 加入试管, 并将试管滑入试管支架。</li>
	<li>在设备上设置所需的电穿孔条件: 人 HSPCs (1600 v, 10 毫秒, 3 脉冲) 或鼠标 HSPCs (1700 V, 20 毫秒, 1 脉冲)。 注意:如果需要, 在对 CD34+ HSPCs 进行实验之前, 在急性髓细胞白血病 (aml) 电池线 (如 HL60) 中使用相同的策略 (使用无抗生素培养基进行 AML 细胞系), 并在以下电穿孔条件下进行试验 sgRNA 效率:缓冲器 R, 1350 V, 35 毫秒, 1 脉冲。</li>
	<li>电穿孔是用一个特殊的电穿孔吸管, 它有一个爪, 闩锁在电穿孔吸管尖端。按住电穿孔吸管, 延长爪, 抓住圆盘内的吸管尖端, 然后坚定地按下吸管, 以确保尖端。</li>
	<li>用吸管将样品上下10次混合。取出10µL, 确保没有气泡, 并将吸管尖端直接插入电穿孔试管的电解缓冲器。尽量不要碰试管的墙。在屏幕上按 "开始"。在 "完整" 信息出现后, 取出吸管, 并将内容放入新的 HSPC 培养基中。</li>
	<li>对所有样品重复。只有在必要的时候, 才能改变样品之间的吸管提示。</li>
</ol>

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无利益冲突披露

本详细协议中描述的 RNP 方法能够有效地在小鼠和人的原发性造血细胞以及悬浮细胞系中进行基因剔除。虽然经常获得高的效率, 但需要考虑几个关键变量。首先, 正确的外显子选择是保证有效的 indel 合并对应于基因敲除的关键。与外显子选择相关的两个重要因素是在转录中跨亚型和外显子位置的守恒。异构体表达式模式是跨单元格类型的变量, 因此, 如果需要跨单元格类型的基因高, 则应针对单元格类型之间不变的外显子。异构体模式可以通过 q PCR 或 RNA 测序数据来验证。对于在成绩单内的外显子位置, 最好是针对早期外显子, 因为 frameshift 突变在终端或倒数第二外显子可能逃脱废话介导的衰变10, 产生的蛋白质具有新颖或未知的功能, 而不是真正的空。
确定 indel 频率是评估高效率和正确解释实验生物学结果的关键步骤。内切酶化验 (如验船师化验) 具有相对快速和易于执行的优点。然而, 它们往往是不准确的, 由于重要的背景信号和结果往往难以重现。此外, 他们也没有提供关于实验中产生的 indels 质量 (例如插入和删除长度) 的信息。限制性的测序提供了一个宝贵的替代方法。诸如潮汐11 (https://tide.nki.nl/) 这样的平台有助于分解的痕迹, 并能准确估计出等位基因的干扰效率, 同时也提供了个体 indels 的频率。主要的缺点是绝对依赖高质量的桑格测序跟踪。高通量扩增子排序克服了大多数这些问题。扩增子库可以从非常低的 DNA 输入开始准备, 高覆盖率确保了可靠的结果。为了降低扩增子测序的成本, 我们通常只使用 1-1.5% 的总读数, 将扩增子库调成更大的顺序运行 (如 RNA 排序)。最后, 为了确认成功的淘汰赛, 我们强烈建议在可能的情况下, 通过西方印迹或流式细胞术评估蛋白质水平。
如前所述, 这里提出的方法是快速和直接的, 代表了一个新的工具来研究基因敲出老鼠和人类 HSPCs。虽然我们的协议提供了使用尖端电穿孔系统进行基因挖空的指令, 但其他系统已经被证明是高效的12,13。
在 RNP 方法方面需要承认两个主要的限制。首先, 正如我们小组所描述的, HSPCs 转与体外转录 sgRNAs 的生存能力可以通过电穿孔显著地损害, 特别是当条件不是最佳6时。如果需要, 商业合成的 sgRNAs (即消除体外转录) 可能会克服这个问题。随着时间的推移, 这些成本越来越低, 可以从几家供应商那里购买。在这种情况下,体外转录可用于生成 sgRNA 的池以进行有效性筛选, 然后可以选择最有效的 sgRNA 进行合成。RNP 方法的第二个主要限制是无法追踪成功转染的细胞, 如在基于病毒的方法中。然而, 在许多实验场景中, Cas9-sgRNA RNPs 获得的高高效率和快速编辑可能会超过这些缺点。我们建议使用高通量的扩增子测序来精确确定每个实验的中断效率。如果预期基因的剔除会赋予增生表型 (即与野生型细胞相比减少或增加增殖), 那么在多个时间点确定 indel 频率可以评估高细胞是否经过浓缩 (即 indel 频率随时间增加) 或 outcompeted (i. e. indel 频率随时间而减少)。
执行体内或体外实验来了解基因功能丧失的生物学效应需要适当的控制。如前所述,体外转录 sgRNAs 可能对细胞有毒, 特别是主要祖细胞6。此外, Cas9 蛋白引起的 DNA 双链断裂可引起基因独立的抗增生反应14。因此, 我们经常在 CRISPR 实验中使用一些控制 sgRNAs, 并推荐在您的细胞类型上表达的细胞表面标记以及未表达的第二个目标基因。
在细胞因子存在的情况下, 人类 HSPCs 的短期培养提高了基因破坏效率6 , 是实现高效高的必要条件。在电穿孔6之前, 我们发现 36-48 小时是文化的理想时期。随着电穿孔, 细胞可以进一步培养, 记住, 越长的文化越高的风险, 失去多向植入能力。因此, 我们通常在电穿孔后6小时进行移植, 并且在电穿孔后不超过24小时。
CRISPR/Cas9 极大地提高了科学家成功编辑哺乳动物细胞基因组的能力。预测在原代造血祖细胞中基因破坏更可行, 可以快速提高 HSPCs 和血液病领域的科学知识。重要的是, 这里描述的协议使它也可以同时产生多个挖空高效率, 允许建模复杂的遗传景观, 往往见于白血病疾病。
虽然本文所描述的协议的重点是基因中断, 它们可能很容易适应基因敲入实验。这可以通过联合交付单链寡核苷酸模板进行同源定向修复6、13 (HDR) 或通过电穿孔后的细胞转导, AAV6 向量包含同源模板12。在 HSPCs 中修复或引入特定突变的能力将促进基因治疗的新的治疗策略, 以及在反洗钱和其他血液疾病中癌症驱动因子突变的扩大研究。虽然人类 HSPCs 中的 HDR 已成功6、12、13, 但使用此策略6很难编辑鼠标 HSPCs。优化人类中的 HDR 协议, 特别是在鼠标 HSPCs 中, 将能够更具体地编辑和开发工具, 以便使用内源标记跟踪编辑过的单元格。
最后, 还有一种设想是, 突变的功能性等位基因的中断可能是先天性和后天造血疾病的潜在治疗方法。在这种情况下, 建议采用无 DNA 的方法, 以避免可能的集成, 从而促进转化。此外, "命中和运行" 方法减少了不想要的非目标效果的可能性。需要作出更多的努力, 以发展具体的运载系统, 为治疗恶性和非恶性血液病开辟新的途径。

