作者要感谢 Prof. Dr. 和 Prof. Dr. Goetz 为他们的投入, 帮助和支持发展 CORALINA 方法, 马西米兰 Wiessbeck 和瓦伦丁鲍曼的有用的评论。这项工作得到了 DFG (STR 1385/1-1) 的支持。

1. Micrococcal 核酸的 DNA 消化
<ol><li>对每一个新的 micrococcal 核酸酶 (MNase) 进行优化反应。 注: 使用的 MNase 的单位数应进行测试 (使用串行稀释,图 2A)。通常, 5-10 ü MNase 消化1µg 纯化基因组或 BAC DNA 下降到 5-100 bp 的范围, 下面描述的条件。</li>
	<li>每反应, 建立了1µL 的 10x MNase 缓冲, 1x 牛血清白蛋白 (BSA), 1 µg 的目标 DNA, 1 µL MNase (0.1-50 单位) 在10µL 反应体积。</li>
	<li>孵育在37° c 为15分钟。</li>
	<li>立即钝化酶, 加入1µL 500 毫米乙二醇双 (β-氨基乙基醚)-n, n, n, n-', 乙酸酸 (EGTA)。</li>
</ol>2. 用聚丙烯酰胺凝胶电泳分离 DNA 片段 (页)
<ol><li>添加样本缓冲/凝胶加载染料的 DNA 样本和负载到一个20% 页的凝胶。为上浆 (5 bp dna 阶梯) 加载合适的 dna 阶梯。不要使凝胶 (每井1µg DNA) 过载。</li>
	<li>运行凝胶在适当的1x 三硼酸盐-EDTA (无) 运行缓冲在 150 V (恒定) 为大约 1.5 h 或直到更低的染料前面 (溴蓝色, 深蓝颜色) 旅行在大约 15 bp, 到达胶凝体的更低的末端。</li>
	<li>使用超灵敏的核酸染色和 UV 光下的可视化来着色凝胶。</li>
	<li>从凝胶图像, 确定最佳浓度的 MNase 消化 DNA 下降到 20-30 bp 的大小。</li>
	<li>通过重复步骤 1.2-2.2, 使用 MNase 的优化浓度来消化目标 DNA。通常, 设置10-12 反应 (即10-12 µg 的总起始材料) 将产生足够的消化 DNA 后, 页凝胶提取, 以进行后续步骤。</li>
	<li>使用无菌手术刀, 切割在标记车道旁边的凝胶, 并只对含有该阶梯的凝胶部分进行染色, 新的1x 的运行缓冲含有1X 的超灵敏核酸染色。可视化的 dna 阶梯和消费 MNase 消化 dna 片段的大小范围内约 18-30 bp 使用刀片。 注意: 避免将 MNase 消化的碎片暴露在 UV 光中。它可以使用蓝色光源 (而不是 uv) 或采取在紫外线下的梯子的图像, 打印它的规模, 并使用它来指导切除 MNase 消化的 DNA 片段)。这一步避免了染色和暴露的 DNA 片段, 以紫外光。在这一步中, 总是使用无菌的一次性手术刀或刀片, 以避免污染。</li>
	<li>将凝胶切片转移到离心管。</li>
	<li>染色的其余部分的凝胶与核酸染色如上所述, 暴露在紫外线和记录一个图像, 以保持一个记录的凝胶切除步骤。</li>
</ol>3. 用粉碎和浸泡法分离页凝胶中的 DNA 片段
注意: 此步骤已从理查德·山姆布鲁克et al.中采用11
<ol><li>准备页面凝胶增溶缓冲剂 (1.88 毫升4米醋酸铵, 150 µL 1 米醋酸镁, 30 µL 0.5 米乙酸酸 (EDTA) (pH 8) 在超纯 H2O 到总容量15毫升)。</li>
	<li>使用无菌吸管尖端将切除的凝胶片粉碎到微离心管的壁上。</li>
	<li>在旋转平台上加入2页增溶缓冲剂, 在37° c 下孵育16小时。</li>
	<li>在离心中以最大速度离心样品1分钟。把上清液转移到一个新的离心管, 注意不要转移任何粉碎的凝胶件。</li>
	<li>增加0.5 卷的页面增溶缓冲剂的凝胶颗粒, 涡旋, 重复离心 (步骤 3.4)。结合清。</li>
	<li>
用标准的苯酚-氯仿萃取提取 DNA 片段。 注意: 如果与皮肤接触或吞食, 苯酚是有毒的。安全预防措施, 如手套, 防护眼镜, 实验室大衣, 并在通风罩工作是至关重要的。根据研究所的规定处理所有含酚废物。<ol>
<li>将一卷 phenol:chloroform:isoamyl 醇 (25:24:1) 添加到样品和涡流中。</li>
		<li>在室温下的台式离心机中, 离心机的最大转速为10分钟 (1.6万 x g)。小心地将上部水相转移到新鲜的离心管。注意不要在移期间携带任何苯酚。</li>
		<li>重复步骤3.6.1 和3.6.2 一次。</li>
		<li>添加少量 (0.2 µL) 的糖原, 0.1 卷3米醋酸钠 (pH 5.2) 和2卷100% 乙醇 (乙醇)。</li>
		<li>漩涡和孵化在-20 ° c 几个小时或过夜。</li>
		<li>用台式离心机在4° c 的最大转速下离心30分钟。</li>
		<li>小心去除上清液, 用70% 乙醇清洗 DNA 颗粒。</li>
		<li>用台式离心机在4° c 的最大转速下离心30分钟。</li>
		<li>除去上清和风干的 DNA 颗粒。确保所有的乙醇已蒸发, 但小心不要过度干燥的颗粒。</li>
		<li>在12µL 的 H2O 中溶解 DNA 颗粒。 注: 页面凝胶提取是非常低效的。每10µg 开始 DNA 消化 MNase, 预期恢复 1-20 ng 纯化后的凝胶提取物。通过在页面凝胶上加载 1/6th (2 µL) 来控制碎片的数量和完整性 (图 2C)。</li>
	</ol>
</li>
</ol>4. MNase 的末端修复, 凝胶纯化的碎片
<ol><li>使用 DNA 钝化试剂盒建立以下反应:10 µL 的纯化 dna 从步骤 3.6.10, 1.5 µL 的10X 钝化缓冲, 1.5 µL 1 毫米 dNTP 混合, 0.6 µL 钝化酶, 1.4 µL 的 H2O。</li>
	<li>孵育在22° c 为30分钟, 然后加热-钝化酵素在70° c 为 10 min。</li>
	<li>添加85µL 的 H2O, 并使用标准苯酚/氯仿萃取和乙醇沉淀 (如3.6 节所述) 执行反应清洁。立即进行连结器结扎。</li>
</ol>5. 链接器生成
注意: 连接需要与3节并行放大, 以便能够立即进行连接器结扎。下面使用的引物序列必须适合于所选的 gRNA 表达载体。这里介绍的是为矢量 pgRNA-pLKO 1。9为放大 5 "连接器从 pgRNA-pLKO 1, 使用底漆序列 5"-链接器 F (TTGGAATCACACGACCTGGA) 和 5 '-链接器 R (CGGTGTTTCGTCCTTTCCAC), 产生 689 bp 增。为放大 3 ' 连接器从 pgRNA-pLKO 1, 使用底漆 3 '-连接器 F: (GTTTTAGAGCTAGAAATAGCAAGTTAAAATA) 和 3 '-链接器 R: (ACTCGGTCATGGTAAGCTCC), 产生 848 bp 增。
