这项工作得到了国家卫生研究院 (R21CA182197 联办)、拉尔夫 W 和格雷斯 m. 肖沃尔特研究信托基金的支持, 并由普渡农业科学和经济发展推广 (AgSEED) 计划提供。P30 CA023168 支持的普渡大学基因组核心设施获得了桑格测序数据。玛丽 Witucki 得到了普渡大学癌症研究中心暑期本科生研究计划的支持, 卡罗尔县癌症协会支持。资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。我们感谢本杰明. 卡特, 艾伦丹宁, 泰勒萨巴托为手稿提供了关键的反馈。我们感谢生物化学系对这项工作的支持, 以及在普渡大学的斑马鱼研究中心, 他们致力于我们的斑马鱼殖民地的照顾和福利。最后, 我们感谢普渡大学基因组核心设施, 以及菲利浦 San 米格尔对服务的贡献。

这项研究是严格按照《国家卫生研究院护理和使用实验动物指南》的建议进行的。该议定书得到了普渡动物保育和使用委员会 (PACUC 号 08-031-11) 的批准。
1. 指南 RNA 生产用模板专用寡核苷酸的设计
<ol><li>选择要在基因编码区域中修改的感兴趣的目标区域。这应该是接近 5 ' 末端的基因, 以产生一个截断蛋白质, 但没有如此接近, 这样, 随后的帧内启动密码子, 使生产的蛋白质与适度的 N 终端截断。 注意: 排除这种可能性的一种方法是扫描帧开始密码子中的基因的下游编码区域。此外, 使用引导 RNA, 目标的大外显子的中间, 可以帮助识别 PCR 引物后评分的结果 indel。</li>
	<li>使用指南 RNA 选择程序 (参见材料表), 在感兴趣的基因组区域中识别潜在的指南站点 (见资料目录)34,35,36。将浏览器数据设置为斑马斑马和 protospacer 相邻母题 (PAM) 序列到 5 '-NGG-3 '。 注意: 理想的 gRNA 序列包含一个 5′ G 在 gRNA 的第一个位置为有效 T7的体外转录。如果在5′位置的 g 处找不到可接受的指南, 则可以将另一指南的5′基修改为 g 或 g, 可以添加到指南 RNA 的5′端, 但这可能会降低切割效率37。为了最大限度地提高切割效率, 最佳导联序列具有 40-80% GC 含量 (较高更好), 在20次位置包含 G,与 PAM 相邻, 但不需要38。一个理想的目标序列的例子: 5′ g (N)18克-3′-NGG (NGG 是 PAM)。除了检查指南 rna 选择程序的结果, 以确定最佳指南 rna 如上文所述, 应注意避免引导 rna 与预测强的偏离目标的影响, 大大复杂化下游分析。特别是, 应排除在编码区域内的具有预测的离靶效应的导 rna, 并将预测的总离目标站点减到最小。</li>
	<li>从指南 RNA 设计工具的输出, 排除 PAM 序列 (5′-NGG-3′);它不用于靶向, 但包括 Cas9 解的识别序列。</li>
	<li>对其余的20核苷酸 (T7), 添加的启动子序列和重叠序列 (区域互补的脚手架寡核苷酸用于合成全长 sgRNAs 提供推荐的体外转录试剂盒) 的顺序表明以下获得 54 nt 寡核苷酸: T7 启动子序列: 5′-TTCTAATACGACTCACTATA-3′;导 RNA 序列 5′-g (N)18克 3′;重叠序列: 5′-GTTTTAGAGCTAGA-3′</li>
	<li>通过使用基于 web 的软件 (参见材料表), 在50–150基对 (bp) 的距离上, 识别出预测切割站点侧面的 PCR 引物 (Cas9 在 PAM 序列上游 3 nt)。 注: 这些将在以后的步骤中用于测量切割效率。如果不使用这些约束确定合适的引物, 则可能需要考虑另一个指南 RNA 站点。</li>
	<li>命令 54 nt 寡核苷酸生产指南 RNA 和 PCR 引物为分析目标站点 (参见材料表)。 注: 作为一个可选的阳性对照, 可能有助于产生一个 sgRNA 靶向一个必要的基因生产色素, 以验证该协议的性能, 使用一个容易得分的视觉表型 (见图 1的代表性结果)。一个共同的目标是基因酪氨酸酶, 使用寡核苷酸 (指南 RNA 序列下划线)39: 5′-TTCTAATACGACTCACTATAGGACTGGAGGACTTCTGGGGGTTTTAGAGCTAGA 3′</li>
</ol>2. 胚胎显微注射用 CRISPR 试剂的制备
<ol><li>
定购商业上可利用的 Cas9 蛋白质 (参见材料表)。 注: 注射 Cas9 mRNA 也可用于生成 indels 斑马鱼40,41, 但斑马鱼胚胎微注射与 Cas9 蛋白已证明是更有效的32,42。<ol>
<li>将 Cas9 蛋白悬浮在所提供的缓冲器中, 生成1毫克/毫升溶液。在-80 °c 的 PCR 管中, 将溶液注入整除数, 以减少冻融循环的次数。为了产生5µL 注射液, 2 µL 1 毫克/毫升 Cas9 溶液, 因此 Cas9 可以 aliquoted 在2µL 整除数使用 PCR 条管。</li>
	</ol>
</li>
	<li>用 sgRNA体外转录试剂盒合成 sgRNA (见材料表)。根据制造商的指示执行体外转录。 注意: 在所有合成、清理和注射液准备步骤中保持 RNase 技术。例如, 使用一次性手套并经常更换, 使用 RNase 认证的管子和小贴士, 干净的表面和吸管与商业上可用的解决办法来净化材质 (见材料表)。</li>
	<li>
利用无 RNase 技术纯化合成的 sgRNA, 采用铵/醋酸沉淀法。 注: 另外, sgRNAs 可以使用各种商业上可用的基于列的 RNA 清理套件, 以相对适中的成本进行纯化。<ol>
<li>添加25µL 5 米醋酸铵和涡旋彻底混合。 注: 醋酸铵溶液是商用的 (见材料表), 或5米溶液可以在房子里添加385.4 毫克的分子级醋酸铵到1毫升的 RNase 水和储存在-20 摄氏度。</li>
		<li>在每个样品中加入150µL 200 无核酸酶乙醇。将反应放在-80 摄氏度的冷藏柜中, 至少20分钟。 注: 样品可以在-80 摄氏度隔夜储存, 但不会显著增加总 RNA 的产量。</li>
		<li>以最大速度 (> 1.6万 x g) 在4°c 离心中离心样品20分钟。</li>
		<li>通过缓慢吹打液体, 确保 RNA 颗粒不受干扰, 仔细去除上清液。</li>
		<li>添加1毫升70% 乙醇 (通过稀释无核酸酶乙醇在 RNase 水中创建), 并通过多次反转, 轻轻地混合管, 以洗涤残余的盐从管。</li>
		<li>重复离心步骤7分钟。</li>
		<li>先用 P1000 吸管吹打, 去掉上清液, 然后用 P200 吸管去除尽可能多的溶液, 而不扰动颗粒。将 RNA 颗粒干燥在清洁的空间, 如层流罩或长凳顶部, 小心避免 RNase 污染, 15 分钟或直到没有更多的液体滴在管内可见。</li>