CRISPR/Cas9 的出现从根本上简化了哺乳动物细胞的基因编辑。早期的 CRISPR/Cas9 协议利用质粒或基于病毒的 Cas9 蛋白传递和 sgRNA1,2,3。这些方法在细胞和动物模型的发展中被证明是革命性的, 并且也成功地应用于原发性造血干细胞和祖细胞 (HSPCs)4,5。然而, 这些方法有许多缺点。首先, 基因中断效率通常仍然低于 50%4,5。虽然这足以在细胞系中产生挖空 (高) 无性系, 但短期文化的必要性使得 HSPCs 不适合这样的策略。其次, 克隆的要求限制了可以在单个实验中测试的 sgRNAs 量, 并生成大而难染的构造。第三, 这些方法迫使科学家放慢周转时间。
为了克服这些限制, 我们的小组在 HSPCs 使用基于 Cas9-sgRNA ribonucleoproteins (RNPs) 的交付6开发并最近发布了一种替代 CRISPR/Cas9 方法。在这个策略中, CRISPR (Cas9 蛋白和体外转录 sgRNA) 的原始成分是预复杂的, 通过电穿孔直接传递到目标细胞 (图 1)。由于 Cas9-sgRNA RNP 复合体的半衰期比转录质粒或病毒核酸的时间短, 与早期方法7相比, 离靶速率较低。此外, RNP 方法增加了消除任何外源 DNA 来源的好处, 它可以随机整合到目标细胞基因组, 导致细胞转化。
该协议是基于基于 RNP 的基因中断实验的简化工作流, 如图 1所示。第一步是为每个 sgRNA 设计和订购底漆。这些引物用于制作 sgRNA DNA 模板, 用于体外转录 (IVT) 以获得 sgRNAs。纯化后的 sgRNAs 与以前购买的 Cas9 蛋白一起孵化, 形成 Cas9-sgRNA RNP 复合物。最后, 将预复合 Cas9-sgRNA RNPs 转成细胞。在电穿孔后, 可以对编辑效率进行测试, 并根据需要对体外/体内实验进行启动。下面对这个创新的实验方法的详细描述可以找到。