<ol><li>PCR-放大适配器序列从 gRNA 表达载体 (使用选择试剂和自定义引物序列, 如果需要)。为 pgRNA-pLKO. 1 建立了以下50µL pcr 反应:25 µL pcr 大师组合, 2.5 µL 底漆 F (10 µM), 2.5 µL 底漆 R (10 µM), 0.1 ng gRNA 表达载体 (pgRNA-pLKO. 1) 在 H2O。</li>
	<li>孵育反应在 thermocycler 使用以下情况: 1 周期98° c 为三十年代, 32 个周期98° c 为十年代, 59 ° c 为十年代, 72 ° c 为三十年代, 1 个周期72° c 为 10 min。</li>
	<li>根据制造商的说明, 用固相可逆固定化珠纯化 PCR 反应。洗在30µL 中的 H2O。</li>
	<li>
要强制连接器定向结扎到最终修复的, MNase 消化的 DNA 片段, 消化连接与适当的限制性酵素 (这里后 III 和 SacII)。设置以下反应:
	<ol>
<li>为消化 5 ' 链接器与后 iii, 使用30µL 纯化 5 ' 链接增从步骤 5.3., 5 µL 的缓冲, 3 µL 的后 III (20 u/µL), 和12µL 的 H 2O。</li>
		<li>为消化 3 ' 连接器与sacII, 使用30µL 纯化 3 ' 链接增从步骤 5.3., 5 µL 的缓冲, 3 µL 的sacii (20 U/µL), 和12µL 的 H2O。</li>
	</ol>
</li>
	<li>孵育消化在37° c 为 3 h。</li>
	<li>添加 DNA 凝胶加载染料和运行限制酶消化1% 琼脂糖凝胶。在 637 bp (5 "链接器摘要") 和 295 bp (3 "链接器摘要") 上对频带进行切除。</li>
	<li>使用凝胶萃取试剂盒净化从切除的凝胶件的 DNA。</li>
</ol>6. 连接器结扎和插入物的放大
<ol><li>
建立一个14µL 结扎反应使用摩尔量的 MNase 消化, 最终修复片段和链接器序列。 注意: 链接器到片段比率可以优化。建议包括无碎片控制 (NFC) 反应。<ol>
<li>使用 5 ng MNase 消化的碎片 (最终修复和纯化), 120 ng 5 ' 链接器 (后 III 摘要, 纯化), 55 ng 3 ' 链接器 (SacII 文摘, 纯化), 1.4 µL T4 连接缓冲, 1.4 µL T4 在 H2O。</li>
	</ol>
</li>
	<li>孵育的结扎反应在16° c 为16小时。不对酶进行热灭活。立即进行尼克翻译。 注: 最终修复的 MNase 消化片段提供了 5 ' 磷酸盐必要的结扎连接, 因为连接器本身是不磷酸化。</li>
	<li>对结扎反应 (和 NFC 反应) 增加以下:25 µL Taq 2X 大师组合 (能尼克翻译), 2.5 µL 底漆 Linker-Minus450-F (10 µM, GGGCAAGTTTGTGGAATTGG), 2.5 µL 底漆 Linker-Plus275-R (10 µM, AAGTGGATCTCTGCTGTCCC) 和6µLH2O。</li>
	<li>包括无模板控件 (NTC)。在 thermocycler 上孵育反应使用以下条件: 1 周期 (尼克翻译):72 ° c 为 20 min, 1 周期:95 ° c 为5分钟, 3-4 个周期:95 ° c 为十五年代, 58 ° c 为十五年代, 72 ° c 为三十年代, 1 个周期:72 ° c 为 5 min。</li>
	<li>采用固相可逆固定化珠进行反应清理, 样品珠比为1:1。洗在40µL 中的 H2O。</li>
	<li>
进一步放大所需的片段 (5 ' 链接器 + MNase 片段 + 3 ' 链接器) 使用适当的 PCR 试剂和以下引物:
	<ol>
<li>使用12.5 µL PCR 大师组合, 1.25 µL 底漆 Linker-Minus450-F (10 µM, GGGCAAGTTTGTGGAATTGG), 1.25 µL 底漆 Linker-Plus275-R (10 µM, AAGTGGATCTCTGCTGTCCC), 2.5 µL 的纯化尼克翻译产品从步骤6.4。7.5 µL 的 H2o 使用以下条件: 1 周期:98 ° c 为三十年代, 10-16 个周期:98 ° c 为十年代, °ç为十年代, 72 c 为十五年代, 1 周期:72 °为10分钟。 注: 如有必要, 可同时设置多个反应, 以确保有足够的 PCR 产物用于后续步骤。为了在琼脂糖凝胶上可视化少量的 PCR 产物, 在步骤6.5 中设置额外的反应, 并将循环次数从15增加到32。这种反应可以作为质量控制, 如果增在15周期之后是不可见的。也包括无模板控件 (NTC)。</li>
	</ol>
</li>
</ol>7. 尺寸选择
注意: 此步骤将 MNase 碎片与正确连接的 5 "和 3" 链接器从片段中分离出来, 其大小为两个 5 "或两个 3" 连接 "。
<ol><li>结合所有15周期 PCR 反应从步骤 6.5, 添加 DNA 加载染料和运行在0.8% 琼脂糖凝胶。使用一个凝胶萃取试剂盒对 869 bp 的突出频段进行切除并纯化 DNA。</li>
	<li>量化的数量的 DNA (例如使用分光光度计)。</li>
</ol>8. 通过吉布森组装将 PCR 扩增片段克隆成 gRNA 表达载体
<ol><li>
准备组件主组合, 如下所示: 注意: 以下步骤适用于吉布森et al.12。<ol>
<li>通过结合3毫升的1米三 (羟甲基) 氨基 (三)-HCl pH 值 7.5, 创建6毫升等温反应缓冲器, 300 µL 1 米氯化镁, 60 µL 100 毫米脱氧三磷酸 (dGTP), 60 µL 100 毫米脱氧三磷酸 (dATP), 60 µL 100 毫米苷三磷酸 (dTTP), 60 µL 100 毫米脱氧三磷酸 (桩), 300 µL 1 M 糖 (德勤), 1.5 g 的聚乙二醇 (PEG)-8000, 300 µL 100 毫米烟酰胺腺嘌呤核苷酸 () 在超纯 H2O。 注: 此缓冲器可 aliquoted, 贮存于-20 ° c。</li>
		<li>结合320µL 的5X 等温反应缓冲器, 创建1.2 毫升的装配主组合, 3 µL 10 u/µL T5 酶, 20 µL 2 u/µL dna 聚合酶, 160 µL 的 40 u/µL dna 连接在超纯 H2o. 使用15µL 的组件主组合与5µL插入. 注: 组件主混合可 aliquoted 并存储在-20 ° c, 在那里它是稳定的一年多, 可以容忍多个冻融循环。T5 酶的选择量是理想的使用长悬垂。</li>
	</ol>
</li>
	<li>
向量骨架消解 注意: 在步骤8.3 中, 请确保将足够的矢量作为输入, 以满足所需的组件反应数量。<ol>
<li>每反应, 增加以下: 1.5 µg gRNA 表达载体 (pgRNA-pLKO. 1), 5 µL 的缓冲, 1.5 µL 的年龄I (5 u/µL), 和38.5 µL h2o. 在37° c 下孵育文摘 2 h。</li>
	</ol>
</li>
	<li>
线性化向量的磷酸。 注意: 这一步是建议的, 但不是绝对必要的。T5 酶在主组合将主要删除年龄我悬在之前的 Taq DNA 连接有机会采取行动。因此, 不需要对向量进行过多的再结扎。<ol>