		<li>并用重悬在30µL 的 RNase 水中的颗粒, 量化的产品 (例如使用分光光度计), 并整除的解决方案, 长期储存在-80 °c 冰箱。 注: 典型浓度范围从800–2,500 µL。</li>
	</ol>
</li>
	<li>
选验证是否使用尿素/页生成了全长 RNA。或者, 使用琼脂糖凝胶来验证 RNA 是否完好无损。 注: 但是, 如果使用琼脂糖凝胶, 必须运行大量的 gRNA, 以可视化 RNA, 长度不能准确地确定。在分析注射试剂后的目标切割效率时, 如果没有或小切口存在, 则应检查 sgRNA 是否降解。<ol>
<li>采用无 RNAse 技术, 将8% 聚丙烯酰胺凝胶与40% 聚丙烯酰胺 (19:1) 和8米尿素结合使用, 用于解决方案和设备43。 注: 可用于清洁设备的商用材料 (见材料表)。</li>
		<li>在凝胶完全凝固后 (约30分钟), 平衡凝胶通过放置在运行缓冲和执行电泳30分钟5伏/厘米。</li>
		<li>混合 300–500 sgRNA 与等量的 2x RNA 凝胶加载染料 (见材料表)。使用 P1000, 清除任何碎片的水井, 吹打运行缓冲区在每个井数次。载入溶液并在10伏/厘米处运行凝胶2.5 小时。 注意: 这里的标记车道有助于可视化 RNA 的长度, 但不是必需的;一般来说, 如果合成了完整的 RNA (图 2), 这是显而易见的。</li>
		<li>使用核酸染色对带进行可视化 (见材料表)。 注: sgRNA 带应显示为单个波段, 而涂抹表示 RNA 降解 (图 2)。</li>
	</ol>
</li>
</ol>3. 将 CRISPR 成分注射到斑马鱼胚胎中
<ol><li>设置育种坦克在注射前的前一天晚上放置的期望男性和女性的数量 (通常2女性和1或2男性) 在一个繁殖槽在地方44的分隔。</li>
	<li>用1.5% 琼脂糖在 1x E3 介质 (见材料表) 中加入0.01% 亚甲基蓝 (杀菌剂), 将35毫升的熔融琼脂糖倒入10厘米培养皿中, 然后轻轻地放置一个塑料模具, 形成楔形槽, 进入解决方案, 敲击模具, 消除气泡。</li>
	<li>允许琼脂糖设置, 并存储在小数量的介质和包裹在石蜡膜, 以防止板材干燥4摄氏度。 注: 注塑板可重复使用几个星期, 直到油井变形或干燥, 或板材开始增长模具。</li>
	<li>在注射的早晨, 解冻纯净的 sgRNA 和 Cas9 蛋白质在冰上。记得用手套处理所有材料, 以防止 RNase 污染和使用无 RNase 的提示和管子。</li>
	<li>通过结合 Cas9 蛋白和 sgRNA 在 2:1 Cas9:sgRNA 的比例, 获得 400 pg/nl Cas9 蛋白和 200 pg/nl sgRNA 的最终浓度, 生成5µL 注射液溶液。在室温下孵化 Cas9/sgRNA 溶液5分钟, 使 Cas9 和 sgRNA 形成 ribonucleoprotein 复合物。添加0.5 µL 2.5% 的酚红色溶液 (见材料表), 和 RNase 的水, 最终体积5µL。 注: 溶液的离子强度已被证明对 Cas9/sgRNA 络合物的溶解度有影响, 因此添加氯化钾可提高 sgRNAs 的切割效率, indel 形成率低28。</li>
	<li>用微拉拔1.0 毫米的玻璃毛细管制成注射针。用新的剃刀刀片或镊子切开刚制成的针的尖端, 以获得一个容易刺穿绒毛膜和蛋黄囊的角度开口。</li>
	<li>将针放在附着在 microinjector 上的机器人上, 并打开空气源。在光镜下使用适用于为特定设备确定的校准的放大倍数, 调整注射压力, 直到针一贯地将 1 nL 溶液喷射到充满矿物油的培养皿中。 注: 针的质量是至关重要的。实践生产针和注入到胚胎的蛋黄囊, 直到这是技能掌握之前, 尝试进一步的实验45。</li>
	<li>去掉分隔线, 让鱼繁殖大约15分钟。 注: 繁殖时间延长会产生更多的胚胎, 但是注射应该完成, 而胚胎在1细胞阶段, 以最大限度地增加 Cas9 切割的机会, 从而减少遗传嵌合体。胚胎可以注射在后期 (2–4细胞阶段), 但这可能会降低生殖传播率的改良的等位基因。</li>
	<li>收集鸡蛋使用过滤器, 并冲洗成一个10厘米培养皿使用 1x E3 培养基与0.0001% 亚甲基蓝。在光显微镜下检查鸡蛋的健康状况, 除去未受精的卵和碎片。</li>
	<li>把10–15胚胎作为 uninjected 控制在一个单独的, 标记的培养皿。</li>
	<li>使用转移吸管, 轻轻地把鸡蛋放在注射板上, 加热到室温。</li>
	<li>在解剖显微镜下, 在2.5X 的放大倍数, 注入 1 nL 的溶液进入每个胚胎的蛋黄囊, 注射共 400 pg 的 Cas9 蛋白和 200 pg sgRNA。 注: 如果需要或必要, 增加切割, 增加 Cas9 蛋白的最终浓度为 800 pg/nl 和 sgRNA 到 400 pg/nl 在注射液中;然而, 这也可能增加离靶切割和/或减少胚胎健康。切割效率也可以增加直接注射到细胞46。然而, 注射到蛋黄袋是技术上更少的要求, 并给予足够的切割生产鱼高生殖传输 (> 70% 的后代含有改良的等位基因)。</li>
	<li>将注入的胚胎返回适当标记的培养皿, 用 1x E3 培养基与亚甲基蓝覆盖, 并将其放入胚胎孵化器中, 设置为28摄氏度。</li>
	<li>在 24 h 后受精 (hpf), 检查注射胚胎的健康, 消除死亡或异常发育的个人和改变媒体 (见图 3)。检查 uninjected 控制的生存率。 注: 当靶向非必需基因时, 预计相对于 uninjected 控制的致死率小于10%。如果在引导注射的人群中观察到的致死性升高水平与 uninjected 控制相比, 它可能表明靶向基因对发展是必不可少的, 或者离靶效应导致发育失败。减少注射 CRISPR 试剂的数量可能是必要的, 或产生一个新的 sgRNA, 减少了靶向效应可能需要。</li>
	<li>将胚胎恢复到孵化器中, 继续将胚胎生长到 72 hpf, 每天改变培养基以保持胚胎健康。</li>
</ol>4. 利用 HMA 对 Indel 形成效率的分析
<ol><li>收集两组五胚胎从注射板生长到 72 hpf 成离心管和收集一组五胚胎从 uninjected 控制。</li>
	<li>通过添加 0.004% MS-222 (tricaine) 和等待2分钟麻醉胚胎。</li>
	<li>要提取 gDNA, 请轻轻地将介质从每个胚组中抽出, 加入45µL 50 毫米氢氧化钠。在95摄氏度孵化出10分钟的胚胎。</li>