<table>
	<tbody>
		<tr>
			<td>Tissue culture plates according to cell numbers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Eppendorf microtubes</td>
			<td>Sigma</td>
			<td>Z606340-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Falcon tubes</td>
			<td>Corning</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease Free Water - DEPC treated</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9915G</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix (2X)</td>
			<td>KAPA Biosystems</td>
			<td>KK2600</td>
			<td></td>
		</tr>
		<tr>
			<td>MinElute PCR purification Kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAse Zap RNAse Decontamination Solution</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA Clean and Concentrator-25</td>
			<td>Zymo</td>
			<td>R1017</td>
			<td></td>
		</tr>
		<tr>
			<td>HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td>New England Biolabs</td>
			<td>E2040S</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R S.p. Cas9 Nuclease 3NLS, 100 &#181;g</td>
			<td>IDT</td>
			<td>1074181</td>
			<td></td>
		</tr>
		<tr>
			<td>40 mm Cell Strainers</td>
			<td>VWR</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>GenDEPOT</td>
			<td>CA008-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS), Regular</td>
			<td>Corning</td>
			<td>35010CV</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator or MACS manual Cell Separation columns</td>
			<td>Miltenyi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System Device</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10mL kit</td>
			<td>ThermoFisher Scientific</td>
			<td>MPK1025</td>
			<td>For larger numbers of cells (see protocol) Neon Transfection System 100 mL kit (# MPK10025) can be used</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>UltraPure 0.5M EDTA, pH 8.0</td>
			<td>ThermoFisher Scientific</td>
			<td>15575020</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stem Cell Technologies</td>
			<td>7851</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>CD34 MicroBead Kit UltraPure human</td>
			<td>Miltenyi Biotec</td>
			<td>130-100-453</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>Stem Span SFEM II</td>
			<td>Stem Cell Technologies</td>
			<td>9605</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>Recombinant Human SCF</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-695</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>Recombinant Human TPO</td>
			<td>Miltenyi Biotec</td>
			<td>130-094-013</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>Recombinant Human FLT3L</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-479</td>
			<td>For human experiments only</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mouse dissection tools</td>
			<td></td>
			<td></td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Mortar and Pestle</td>
			<td></td>
			<td></td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Biotin-conjugated anti c-kit antibody</td>
			<td>eBioscience</td>
			<td>13-1171-82</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Anti-Biotin MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-485</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>X VIVO-15, with L-Glutamine, Genatmycin and Phenol Red</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Mouse IL-6</td>
			<td>Peprotech</td>
			<td>216-16</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Mouse TPO</td>
			<td>Peprotech</td>
			<td>315-14</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Mouse IL-3</td>
			<td>Peprotech</td>
			<td>213-13</td>
			<td>For mouse experiments only</td>
		</tr>
		<tr>
			<td>Mouse SCF</td>
			<td>Peprotech</td>
			<td>250-03</td>
			<td>For mouse experiments only</td>
		</tr>
	</tbody>
</table>

highly efficient gene disruption of murine and human hematopoietic progenitor cells by crispr/cas9