<li>加入2.5 µL 的虾碱性磷酸酶 (rSAP, 1 U/µL)。</li>
		<li>孵育在37° c 为30分钟, 然后通过孵化在65° c 为5分钟钝化酶。</li>
	</ol>
</li>
	<li>进行 DNA 纯化 注意: 建议执行琼脂糖凝胶提取步骤。或者, 可以使用柱形或磁珠净化。重要的是要检查, 矢量消化是完整的, 如琼脂糖凝胶电泳。为了比较, 未消化的向量应该并行运行。</li>
	<li>对样品中纯化的消化载体的数量进行量化。</li>
	<li>用2倍的摩尔数来设置装配, 将刀片插入到向量中。每20µL 反应使用 100 ng 向量 (年龄I 消化从步骤 8.5), 12.2 ng 插入 (从步骤 7.1), 15 µL 的装配大师混合从步骤8.1.2。在 H2O 中。</li>
	<li>孵育在50° c 为1小时。</li>
	<li>用柱纯化净化反应。重的 DNA 在75µL 的 H2O (或适当的体积取决于电穿孔的规模)。 注意: 转化效率很大程度上依赖于 DNA 纯度。其他纯化步骤 (如苯酚/氯仿萃取) 可能提高效率。</li>
</ol>9. 电子 TG1 的制备大肠杆菌细胞
<ol><li>确保所有的离心机瓶和烧瓶在灭菌前漂洗和随后的蒸馏水中无洗涤剂。 注意: 这一步有助于消除任何可能影响转化效率的杂质。水应在使用前立即丢弃。另外, 使用一次性离心瓶可能是可取的。</li>
	<li>确保用于制备 electrocompetent 细胞的离心瓶、试管和溶液在使用前结冰。最好在寒冷的房间中进行以下步骤, 以尽量减少可能影响转换效率的温度波动。</li>
	<li>准备2TY 介质。对16克 bacto 胰, 10 克酵母提取物, 和5克氯化钠, 加入蒸馏水到1升, 混合和高压釜。室温贮存介质。</li>
	<li>准备2份琼脂涂层生物测定皿和 10 cm 培养皿, 含有适当的抗生素。贮存盘在4° c。 注: 抗生素的选择取决于 gRNA 表达载体的性质, 使用100µg/毫升氨苄西林 pgRNA-pLKO. 1。</li>
	<li>从甘油 TG1 细胞中刮出, 接种10毫升的2TY 培养基 (无抗生素)。</li>
	<li>孵育文化在37° c 隔夜 (~ 16 h) 以震动在225转每分钟。</li>
	<li>接种1升2TY 培养基 (不含抗生素) 与10毫升夜间培养 (1/100 稀释), 并平分它在两个2升烧瓶 (包含挡板)。</li>
	<li>孵育文化在37° c, 225 转每分钟直到 OD600 毫微米0.55 被到达 (近似地在 1.5-2 h 以后)。使用分光光度计定期检查 OD600 nm。</li>
	<li>冰镇文化冰上30分钟。</li>
	<li>在四500毫升的离心机瓶 (冰前冷冻) 之间平分养殖。</li>
	<li>离心机为15分钟在 4000 x g 在4° c 在一个预冷离心机。</li>
	<li>醒酒清, 并添加1卷 (即250毫升) 前冷冷无菌蒸馏 H2O 到每一个离心机瓶。通过旋转或倒置瓶子 (如有必要, 通过轻柔的移) 重细菌颗粒。 注: 通过先加入少量的水, 更容易重颗粒。确保颗粒最终完全悬浮。</li>
	<li>离心机为15分钟在 4000 x g 在4° c。</li>
	<li>重复洗涤两次 (步骤9.12 和 9.13)。除去上清液。迁当细菌颗粒在洗涤后变得越来越疏松时要小心。</li>
	<li>重在50毫升的无菌, 冰冷的10% 甘油, 并转移到一个预冷50毫升离心管的颗粒。</li>
	<li>离心机细胞15分钟在 ~ 4000 x g 在4° c。小心地除去上清液。</li>
	<li>在2毫升的冰冷无菌10% 甘油中轻轻重细菌。</li>
	<li>如果细胞被立即用于电穿孔, 请保持冰。 注: 在0.5 毫升的干冰乙醇浴中, 细胞可在50µL 的等分中冷冻, 并贮存在-80 ° c, 但不推荐这样做。</li>
</ol>10. TG1 Electrocompetent 的电穿孔大肠杆菌细胞
注: 电穿孔是综合图书馆产生的瓶颈之一。为了保持图书馆的代表性, 建议在必要的/切实可行的情况下进行许多个人电穿孔反应, 并执行下面描述的质量控制步骤 (10.6. 和 10.8)。
<ol><li>分的纯化反应 (从步骤 8.8) 到无菌和预冷 PCR 管, 并保持在冰 (1 µL 每管)。冷却 1 mm 间隙电穿孔试管在冰上。</li>
	<li>添加25µL 的新鲜制备的 TG1 细胞直接到一个分的 DNA, 并立即转移到一个电穿孔试管的混合物。轻拍或轻击试管, 以确保细胞/DNA 混合物沿试管室的长度分布 (没有任何被困的空气或气泡)。</li>
	<li>将试管放在滑动室中, 并启动适当的电穿孔程序 (例如, 1 脉冲1.8 伏 (EC1))。 注: 时间常数应介于5.7 和6.0 毫秒之间. 万一 electroporator 弧线, 轻弹试管, 确保室内没有气泡, 再试一次。</li>
	<li>立即添加975µL 室温2TY 介质到试管。</li>
	<li>使用转移吸管, 移动 electroporated 细菌到50毫升管。重复步骤10.2。在一个50毫升的管子里收集所有与一个图书馆转换的细胞。</li>
	<li>记录总卷。</li>
	<li>
质量控制步骤
	<ol>
<li>为了量化新生成的电性细胞的能力, 使用定义数量的未切割质粒 DNA (如 10 pg pUC19 控制质粒) 进行单独的电击反应。把这个转到1.5 毫升的离心管 注: 该能力应至少 1010菌落形成单位 (cfu) 每µg DNA。新鲜的细胞通常表现得比这更好。</li>
	</ol>
</li>
	<li>孵育转化细菌在37° c 60 分钟晃动 225 rpm。</li>
	<li>
质量控制步骤:
	<ol>
<li>在一个10厘米琼脂平板上, 用适当的抗生素选择, 将少量的细菌转化为库 (例如, 10 µL 10-100 倍稀释)。 注意: 了解总的文化量 (从步骤 10.6), 得到的菌落数可以用来估计整个图书馆的菌落总数。20-30 图书馆的折叠表示法理想地应该被维护。</li>
	</ol>
</li>
	<li>将细菌的其余部分分散到2TY 的琼脂涂层生物测定皿中, 其中含有适当的抗生素选择。 注: 该培养液的体积可通过离心在 ~ 4000 x g 为10分钟 (或直到一个可见的颗粒形成, 上清看起来清晰) 减少。这减少时间板材需要烘干在传播以后文化。使用平板而非液态文化可以最大限度地减少个体群体的不成比例的增长。13
</li>
	<li>在一个额外的10厘米琼脂的 pUC19 控制电穿孔反应适当的体积, 适当的抗生素选择。</li>
	<li>在37° c 的夜间孵育琼脂板 (16 小时)。</li>
	<li>计数获得的控制板上的殖民地 (pUC19 和控制库板) 估计 CFU/µg DNA 的细菌以及图书馆的复杂性。</li>
</ol>11. 质粒 DNA 的提取
<ol><li>将10毫升 2-TY 介质添加到隔夜板上, 用一次性的吊具将细菌层刮掉, 然后用50毫升的管子将其收集起来。重复几次, 直到所有的盘子看起来都是干净的。</li>