	<li>从热源中取出胚胎, 冷却至室温。添加5µL 1 米的三盐酸 pH 值 = 8, 并大力涡流样品 (5–10 s)。将溶液以最大速度 (> 1.6万 x g) 在室温离心中离心处理3分钟. 将上清液转移到一个干净的, 标记的管, 并存储 gDNA 在-20 °c 的 DNA 长达6月。 注: 通过使用本协议, 将生成大约 900 bp 和较小的 DNA 片段。</li>
	<li>使用预先设计的引物, 在预测的切割点两侧, 使用2µL 的 gDNA 从每个样品 (包括 uninjected 控制) 中建立50µL PCR 反应。</li>
	<li>使用 pcr 清理试剂盒 (见材料表) 纯化 pcr 产品, 洗脱30µL 水或洗脱缓冲器中的样品, 并使用分光光度计量化 DNA。</li>
	<li>Reanneal 所有30µL 的每一个纯化的 PCR 产品通过放置在一个可浮动机架在沸腾的水浴 (大约150毫升在500毫升烧杯)。3分钟后, 关闭热源, 让溶液冷却到室温, 约1小时。 注意: 这一步首先变性 dna, 然后允许链 reanneal 随机生成可能的 heteroduplex 带, 或不匹配的双链 DNA, 含有多态性由 CRISPR 诱变创造, 因此有一个改变电泳机动性比 homoduplexes。最后一个周期的 PCR 或坡道下降程序在 thermocycler 也可以用来 reanneal 产品47,48, 但使用的沸腾浴可能会产生提高 heteroduplex 产品的分辨率。</li>
	<li>添加5µL 的6x 负载染料 (见材料表) 到 reannealed PCR 解决方案。</li>
	<li>用30% 聚丙烯酰胺 (29:1) 浇铸15% 聚丙烯酰胺/凝胶。凝胶设置后, 将其放入电泳装置中, 运行缓冲器。使用 P1000, 清除任何碎片的水井, 如残留盐或凝胶碎片, 可以通过轻轻吹打缓冲区向上和向下多次进入井中来阻碍油井。</li>
	<li>负载 500 reannealed PCR 产品, 并加载一个控制 (样本从 uninjected 鱼) 旁边的每一套 sgRNA 样品。用150伏的凝胶2.5 小时或直到染料前面在凝胶的底部。</li>
	<li>使用核酸染色 (如溴溴或 SYBR 绿) 可视化带 (见材料表))。</li>
	<li>检查每个控制和 CRISPR 注入的胚池的带模式。 注: 在注入与 uninjected 车道上运行较慢的多个波段的外观表明了新型 heteroduplex 产品的形成 (图 4)。新的 heteroduplex DNA 的存在表明 indels 是由 CRISPR 注射产生的。homoduplex 带强度在大约50% 或更高的注入溶液中的减少通常足以导致足够的生殖传输。此外, 在 uninjected 鱼中有时会发现额外的带, 不应该被认为是 heteroduplex 带, 当观察到注射鱼。</li>
	<li>选择相对于每个目标的 uninjected 控制的最高切割效率的注射;这些注射的胚胎可以用来生长鱼, 寻找 indels 导致过早停止密码子。 注: 为高效切割 (减少 homoduplex 带约 > 50% 强度), 筛选20–30成年鱼应足以获得生殖传播。 对于 sgRNAs 减少切割, 可能需要更多的成年鱼, 以增加识别鱼的可能性, 显示生殖传输的修饰等位基因含有过早停止密码。如果没有观察到 indel 的形成, 可能需要重新设计 sgRNA 到基因的不同区域。如果没有观察到 heteroduplex 带的形成, sgRNA 可能已经退化, sgRNA 质量应使用尿素/页凝胶进行验证。</li>
</ol>5. 敲出线的识别和传播
<ol><li>要识别潜在的创始人母鱼, 请执行成年 F0 鱼 (生长到大约2.5–3月) 的尾部片段, 以确定 indels 的存在。麻醉鱼在0.62 毫米 tricaine (参见材料表), 然后使用干净, 锋利剃刀刀片去除大约 1/2-3/4 尾巴鳍。</li>
	<li>把尾巴放在45µL 50 毫米的氢氧化钠上, 然后把鱼送回回收箱。按照所述执行 gDNA 提取 (步骤 4.2–4.4)。一旦鱼恢复正常的游泳行为, 把鱼放回流动的系统水, 直到 indel 的性质被确定。 注意: 确保单个鱼是可识别的, 并能与尾部夹的结果适当匹配是至关重要的。</li>
	<li>如所述 (步骤 4.5–4.9), 请在尾部剪辑 gDNA 上执行 HMA, 以确定 CRISPR 注入是否修改了鱼 (图 5)。为了确定一个创始人鱼, 繁殖的成人, 展示 heteroduplex 带从尾部 gDNA 到野生型鱼类。收集胚胎并将其生长到 72 hpf。</li>
	<li>在 72 hpf, 收集10胚胎和放置每个胚胎在一个单独的管。执行 gDNA 提取如上文所述 (步骤 4.2–4.4) 使用11.25 µL 50 毫米氢氧化钠和1.25 µL 1 米三 pH = 8。</li>
	<li>重复 PCR 和电泳, 以确定 heteroduplex 带如上文所述 (步骤 4.7–4.13), 以确定 indel 等位基因已传给这一代 (图 6)。</li>
	<li>根据使用 HMA 观察到的单个胚胎中的透射率, 生长平均的20–30胚胎获得的每个 CRISPR 线的利益成年。 注: 这个数字可能会增加或减少, 这取决于生殖传输的频率。</li>
	<li>执行成人 F1 鱼的尾部剪辑, 以确定 indels 的存在, 如所述 (步骤 5.1–5.2)。如所述 (步骤 4.5–4.11), 在尾部剪辑 gDNA 上执行 HMA, 以确定鱼是否带有 indel (图 7)。</li>
	<li>
对于含有 heteroduplex 带的鱼, 准备 DNA 进行测序分析。
	<ol>
<li>使用新的引物进行 PCR, 以放大300–600的 bp pcr 产品围绕切割站点。</li>
		<li>使用 PCR 纯化试剂盒来清除 dna, 并在30µL 中洗脱. 检查琼脂糖凝胶上的 dna, 以确保有一个带存在。 注: 某些 heteroduplex 带可能出现在琼脂糖凝胶上, 并显示为一个小涂片或双带刚好高于 PCR 产品的预期大小。</li>
	</ol>
</li>
	<li>如果对三 indels 进行分析, 请使用 indel 序列化 PCR 产品, 并使用生物信息学工具49确定序列。否则, 使用产品分析 (见材料表) 的多 PCR 产物序列的确定更经济。</li>
	<li>通过使用生物信息学工具分析序列, 确定是否已获得过早停止密码子 (见材料表)。</li>
	<li>将含有所需突变等位基因的鱼放入新的水箱中, 并标明合适的标签。</li>
	<li>为将来的基因分型需要设计特定 indel 等位基因的 PCR 引物。这些引物应跨越变异序列, 而不是放大野生类型序列。</li>
	<li>在十字异型斑马鱼产生一个分离的人口, 将包含25% 条淘汰线。</li>
</ol>