在造血干细胞 (HSCs) 领域的研究进展得到了基因上的原代祖细胞和动物模型等方法的辅助。完整的基因消融在 HSCs 需要产生的剔除小鼠, HSCs 可以被隔离, 和基因消融在初级人类 HSCs 是不可能的。病毒转导可用于击倒的方法, 但这些都是由可变的功效。一般而言, 人类和小鼠造血细胞的基因操纵受到效率低下和时间和成本承诺的影响。最近, CRISPR/Cas9 极大地扩大了对哺乳动物细胞 DNA 的设计能力。然而, CRISPR/Cas9 在造血细胞中的应用具有挑战性, 主要是由于它们的转染效率低、质粒化方法的毒性以及基于病毒的协议的缓慢周转时间。
本文提供了一种快速的方法来执行 CRISPR/Cas9-mediated 基因编辑的小鼠和人造血干细胞和祖细胞的淘汰赛效率高达90%。这种方法采用了 ribonucleoprotein (RNP) 交付策略, 并简化了三天的工作流。使用 Cas9-sgRNA RNP 允许一个命中和运行的方法, 不引入外源 DNA 序列的基因组编辑细胞和减少目标的影响。基于 RNP 的方法是快速和直接的: 它不需要克隆 sgRNAs, 病毒制备或特定的 sgRNA 化学修饰。通过这项协议, 科学家们应该能够在一周内成功地生成一个人对原发性造血细胞感兴趣的基因的挖空, 包括对寡核苷酸合成的停机时间。此方法将允许更广泛的用户组根据需要调整此协议。