	<li>使用质粒最大试剂盒提取 DNA (每个生物检测板需要2-3 列)。</li>
</ol>

<ol>
	<li>Mali, P., Esvelt, K. M., Church, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9+as+a+versatile+tool+for+engineering+biology.">Cas9 as a versatile tool for engineering biology.</a> Nat Methods. 10 (10), 957-963 (2013).</li><li>Yang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+generation+of+mice+carrying+reporter+and+conditional+alleles+by+CRISPR/Cas-mediated+genome+engineering.">One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering.</a> Cell. 154 (6), 1370-1379 (2013).</li><li>Stricker, S. H., Koferle, A., Beck, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+profiles+to+function+in+epigenomics.">From profiles to function in epigenomics.</a> Nat Rev Genet. 18 (1), 51-66 (2017).</li><li>Dominguez, A. A., Lim, W. A., Qi, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+editing:+repurposing+CRISPR-Cas9+for+precision+genome+regulation+and+interrogation.">Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation.</a> Nat Rev Mol Cell Biol. 17 (1), 5-15 (2016).</li><li>Shalem, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-scale+CRISPR-Cas9+knockout+screening+in+human+cells.">Genome-scale CRISPR-Cas9 knockout screening in human cells.</a> Science. 343 (6166), 84-87 (2014).</li><li>Wang, T., Wei, J. J., Sabatini, D. M., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+screens+in+human+cells+using+the+CRISPR-Cas9+system.">Genetic screens in human cells using the CRISPR-Cas9 system.</a> Science. 343 (6166), 80-84 (2014).</li><li>Koike-Yusa, H., Li, Y., Tan, E. P., Velasco-Herrera Mdel, C., Yusa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+recessive+genetic+screening+in+mammalian+cells+with+a+lentiviral+CRISPR-guide+RNA+library.">Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.</a> Nat Biotechnol. 32 (3), 267-273 (2014).</li><li>Hart, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+CRISPR+Screens+Reveal+Fitness+Genes+and+Genotype-Specific+Cancer+Liabilities.">High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.</a> Cell. 163 (6), 1515-1526 (2015).</li><li>Koferle, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CORALINA:+a+universal+method+for+the+generation+of+gRNA+libraries+for+CRISPR-based+screening.">CORALINA: a universal method for the generation of gRNA libraries for CRISPR-based screening.</a> BMC Genomics. 17 (1), 917-940 (2016).</li><li>Lane, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatically+Generated+CRISPR+Libraries+for+Genome+Labeling+and+Screening.">Enzymatically Generated CRISPR Libraries for Genome Labeling and Screening.</a> Dev Cell. 34 (3), 373-378 (2015).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+DNA+fragments+from+polyacrylamide+gels+by+the+crush+and+soak+method.">Isolation of DNA fragments from polyacrylamide gels by the crush and soak method.</a> CSH Protoc. (1), (2006).</li><li>Gibson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+overlapping+DNA+fragments.">Enzymatic assembly of overlapping DNA fragments.</a> Methods Enzymol. 498, 349-361 (2011).</li><li>Elsaesser, R., Paysan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liquid+gel+amplification+of+complex+plasmid+libraries.">Liquid gel amplification of complex plasmid libraries.</a> Biotechniques. 37 (2), 200-202 (2004).</li></ol>