作者没有什么可透露的。

本协议描述了利用 CRISPR-Cas9 技术生产斑马鱼脊椎动物模型中基因挖空的方法。一些协议以前被描述进行 CRISPR 介导的基因组工程学在斑马鱼15,25,26,50,51,52。该协议建立在以前的努力基础上, 结合了一些简单而重现性一致的实验技术, 特别是多异型鱼的 HMA 和, 以创建一个简单、经济和实验性强的协议。CRISPR 介导的对斑马鱼的诱变, 适用于配备有一系列培训和经验的人员的实验室, 以及教学实验室。
本议定书包括了指南 rna 的设计和合成建议。指南 RNA 设计的一个主要考虑是最小化的离靶效应。开发了几种预测算法, 允许 CRISPR 用户使用友好的图形界面访问计算工具, 同时预测目标指南的活动和离目标效果34的几率, 35,36。斑马鱼系统的一个特定优势是降低了离靶效果的比率, 因为 Cas9 被注入胚胎, 因此表达是瞬态的, 这已经显示在小鼠, 以减少离靶效应53。然而, 在斑马鱼54中已经证明了非靶向效应的发生。控制离靶效果的一个方法是, 表型创建者斑马鱼是由两个独立的指南 rna 产生的, 目标相同的基因, 因为这些指南将很可能会影响不同的目标地点。在本协议中没有描述的减少目标效果的另一种方法是使用突变的 Cas9, 在目标 DNA 上产生单链断裂, 并以高效率修复。配对 DNA 刻痕在彼此附近互相是互补的对相反的链导致有效的 indel 形成在期望的轨迹并且最小化目标作用55,56。
除了有不同的率的非目标效应, 不同的 sgRNAs 可以有不同的率的突变的预期目标57,58,59。该协议使用 HMA 分析 sgRNA33、40的 heteroduplex 带的诱变效果。Heteroduplex 带是由 PCR 生成的 DNA 链的杂交产生的, 含有不匹配, 可以很容易地解决使用凝胶电泳。与通常用于测量 indel 形成的其他方法不同, 如 T7 限制性法或高分辨率熔体分析25、26, HMA 不需要昂贵的酶来切割不匹配的 DNA, 也不需要PCR 熔体曲线的复杂分析。重要的是, 使用 HMA 来验证注射的人群中 indel 形成的高比率, 也使调查人员能够尽量减少随后生产出的排线所需的鱼数, 从而降低了识别突变的成本。期望的特征。
生成基于 CRISPR 的 indels 相对容易, 可以同时创建多个基因的多个等位基因。基于 Web 的软件可用于分析异型鱼的单一突变, 利用 PCR 产品49的基因测序。在分析了三或更多 CRISPR 变异的等位基因的情况下, indel 的性质可能更具成本效益, 以描述 indel 的性质, 因为这种方法允许有多达50种不同的等位基因的池在 o(见材料表)60,61,62。这种规模经济在本科实验室设置中可能特别有用。
总之, 本协议提供了重现性生成高质量 CRISPR 试剂 (特别是 sgRNA) 的分步指导, 这样就需要创建和分析更少的成年鱼, 以成功地识别出感兴趣的突变等位基因, 这还可以减少生成所需行的时间和成本。重要的是, 这项议定书的设计, 使它可以适用于有限资源的实验室, 生产突变的斑马鱼以负担得起的方式。此外, 我们发现这种方法适合本科生, 从而扩大了对 CRISPR 基因组编辑的亲身体验有兴趣的本科生的教育和培训机会。