本协议的目标是使用 Cas9-sgRNA RNPs 在小鼠和人类 HSPCs 中进行基因敲除 (KO)。该工作流简单快捷, 允许在不到一周的时间内完成实验设计, 以评估高效率。
与质粒或基于病毒的方法相比, 我们的协议的成功建立在 sgRNA RNPs 和电穿孔的结合上。这种无克隆策略大大缩短了获取组件到染所需的时间。此外, 电穿孔允许转染多达95% 的细胞, 最大限度地提供 RNPs. sgRNAs 可以通过体外转录 (IVT) 在实验室综合升华, 而纯化的 Cas9 蛋白质可以从几家公司购买 (参见协议)。sgRNA IVT 的 DNA 模板使用 sgRNA 前进底漆 (图 1A、B) 和通用脚手架转速底漆 (图 1B -见协议) 进行短 PCR。然后将 PCR 产品用作 IVT 的模板 (图 1C)。Cas9-sgRNA RNP 复合体生成的孵化时间为 15-30 分钟 sgRNAs 和 Cas9 (图 1C)。最后, sgRNA RNPs 转进入细胞。然后, 编辑的 HSPCs 可以用于同时执行体外和体内实验。
小鼠细胞基因分裂
为了证明 Cas9-sgRNA RNP 电穿孔策略的有效性, 从小鼠无所不在表达 gfp 或 tdTomato 的 c 试剂盒 + HSPCs 与转对 gfp 或 sgRNA tdTomato。在针对 gfp 设计的三 sgRNA 中, gfp sg1 最有效地执行, 显示大约 70-75% 中断的 gfp 表达式, 由流式细胞仪 (图 2A, B) 确定。为了量化基因中断频率在基因组水平, 转细胞的基因组 DNA 被隔离, 并 T7 限制性 I 型 (T7EI) 检测。如图 2C所示, T7EI 检测发现多达 60% indel 形成。请注意, T7EI 化验往往低估了 indels 的频率。我们还产生了 sgRNA 反对 tdTomato 和转 HSPCs 表达 tdTomato 从 Rosa26 的轨迹。如图 2D所示, tdTomato sg1 有效地消融了 tdTomato 的表达式。
人类细胞中的基因分裂
在这里, 以单一的 sgRNA 方法获得有效的干扰, 以 CD45, 一个细胞表面标记表达的造血细胞, 显示。设计了三 sgRNAs 靶向不同外显子的 CD45 (CD45 sg1、sg2 和 sg3)。sgRNAs 的效率在 HL60 AML 细胞线 (图 3A) 中进行了测试。用流式细胞仪对蛋白高进行电穿孔5天的评估。CD45 sg1 是最有效的 (98% 高效率-图 3A), 并被用于进一步的实验。图 3B显示了使用 CD45 sg1 的主要人类 CD34+ 祖细胞中的 CD45 高, 经电穿孔5天的流式细胞仪评估。虽然 CD34 表达不受影响, CD45 表达在大约80% 的细胞中丢失。200.000 个经编辑的原 CD34+ 细胞轨道在电穿孔6小时后移植到核型核型小鼠体内。骨髓和脾细胞移植3月后收获。编辑过的单元格 (HLA-ABC 阳性, CD45 阴性, 突出显示为红色) 只能在 sgRNA 处理的样本中检测到, 而不是仅在 Cas9 控件中 (图 3C)。
如果需要, 可以使用两个针对附近序列的 sgRNAs 来生成删除。这种方法允许用 PCR 快速估计编辑效率, 并且经常产生更高的干扰效率。图 3D显示在DNMT3A的外显子10中有效地生成 58 bp 删除。200.000 经编辑的 CD34+ 细胞在电穿孔6小时后移植到核组鼠体内。在注射 (图 3E) 后5月内, 嫁接核型核组织小鼠的外周血中明显检测到58的 bp 缺失, 确认了具有长期植入能力的 CD34+ 细胞的编辑。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57278/57278fig1.jpg"> 图 1: 生成 sgRNA RNPs 的快速简便方法。A) sgRNA Fwd 底漆的表示法。sgRNA 前进底漆是一种 DNA 寡核苷酸, 含有 T7 启动子、protospacer 序列以及与 sgRNA 支架重叠的序列。在 3 ' 末端, 用红色突出显示, 是 "ATAGC" 序列, 需要添加到 sgRNA 前进底漆序列 CRISPRscan 获得。B)为获取 IVT 模板而执行的重叠 PCR 的示意图表示形式。sgRNA 前进底漆和通用脚手架转速底漆之间的重叠, 可以通过 PCR 的扩展和扩增。C)卡通描述 IVT 反应和 sgRNA RNP 预络。PCR 产品被纯化并用作 IVT 的模板。IVT 生成大量的 sgRNAs, 可以在多个复制中使用 (参见协议)。sgRNA RNPs 的孵化 sgRNA 纯化从 IVT 和以前购买的 Cas9 蛋白。<a href="https://cloudflare.jove.com/files/ftp_upload/57278/57278fig1large.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/57278/57278fig2.jpg"> 图 2: 有效的基因在小鼠造血祖细胞中的破坏。A) sgRNA 靶向 gfp (gfp sg1) 与 Cas9 蛋白转成小鼠 c 套件 + HSPCs 表达 GFP, 并通过流式细胞术24小时后电穿孔分析。B)不同数量的 GFP sg1 与1µg Cas9 蛋白和转复合。只要 200 ng 的 gfp sg1 有效地消融 gfp 的表达。C) T7EI 对从 B 中使用的细胞中分离出的基因组 DNA 进行的检测。PCR 扩增子跨越 Cas9-sgRNA 裂场被稀释1:4 在1x 缓冲2和慢慢杂交在热循环仪。杂交片段然后消化与 1.25 U T7 限制性 I 10 分钟在37°c。用聚丙烯酰胺凝胶电泳分离消化片段。利用 ImageJ 软件对各车道的带强度进行了分析。劈裂率的计算方法是 uncleaved 带的强度比。D) c 套件 + HSPCs 从表达 tdTomato 的小鼠中分离出来, 转 Cas9 蛋白配合 sgRNA 对 tdTomato。流式细胞术24小时后进行电穿孔显示, 约74% 的细胞删除 tdTomato。R26 = Rosa26。* p < 0.05, ** p < 0.01, *** p < 0.001。此数字已从冈德里et中进行了调整。6<a href="https://cloudflare.jove.com/files/ftp_upload/57278/57278fig2large.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/57278/57278fig3.jpg"> 图 3: 人类造血细胞的有效基因破坏。A)三 sgRNAs 靶向 CD45 (CD45 sg1、sg2 和 sg3) 是在 HL60 AML 细胞线上设计和测试的 (见电穿孔条件协议)。流式细胞术在电穿孔5天后进行。CD45 sg1 被选为三 sgRNAs (高效率 98%) 中最有效的.B) CD45 CD34+ HSPCs 使用 CD45 sg1 的主要人脐血。CD45 的损失可以在大约80% 的细胞中检测到。请注意, 编辑后 CD34 表达式不变。C)编辑的人 HSPCs 可以有效地嫁接到核供应国小鼠。人核细胞显示 Cas9-only 嫁接小鼠正常的 HLA+/CD45+ 表型, 而在小鼠嫁接 CD45-sg1 处理 CD34+ 细胞, HLA+/CD45+ 和 HLA+/CD45-人口的观察表明植入的人 CD45 高细胞。D) 2% 琼脂糖凝胶, 显示 58 bp 删除 2 sgRNAs (双导方法) 的外显子10的DNMT3A在人类 HSPCs 从3不同的脐带血 (CB1, CB2, CB3)。在电穿孔3天后进行 PCR。E) 2% 琼脂糖凝胶, 表明58的 bp 删除5月后, 移植到核供应集团的小鼠持续。此数字已从冈德里et . 中进行了修改。6<a href="https://cloudflare.jove.com/files/ftp_upload/57278/57278fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>寡 核苷酸</td>
			<td colspan="3">序列</td>
			<td>注意</td>
		</tr>
<tr>
<td>通用转速脚手架底漆</td>
			<td colspan="3">gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgct</td>
			<td>重叠 PCR 通用反向底漆</td>
		</tr>
<tr>
<td>hCD45 sgRNA 1</td>
			<td colspan="3">taatacgactcactataGGTGCTGGTGTTGGGCGCACgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>hCD45 sgRNA 2</td>
			<td colspan="3">taatacgactcactataGGGAGCAAGTGAGGATCCTCgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>hCD45 sgRNA 3</td>
			<td colspan="3">taatacgactcactataGGGATGCTTGTTCCCTTCAGgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>hDNMT3A Ex10 sg1</td>
			<td colspan="3">taatacgactcactataGGACACTGCCAAGGCCGTGGgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>hDNMT3A Ex10 sg2</td>
			<td colspan="3">taatacgactcactataGGGTGGCCAGCAGCCGCGCGgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>Rosa26 sgRNA</td>
			<td colspan="3">taatacgactcactataGGACACTGCCAAGGCCGTGGgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>GFP sg1</td>
			<td colspan="3">taatacgactcactataGGGCGAGGAGCTGTTCACCGgttttagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
<tr>
<td>tdTomato sg1</td>
			<td colspan="3">taatacgactcactataGGGGTACAGGCGCTCGGTGGgtttaagagctagaaatagc</td>
			<td>sgRNA 的交叉 PCR 引物序列</td>
		</tr>
</tbody></table>表 1:sgRNA 前进底漆的完整序列, 用于实验和通用转速底漆.