作者没有什么可透露的。

CORALINA 可用于生成大规模的 gRNA 库由控制核酸消化的目标 DNA 和大量克隆产生的双滞留片段。统计推断表明, 许多超过 107的单个 gRNA 序列已经成功地使用了9的协议进行了克隆。CORALINA 可以通过多种方式进行自定义。模板 DNA 的选择定义了目标区域和生成库的最大复杂性。使用此协议, CORALINA 库以前是由人类和小鼠基因组 DNA9生成的。代表性的结果描述了从纯化 BAC DNA 的 CORALINA 库的生成。通过选择 gRNA 表达式向量和链接器序列可以实现进一步的自定义。我们以前测试了三不同的连接器长度对吉布森组件, 效率在9中的变化不大。
由于其起源于大量消化的 DNA, protospacer 的 CORALINA gRNAs 通常不完全 20 bp 长度, 但显示长度分布与一个平均值, 这取决于两个参数的 MNase 消化和大小的切除从页面凝胶s.图 2B和C中所示的代表性示例描述了19和 27 bp 之间的中间长度的片段。根据我们的经验, 所生成的 gRNA protospacer9忠实地保存了片段的长度。虽然由于结果 gRNAs 的离靶速率更高, 所以应避免片段少于 20 bp, 但较长的碎片可能对下游应用的问题更少, 因为它已经证明, gRNAs 与 protospacers 只要 45 bp 仍然功能9。
CORALINA 协议中最关键的两个步骤是 MNase 分解片段的大小选择和克隆步骤。生成的片段太短 (例如低于 18 bp) 或合并太多的空 gRNA 表达式向量将使库变得无用。因此, 最重要的是优化 MNase 消化步骤 (图 2A), 以监视切除 (图 2B, C), 检查 gRNA 向量主干的完全消化, 并在整个协议中包括无片段控件。还必须特别注意保护 gRNA 图书馆的代表性。一般情况下, 图书馆产生的一个普遍瓶颈是质粒向细菌的有效转移。因此, 大量的细菌具有优良的能力和大量的个人电穿孔事件是必要的, 以实现大量的 gRNA 克隆。
gRNA 图书馆生产的新战略将是必要的, 以便在未来几十年收获 CRISPR 的筛选方法的全部潜力。有一个巨大的需求, 成本效益, 简单和可定制的方法, 以产生大规模的图书馆, 一个先决条件, 使筛选适合于更多的模型系统和不同 CRISPR 的工程方法。CORALINA 正在向这个方向迈出第一步。潜在的用途是多方面的, 特别是产生综合的基因组库、不太常见的模型系统的 cDNA 文库、高度集中的库和实验性的集 CRISPR 蛋白 (不同的 PAM要求) 结合使用。
与其他方法不同, CORALINA 从输入 DNA 中生成所有可能的 gRNAs。然而, 该方法的一个缺点是, gRNAs 缺乏所需的 PAM 序列也包括在图书馆, 这一功能, 它与第二个酶的方法共享 gRNA 图书馆生成, CRISPR 吃 (表 1)。gRNA 库生成的理想方法的选择取决于计划的筛选实验的规范, 特别是目标区域的性质 (基因、调控、基因) 和大小 (单个轨迹、多个区域、全基因组)。当要分析大量非编码或管理区域时, 如果有不完整或不可靠的序列信息 (奇异的模型系统、种类的混合物 (例如微生物) 或实验获得的输入), 如果不同的 CRISPR 酶合并或如果饱和分析是对一个短的和定义的轨迹 (如代表的巴克斯)。

细菌 CRISPR/Cas9 系统作为分子靶向工具的适应, 引起了分子生物学的最新革命。在定义的基因组位置上, 从没有这么容易操作染色质。CRISPR 的常见应用包括靶向基因突变1, 基因组工程2, 基因编辑3, 转录激活和基因沉默4。CRISPR 系统的一个特别的优点是, 它的应用并不局限于研究良好的候选网站, 因为 gRNA 的库会使不太偏颇的屏幕成为可能。这些都有助于在没有任何先验实验知识的情况下发现基因组中的功能基因座。然而, gRNA 图书馆的建设目前大多是基于寡核苷酸合成, 并有有限的选择, 购买 gRNA 库, 不是人类或鼠标起源或目标地区以外的开放阅读框架。因此, 虽然 CRISPR 屏幕已经证明了难以置信的强大的5,6,7,8, 但它们的全部潜能尚未被利用。
克服经典 gRNA 生成方法的局限性, 提出了两种新的策略。两者都是基于受控酶消化的目标 DNA, 而不是依赖于定制的寡核苷酸合成。当 CORALINA9使用 micrococcal 核酸时, 目前唯一可用的替代方法, CRISPR-吃10, 利用限制酶 (HpaII, ScrFi,博鳌i 和夫人i)。重要的是, 这两种技术都可以应用于任何输入 DNA, 作为 gRNA protospacer 序列的来源。虽然 CRISPR 吃法采用的策略, 以减少克隆 gRNAs 的数量, 其目标网站没有遵循要求的 S. 化脓 PAM (protospacer 相邻的动机), 它只产生一小部分的所有可能的功能 gRNAs给定区域。CORALINA, 另一方面, 能够产生所有潜在的 gRNAs 的源序列, 但也包含了更高的分数非功能指南。通过受控核酸活动的 gRNA 库生成, 可以为任何物种、任何 Cas9-protein 或-效应系统的综合 gRNA 库的生产提供简单且经济高效的方式。此外, CORALINA 可以适应自定义, 因为适当的输入和向量选择定义了库类型、大小和内容。这里提供了一个详细的协议, 可以用于从不同的 DNA 来源 (图 1) 生成全面的 gRNA 库, 包括细菌人工染色体 (巴克斯) 或基因组 dna9。通过将 CORALINA 协议应用于 BAC DNA, 得出了该协议的代表性结果。

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">500 mM EGTA</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:140px;">03777-10G</td>
			<td style="width:272px;">1.4., Inactivation of Mnase</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Novex Hi-Density TBE Sample Buffer</td>
			<td style="width:139px;">Thermo Fisher Scientific</td>
			<td style="width:140px;">LC6678</td>
			<td style="width:272px;">2.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Novex&#174; TBE Gels, 20%, 10 well</td>