在真核生物中, 分子机械的保护是利用模型有机体进行研究的力量。这些模型系统中的许多都有助于使用反向遗传方法, 如靶向基因挖空, 以描述基因产品对生物或疾病过程的贡献。像斑马鱼这样的生物体中的基因干扰技术历来依赖于对 DSBs1、2的不精确修复导致的 frameshift 突变的定向引入。当一个争端研究小组引入到基因组中时, DNA 损伤通过两个途径之一来修复, 在几乎所有细胞类型和有机体中普遍存在: 非同源端连接 (NHEJ) 和同源定向修复 (HDR)3,4.NHEJ 机械的不精确性质经常产生不同长度的 indels5,6,7,8,9。在基因编码序列中引入 frameshift 突变, 可以产生一个过早停止密码子, 这往往使基因无法正常地呈现。
早期的基因组工程战略在斑马鱼促进 indels 包括 meganucleases, 锌手指核酸和转录激活剂类似的效应核酸, 所有这些都利用 DNA 蛋白相互作用的目标核酸酶到特定的基因组目标, 它引入了争端的10,11,12,13,14,15。然而, 这些技术往往很难应用, 因为需要费力和复杂的工程, 以生成一个核酸酶的目标 DNA 序列的兴趣。与以前的策略不同, 基于 CRISPR 的基因编辑并不依赖于蛋白质-DNA 相互作用来进行靶向。相反, CRISPR 相关 (Cas) 限制性是通过 RNA 指南, 使用核苷酸基配对相互作用的目标基因组站点的兴趣16,17,18,19 ,20,21。由于简单的设计一个 RNA 指南与期望的基础配对相互作用的目标, 这是相对容易的靶向 Cas 限制性到所需的轨迹。特别是 II. 型 CRISPR 系统已被广泛开发为基因组编辑应用, 由于几个有利的特点, 包括使用单一多域 Cas 核酸酶 (Cas9), 需要与 DNA 的相互作用, 以刺激限制性活动和使用一个单一的指南 RNA (sgRNA) 靶向它的同源 DNA 序列18。对同源 sgRNA 的靶向序列要求有很好的理解19, 而所需的 sgRNA 很容易通过体外转录产生。CRISPR/Cas9 方法的简单性和健壮性极大地促进了斑马鱼和其他多种生物的靶向基因修饰。
由于开发基于 CRISPR 的试剂, 在斑马鱼中进行靶向基因组编辑的能力增强, 这大大增加了研究诸如中枢神经发育等脊椎动物的过程的机会。系统。斑马鱼基因组包含了在人类基因组中发现的70% 蛋白质编码基因的 orthologs, 以及84% 与人类疾病相关的基因22。斑马鱼的发展展示了几个关键品质, 增强其在逆向遗传学研究中的应用: 胚胎被放置在大的离合器, 外部发展的母亲使他们服从基因操作的显微注射, 和成年斑马鱼性成熟3月的年龄, 允许快速繁殖所需的线23。
许多协议可用于描述在斑马鱼24、25、26、27、28、29 中生成和识别 CRISPR 衍生 indels 的各种方法. ,30,31。然而, 这些程序中的许多都是时间密集型的, 需要获得昂贵的设备, 而且对于具有有限专门知识的实验室来说, 这是很有挑战性的。这里描述的步骤提供了一个简单, 健壮, 经济的 CRISPR/Cas9-strategy, 以工程师斑马鱼淘汰赛线。该协议描述了使用一种高效的试剂盒合成 sgRNAs 使用 DNA 寡核苷酸 (寡核苷酸), 类似于以前描述的其他方法32。所描述的协议包括两个步骤, 特别是大大简化分析 CRISPR 变异线: 逐步使用 PCR 为基础的 HMA33 , 以方便地确定存在的基因组修改, 并排序分析异型斑马鱼以一种经济的方式快速、容易地确定多种 indels 的性质。此外, 还包括分步指导, 用于健壮的选择、可靠的生产和引导 rna 的注入。这里提供的步骤举例说明了一个健壮的、相对低廉的协议, 使实验室人员能够提供一系列专门知识, 有助于鉴定斑马鱼中的基因挖空。