Watch this Scientific Journal Video about Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9 at JoVE.com

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

本文介绍了一种快速 CRISPR/Cas9-mediated 基因在小鼠和人原发性造血细胞中的破坏的协议。Cas9-sgRNA ribonucleoproteins 是通过电穿孔与 sgRNAs 产生通过体外转录和商业 Cas9。在有限的时间和财务成本下实现了高编辑效率。

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

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Genetics

13.3K 次观看.  Baylor College of Medicine. 本文介绍了一种快速 CRISPR/Cas9-mediated 基因在小鼠和人原发性造血细胞中的破坏的协议。Cas9-sgRNA ribonucleoproteins 是通过电穿孔与 sgRNAs 产生通过体外转录和商业 Cas9。在有限的时间和财务成本下实现了高编辑效率。

视频: Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

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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 ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

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 ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

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

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

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

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

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

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 ...

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

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

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

Preparation and Gene Modification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, underlies the only known cure of human immunodeficiency virus (HIV-1) infection, and shows immense promise in the treatment of genetic diseases such as beta thalassemia. Our group has developed a protocol to model HSPC gene therapy in nonhuman primates (NHPs), allowing scientists to optimize many of the same reagents and techniques that are applied in the clinic. Here, we describe methods for purifying CD34+ HSPCs and long-term persisting hematopoietic stem cell (HSC) subsets from primed bone marrow (BM). Identical techniques can be employed for the purification of other HSPC sources (e.g., mobilized peripheral blood stem cells [PBSCs]). Outlined is a 2 day protocol in which cells are purified, cultured, modified with lentivirus (LV), and prepared for infusion back into the autologous host. Key readouts of success include the purity of the CD34+ HSPC population, the ability of purified HSPCs to form morphologically distinct colonies in semisolid media, and, most importantly, gene modification efficiency. The key advantage to HSPC gene therapy is the ability to provide a source of long-lived cells that give rise to all hematopoietic cell types. As such, these methods have been used to model therapies for cancer, genetic diseases, and infectious diseases. In each case, therapeutic efficacy is established by enhancing the function of distinct HSPC progeny, including red blood cells, T cells, B cells, and/or myeloid subsets. The methods to isolate, modify, and prepare HSPC products are directly applicable and translatable to multiple diseases in human patients.

The goal of this protocol is to isolate nonhuman primate CD34+ cells from primed bone marrow, to gene-modify these cells with lentiviral vectors, and to prepare a product for infusion into the autologous host. The total protocol length is approximately 48 h.

非人类灵长类动物造血干细胞和祖细胞的制备及基因修饰

Hematopoietic stem and progenitor cell (HSPC) transplantation has been a cornerstone therapy for leukemia and other cancers for nearly half a century, ...

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 ...