			<td style="width:139px;">Thermo Fisher Scientific</td>
			<td style="width:140px;">EC6315BOX</td>
			<td style="width:272px;">2.1., pre-made 20 % PAGE gel</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">O&#39;RangeRuler 5 bp DNA Ladder,</td>
			<td style="width:139px;">Thermo Fisher Scientific</td>
			<td style="width:140px;">SM1303</td>
			<td style="width:272px;">2.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Novex&#174; TBE Running Buffer</td>
			<td style="width:139px;">Thermo Fisher Scientific</td>
			<td style="width:140px;">LC6675</td>
			<td style="width:272px;">2.1., PAGE gel running buffer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Disposable scalpel, sterile</td>
			<td style="width:139px;">VWR&#160;</td>
			<td style="width:140px;">233-5363&#160;</td>
			<td style="width:272px;">2.3., other equivalent reagents may be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">SYBR Green I nucleic acid stain (1000x concentrate in DMSO)</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td style="width:140px;"> 
			S9430</td>
			<td style="width:272px;">2.3. +2.5., also available from Thermo Fisher Scientific (S7563)</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:229px;">UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td>
			<td style="width:139px;">Thermo Fisher Scientific</td>
			<td style="width:140px;">15593-031</td>
			<td style="width:272px;">3.6.1. + 4.3., other equivalent reagents may be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Glycogen</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;">10901393001</td>
			<td style="width:272px;">3.6.4., other equivalent reagents may be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">3M Sodium acetate , pH5.2</td>
			<td style="width:139px;">Thermo Fisher Scientific</td>
			<td style="width:140px;">&#160; R1181</td>
			<td style="width:272px;">3.6.4., other equivalent reagents may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Ethanol&#160;</td>
			<td style="width:139px;"></td>
			<td style="width:140px;"></td>
			<td style="width:272px;">3.6.4. + 9.1.8., molecular biology grade</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Quick blunting kit&#160;</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">E1201</td>
			<td style="width:272px;">4.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">ammomium acetate</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;"> 
			A1542</td>
			<td style="width:272px;">3.1., other equivalent reagents may be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">magnesium acetate</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;"> 
			M5661</td>
			<td style="width:272px;">3.1., other equivalent reagents may be used</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">0.5 M EDTA (pH 8.0)</td>
			<td style="width:139px;">VWR</td>
			<td style="width:140px;">&#160; MOLEM37465520 (or Promega V4231)</td>
			<td style="width:272px;">2.2. + 3.1., other equivalent reagents may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Agencourt AMPure XP beads</td>