<table>
	<tbody>
		<tr>
			<td>CRISPOR</td>
			<td></td>
			<td></td>
			<td>sgRNA analysis software. http://crispor.tefor.net/</td>
		</tr>
		<tr>
			<td>Breaking-Cas</td>
			<td></td>
			<td></td>
			<td>sgRNA analysis software. https://omictools.com/breaking-cas-tool</td>
		</tr>
		<tr>
			<td>ChopChop</td>
			<td></td>
			<td></td>
			<td>sgRNA analysis software. https://chopchop.rc.fas.harvard.edu/</td>
		</tr>
		<tr>
			<td>Primer 3</td>
			<td></td>
			<td></td>
			<td>PCR primer design. http://bioinfo.ut.ee/primer3-0.4.0/</td>
		</tr>
		<tr>
			<td>Rnase free technique</td>
			<td></td>
			<td></td>
			<td>http://genomics.no/oslo/uploads/PDFs/workingwithrna.pdf</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td>Any brand</td>
			<td></td>
			<td>Synthesis of guide RNAs. Thermo Fisher and Qiagen both carry suitable water.</td>
		</tr>
		<tr>
			<td>Rnase-free ethanol</td>
			<td>Any brand</td>
			<td></td>
			<td>Molecular biology grade ethanol is Rnase free. Fischer, VWR sell suitable ethanol.</td>
		</tr>
		<tr>
			<td>Cas9 Protein</td>
			<td>PNA Bio</td>
			<td>CP01</td>
			<td>Cas9 protein with nuclear localization signal.</td>
		</tr>
		<tr>
			<td>Target specific oligos</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td>Synthesize guide RNAs.</td>
		</tr>
		<tr>
			<td>PCR primers flanking guide RNA cut site</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td>Use for heteroduplex analysis, 100-300 bp product.</td>
		</tr>
		<tr>
			<td>PCR primers flanking guide RNA cut site</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td>Use for sequencing CRISPR-alleles,300-600 bp product recommended.</td>
		</tr>
		<tr>
			<td>EnGen sgRNA Synthesis Kit</td>
			<td>New England BioLabs</td>
			<td>E3322</td>
			<td>In vitro transccribe guide RNAs.</td>
		</tr>
		<tr>
			<td>10X TBE</td>
			<td>Thermo Fisher</td>
			<td>B52</td>
			<td>RNase-free buffer for electrophoresis of nucleic acids.</td>
		</tr>
		<tr>
			<td>6X Load Dye</td>
			<td>New England BioLabs</td>
			<td>B7025S</td>
			<td>Loading DNA for electropheresis.</td>
		</tr>
		<tr>
			<td>5M Ammonium acetate</td>
			<td>Thermo Fisher</td>
			<td>AM9071</td>
			<td>Clean up reagent for purification of guide RNAs.</td>
		</tr>
		<tr>
			<td>Ethanol (200 proof, nuclease-free)</td>
			<td>Any brand</td>
			<td></td>
			<td>Clean up reagent for purification of guide RNAs. Molecular grade materials are RNase free.</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Any brand</td>
			<td></td>
			<td>For synthesis and purification of guide RNAs.</td>
		</tr>
		<tr>
			<td>RNase away</td>
			<td>Thermo Fisher</td>
			<td>10328011</td>
			<td>Eliminates RNase contamination from surfaces, equipment, glassware.</td>
		</tr>
		<tr>
			<td>DNA Ladder, 100 bp</td>
			<td>New England BioLabs</td>
			<td>N0467S</td>
			<td>Molecular weight standards for gel electrophoresis of DNA.</td>
		</tr>
		<tr>
			<td>DNA Ladder, 1 kb</td>
			<td>New England BioLabs</td>
			<td>N0552S</td>
			<td>Molecular weight standards for gel electrophoresis of DNA.</td>
		</tr>
		<tr>
			<td>RNA Ladder</td>
			<td>New England BioLabs</td>
			<td>N0364S</td>
			<td>Single strand RNA marker to verify that full length sgRNA has been synthesized</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Thermo Fisher</td>
			<td>17919</td>
			<td>Casting polyacrylamide gel.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td>Casting polyacrylamide gel.</td>
		</tr>
		<tr>
			<td>40% Polyacrylamide (19:1)</td>
			<td>BioRad</td>
			<td>161-0154</td>
			<td>Casting denaturing polyacrylamide gel.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378-100G</td>
			<td>Casting denaturing polyacrylamide gel.</td>
		</tr>
		<tr>
			<td>RNA Gel Loading Dye (2x)</td>
			<td>Thermo Fisher</td>
			<td>R0641</td>
			<td>Loading guide RNA onto denaturing gel for electrophoresis.</td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma-Aldrich</td>
			<td>E1510-10ML</td>
			<td>Visualization of nucleic acids.</td>
		</tr>
		<tr>
			<td>SYBER Green</td>
			<td>Thermo Fisher</td>
			<td>S7563</td>
			<td>Alternate method to ethidium bromide for detection of dsDNA in agarose or polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td>RNA clean up, PCR purification.</td>
		</tr>
		<tr>
			<td>MyTaq Polymerase</td>
			<td>Bioline</td>
			<td>BIO-21105</td>
			<td>For amplification of DNA using PCR.</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>Any brand</td>
			<td></td>
			<td>PCR amplification.</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Any brand</td>
			<td></td>
			<td>NanoDrop or related product to quantify the amout of DNA/RNA.</td>
		</tr>
		<tr>
			<td>E3 embryo media</td>
			<td>Made in house</td>
			<td></td>
			<td>Media for raising embryos and making injeciton plate. Recipe: http://cshprotocols.cshlp.org/content/2011/10/pdb.rec66449</td>
		</tr>
		<tr>
			<td>Methylene Blue</td>
			<td>Sigma-Aldrich</td>
			<td>M9140</td>
			<td>Added to 1x E3 media to prevent fungal growth on embryos.</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Thermo Fisher</td>
			<td>FB0875711Z</td>
			<td>Store embryos, cast injection plate.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Denville</td>
			<td>CA3510-8</td>
			<td>Casting injection plate, agarose gels.</td>
		</tr>
		<tr>
			<td>Microinection mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td>To create wells to hold embryos during injection.</td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290&#160;</td>
			<td>Dye for visualization of injection.</td>
		</tr>
		<tr>
			<td>Mineral Oil</td>
			<td>Sigma-Aldrich</td>
			<td>M5904-5ML</td>
			<td>To calibrate needle injection volume.</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Any brand</td>
			<td></td>
			<td>Moving embryos.</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Thermo Fisher</td>
			<td>11295-10</td>
			<td>Cutting injection needle, tail clipping adult fish.</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Any brand</td>
			<td></td>
			<td>Maintaining embryos at 28.5 oC.</td>
		</tr>
		<tr>
			<td>Verticle pipette puller</td>
			<td>David Kopf Instruments</td>
			<td>700C</td>
			<td>Geneate needles for injection. Additional needle pulling instructions: http://cshprotocols.cshlp.org/content/2006/7/pdb.prot4651.long</td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>Sutter Instruments</td>
			<td>BF100-58-10</td>
			<td>Geneate needles for injection.</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>Load solution into injection needles.</td>
		</tr>
		<tr>
			<td>Microinjector</td>
			<td>World Precision Instruments</td>
			<td>PV 820</td>
			<td>Injecting embryos.</td>
		</tr>
		<tr>
			<td>Disecting microscope</td>
			<td>Leica</td>
			<td></td>
			<td>Injecting embryos.</td>
		</tr>
		<tr>
			<td>30% Polyacylamide (29:1)</td>
			<td>BioRad</td>
			<td>161-0156</td>
			<td>Heteroduplex gel casting.</td>
		</tr>
		<tr>
			<td>MS-222 (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A-5040</td>
			<td>Anesthetize zebrafish for tail clipping and gDNA extraction from embryos. Recipe: https://wiki.zfin.org/display/prot/TRICAINE</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any brand</td>
			<td></td>
			<td>Casting injection plate, agarose gels.</td>
		</tr>
		<tr>
			<td>Scale</td>
			<td>Any brand</td>
			<td></td>
			<td>Casting injection plate, agarose gels.</td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td>Any brand</td>
			<td></td>
			<td>For all aspects of the protocol.</td>
		</tr>
		<tr>
			<td>N2</td>
			<td>Any brand</td>
			<td></td>
			<td>To expell liquid from the capillary for embryo injection.</td>
		</tr>
		<tr>
			<td>1,000 &#181;L&#160; tips</td>
			<td>Any brand</td>
			<td></td>
			<td>For all aspects of the protocol.</td>
		</tr>
		<tr>
			<td>200 &#181;L tips</td>
			<td>Any brand</td>
			<td></td>
			<td>For all aspects of the protocol.</td>
		</tr>
		<tr>
			<td>10 &#181;L tips</td>
			<td>Any brand</td>
			<td></td>
			<td>For all aspects of the protocol.</td>
		</tr>
		<tr>
			<td>PCR strip tubes</td>
			<td>Any brand</td>
			<td></td>
			<td>For all aspects of the protocol.</td>
		</tr>
		<tr>
			<td>Micropipettes</td>
			<td>Any brand</td>
			<td></td>
			<td>For all aspects of the protocol.</td>
		</tr>
		<tr>
			<td>NGS Sequencing platform: Wideseq</td>
			<td></td>
			<td></td>
			<td>If your institution does not offer this servicce visit: https://www.purdue.edu/hla/sites/genomics/wideseq-2/ (External cost: $35).</td>
		</tr>
		<tr>
			<td>Software for sequence analysis</td>
			<td></td>
			<td></td>
			<td>For Sanger sequencing of heterozygous fish. Visit: http://yosttools.genetics.utah.edu/PolyPeakParser/</td>
		</tr>
		<tr>
			<td>Software for sequence analysis</td>
			<td></td>
			<td></td>
			<td>SnapGene Viewer, Sequence Scanner for analysis of NGS sequencing.</td>
		</tr>
	</tbody>
</table>