			<td style="width:139px;">Beckman coulter</td>
			<td style="width:140px;">A63881</td>
			<td style="width:272px;">5.3. + 6.5.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Gel extraction kit</td>
			<td style="width:139px;">QIAGEN</td>
			<td style="width:140px;">28704</td>
			<td style="width:272px;">5.7.+ 7.1. +8.4., other equivalent reagents may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">concentrated T4 DNA ligase</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">&#160;M0202T</td>
			<td style="width:272px;">6.1.+ 8.1.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Long Amp Taq 2X Master Mix&#160;</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">M0287S</td>
			<td style="width:272px;">6.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Phusion High-Fidelity PCR Master Mix with HF Buffer</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">M0531S</td>
			<td style="width:272px;">5.1. + 6.6., other equivalent reagents may be used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">HindIII</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">R0104S</td>
			<td style="width:272px;">5.4.1.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">SacII</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">R0157S</td>
			<td style="width:272px;">5.4.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">AgeI</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">R0552S</td>
			<td style="width:272px;">8.2.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Tris base</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;">93362</td>
			<td style="width:272px;">8.1.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">2M MgCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;">93362</td>
			<td style="width:272px;">8.1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">dGTP,dATP, dCTP, dTTP</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">N0446S</td>
			<td style="width:272px;">8.1.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">DTT</td>
			<td style="width:139px;">Sigam</td>
			<td style="width:140px;"> 
			DTT-RO</td>
			<td style="width:272px;">8.1.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">PEG-8000&#160;</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;"> 
			P5413</td>
			<td style="width:272px;">8.1.1.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">NAD</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;"> 
			N6522</td>
			<td style="width:272px;">8.1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">T5 exonuclease</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">M0363S</td>
			<td style="width:272px;">8.1.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Phusion DNA polymerase&#160;</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">M0530S</td>
			<td style="width:272px;">8.1.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Taq DNA ligase</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">M0208L</td>
			<td style="width:272px;">8.1.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">rSAP</td>