efficient production and identification of crispr/cas9-generated gene knockouts in the model system danio rerio

化脓链球菌的聚类、定期 interspaced、短、回文重复 (CRISPR) 系统的特性使得开发一个可定制的平台能够快速地在各种包括斑马鱼在内的生物。基于 CRISPR 的基因组编辑使用单一的指南 RNA (sgRNA) 靶向一个 CRISPR 相关 (cas) 限制性的基因组 DNA (gDNA) 目标的兴趣, 其中 Cas 限制性产生双股断裂 (争端)。通过容易出错的机制修复 DSBs 导致插入和/或删除 (indels)。这可能导致 frameshift 突变, 通常在编码序列中引入一个过早停止密码子, 从而产生蛋白质-零等位基因。基于 CRISPR 的基因组工程学只需要少量的分子成分, 通过显微注射就可以很容易地引入斑马鱼胚胎。本协议描述了用于为斑马鱼注射生成 CRISPR 试剂的方法, 并确定了 CRISPR 修饰基因生殖传播的鱼类。这些方法包括 sgRNAs 的体外转录、CRISPR 试剂的显微注射、在靶点诱导的 indels 的识别, 使用一种称为 heteroduplex 移动性试验 (HMA) 的 PCR 方法, 并描述 indels使用低吞吐量和强大的下一代测序方法, 可以分析从异型鱼收集的多个 PCR 产品。该议定书得到简化, 以尽量减少所需的鱼数和进行分析所需的设备类型。此外, 本议定书的目的是让实验室个人使用包括本科生在内的所有级别的经验, 使这一强有力的工具能够在经济上由任何感兴趣的研究小组执行 CRISPR斑马鱼基因组的改良。

本协议中描述的实验方法可以利用 CRISPR/Cas9 技术高效、经济高效地生产斑马鱼的淘汰线。本文中包括以下数字, 以便于解释和排除使用本协议获得的结果。通过 CRISPR 试剂的成功生产和显微注射, 可以分析斑马鱼胚胎的显性表型和 indel 的 HMA。一个有用的控制, 以可视化的成功的 CRISPR 实验是使用 sgRNA 描述在步骤1.5 的目标的色素生成的基因酪氨酸酶.酪氨酸酶Cas9-induced indel 的形成导致色素丧失, 容易获得 48 hpf (图 1)。另一项有帮助的控制, 以确保制备注射 CRISPR 试剂是成功的, 是验证全长 (120 nt) sgRNA 已合成的变性聚丙烯酰胺凝胶 (图 2, 车道1和 2)。如果 rna 已经退化, 它可能会出现作为涂片, 例如3车道 (图 2) 显示退化的 RNA 不适合注射。
为了分析 CRISPR-Cas9 所针对的基因 indel 的形成频率, 不产生酪氨酸酶等显性表型, HMA 分析是一种简单可靠的方法。sgRNA/Cas9 注射的胚胎分析使用 HMA 结果形成 heteroduplex 带, 并降低强度的 homoduplex 带 (图 4)。该议定书进一步利用了 heteroduplex 带的存在, 以确定潜在的杏仁胚胎和成人的创始人鱼 (图 4和图 5), 以分析创始人的生殖传输效率 (图 6), 并验证异型 F1 鱼中是否存在 indel (图 7)。含有 indel 的异型鱼是候选品, 用于确定 indel 的性质, 并确定目标基因编码区域是否存在过早停止密码子。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56969/56969fig1.jpg"> 图 1: 斑马鱼胚胎在单细胞阶段注射 sgRNA 靶向酪氨酸酶时表现出色素缺陷.(A) 野生型, uninjected 胚胎在 48 hpf 和 (B) 注射胚胎在 48 hpf。<a href="https://cloudflare.jove.com/files/ftp_upload/56969/56969fig1large.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/56969/56969fig2.jpg"> 图 2: 用合成试剂盒对 sgRNA 进行体外转录.根据 sgRNA 合成试剂盒的说明, 寡核苷酸的体外转录合成。500 ng 的 RNA 是运行在尿素/页凝胶如所述。sgRNA 在车道1和2显示一个带对应于全长, 原封 120 nt RNA。sgRNA 在3车道显示一个退化的 RNA 样品不适合注射。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56969/56969fig3.jpg"> 图 3: 比较 24 hpf 注射胚胎的健康状况.活胚 (a) 发育为 24 hpf, 很容易与流产的胚胎 (B) 区别开来。胚胎, 如 (B) 或有急剧改变的特点 (A), 如脊柱曲率或改变头部和眼睛发育应从盘子中删除。<a href="https://cloudflare.jove.com/files/ftp_upload/56969/56969fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56969/56969fig4.jpg"> 图 4: Heteroduplex sgRNA-Cas9 杏仁斑马鱼胚胎的流动性测定.在 72 hpf 收集了每样5个胚胎的水池, 提取了 gDNA。Heteroduplex 分析进行了描述, 样品被加载平均 500 DNA。车道: M = 100 bp 标记;1 = uninjected 控制;2 = 射入样品 1;3 = 注塑样品2。预期频带大小 = 98 bp。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56969/56969fig5.jpg"> 图 5: Heteroduplex 移动性测定 gDNA 从一个成年 CRISPR 注射斑马鱼的尾部提取.注射了 sgRNA 和 Cas9 蛋白的胚胎生长到成年期 (3 月)。鱼 B 和 C 展示 heteroduplex 带, 随后养殖以确定生殖传播的 indels;鱼 A 在随后的分析中没有被使用, 因为它不表现出一个积极的 heteroduplex 带。车道: 1 = 野生型控制;2 = 鱼 A;3 = 鱼 B;4 = 鱼 c 期望的带大小 = 98 bp.<a href="https://cloudflare.jove.com/files/ftp_upload/56969/56969fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56969/56969fig6.jpg"> 图 6: Heteroduplex 移动性检测由 F0 CRISPR 注射斑马鱼, 以确定生殖传播 indels 的野生型鱼类产生的单一胚胎.斑马鱼交配, F1 胚胎生长为72小时的单胚, 并进行 heteroduplex 分析的描述。车道: 1 = 野生型控制;2-10 = 每条车道的一个 F1 胚。该凝胶显示, 7 的10胚胎显示一个积极的 heteroduplex 带, 表明生殖传输率70% 的 indel。预期频带大小 = 98 bp.<a href="https://cloudflare.jove.com/files/ftp_upload/56969/56969fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56969/56969fig7.jpg"> 图 7: Heteroduplex F1 斑马鱼尾夹的移动性测定.HMA 对成年 F1 鱼进行了鉴别 indels。展示了阳性 heteroduplex 带的鱼进行 PCR 扩增, 并提交广泛的测序分析, 以确定突变的性质。(A) 车道: 1 = 野生型控制;2 = 鱼 A (4 bp 删除, 1 bp 不匹配)。(B) 这些 F1 鱼是从同一创始人那里发现的, 但却显示出不同的 heteroduplex 模式, 表明生殖从一个创始人那里传送多个修饰的等位基因。车道: 1 = 野生型控制;2 = 鱼 B (4 bp 插入, 7 bp 不匹配), 3 = 鱼 C (4 bp 删除, 4 bp 不匹配)。这些 indels 在目标基因的编码序列中创建了一个过早停止密码子, 由<a href="https://cloudflare.jove.com/files/ftp_upload/56969/56969fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio at JoVE.com

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

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

斑马斑马(斑马鱼) 在模型系统中的靶向基因组编辑由于 CRISPR 方法的出现而大大促进。在这里, 我们描述了一个简化的, 健壮的协议, 以生成和鉴定 CRISPR 衍生的无意义的等位基因, 包括 heteroduplex 移动性检测和识别突变使用下一代测序。

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

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Research

JoVE Journal

Genetics

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

21.7K 次观看.  Purdue University. 斑马斑马(斑马鱼) 在模型系统中的靶向基因组编辑由于 CRISPR 方法的出现而大大促进。在这里, 我们描述了一个简化的, 健壮的协议, 以生成和鉴定 CRISPR 衍生的无意义的等位基因, 包括 heteroduplex 移动性检测和识别突变使用下一代测序。

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

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

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 ...

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 ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

杆状病毒基因治疗应用的生产与纯化

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

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. ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms to questions surrounding insect's interactions with their host plants. Genomic resources are available for several aphid species, and with advances in the next-generation sequencing, transcriptomic studies are being extended to non-model organisms that lack genomes. Furthermore, aphid cultures can be collected from the field and reared in the laboratory for the use in organismal and molecular experiments to bridge the gap between ecological and genetic studies. Last, many aphids can be maintained in the laboratory on their preferred host plants in perpetual, parthenogenic life cycles allowing for comparisons of asexually reproducing genotypes. Aphis nerii, the milkweed-oleander aphid, provides one such model to study insect interactions with toxic plants using both organismal and molecular experiments. Methods for the generation and maintenance of the plant and aphid cultures in the greenhouse and laboratory, DNA and RNA extractions, microsatellite analysis, de novo transcriptome assembly and annotation, transcriptome differential expression analysis, and qPCR verification of differentially expressed genes are outlined and discussed here.

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

The aphid Aphis nerii colonizes on highly-defended plants in the dogbane family (Apocyanaceae) and provides numerous opportunities to study plant-insect interactions. Here, we present a series of protocols for the maintenance of plant and aphid cultures, and the generation and analysis of molecular and -omic data for A. nerii.

nerii 蚜生物培养和基因表达的维持--植物-昆虫相互作用的非模型系统

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System

Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.

使用CRISPR/Cas9系统将基因挖空直接引入锥虫瘤的肌瘤阶段

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas&#8217; disease mainly in Latin America. In order to identify a novel drug target ...