			<td style="width:139px;">New England Biolabs</td>
			<td style="width:140px;">M0371S</td>
			<td style="width:272px;">8.3.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">TG1 competent cells</td>
			<td style="width:139px;">Lucigen</td>
			<td style="width:140px;">60502-1</td>
			<td style="width:272px;">9.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">1mm gap electroporation cuvettes&#160;</td>
			<td style="width:139px;">VWR</td>
			<td style="width:140px;">732-2267&#160;</td>
			<td style="width:272px;">10.2.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Bio-Assay Dish (Polystyrene, 245 mm x 245 mm x 25 mm)</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:140px;">DIS-988-010M&#160;</td>
			<td style="width:272px;">9.4.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">NaCl</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;">S7653&#160;</td>
			<td style="width:272px;">9.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Bacto-tryptone</td>
			<td style="width:139px;">BD</td>
			<td style="width:140px;">211705</td>
			<td style="width:272px;">9.3.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Yeast extract</td>
			<td style="width:139px;">BD</td>
			<td style="width:140px;">212750</td>
			<td style="width:272px;">9.3.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Agar</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;"> 
			A1296</td>
			<td style="width:272px;">9.4.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:229px;">Glycerol</td>
			<td style="width:139px;">Sigma</td>
			<td style="width:140px;"> 
			G5516</td>
			<td style="width:272px;">9.17.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">MNAse</td>
			<td style="width:139px;">New England Biolabs</td>
			<td>M0247S</td>
			<td>1.1.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Nanodrop</td>
			<td>Thermo Fisher Scientific</td>
			<td>ND-2000</td>
			<td>throughout</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Micropulser</td>
			<td style="width:139px;">Biorad</td>
			<td>165-2100</td>
			<td>10.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Electroporation cuvettes</td>
			<td style="width:139px;">Biorad</td>
			<td>732-2267&#160;</td>
			<td>10.2.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250 ml centrifuge tubes</td>
			<td>Corning</td>
			<td>430776</td>
			<td>9.1-9.9.</td>
		</tr>
	</tbody>
</table>

a universal protocol for large-scale grna library production from any dna source

CRISPR/Cas9 系统在基因组和基因工程中的普及源于它的简单性和适应性。一个效应器 (Cas9 核酸或核酸死 dCas9 融合蛋白) 的目标是一个小的合成 rna, 称为指南 rna, 或 gRNA 的基因组中的特定站点。CRISPR 系统的二部性质, 使其在筛选方法中的使用, 因为含有数千个个体 gRNAs 的表达盒的质粒库可以用来在一个实验中审问许多不同的地点。
到目前为止, 图书馆建设的 gRNA 序列几乎完全是由寡核苷酸合成产生的, 这就限制了库中序列的可实现复杂性, 并且相对成本较高。这里, 一个详细的协议为 CORALINA (全面 gRNA 图书馆世代通过被控制的核酸活动), 一个简单和经济有效的方法生成高度复杂 gRNA 图书馆基于酵素消化输入脱氧核糖核酸, 描述。由于 CORALINA 库可以从 DNA 的任何来源生成, 因此存在大量的定制选项, 使得基于 CRISPR 的各种屏幕都能实现。

使用现有的协议, CORALINA gRNA 库已由人类和小鼠基因组 dna9和 BAC dna (图 1) 生成。为了生成适合克隆的 gRNA 表达载体的输入 DNA 片段, 必须确定控制核酸消化的最佳条件。在图 2A中描述了优化 micrococcal 核酸消化的一个典型结果。核酸 (0.1、2、3、4、4.5 或5单位) 的数量不足, 在所需的尺寸范围 (10-100 bp) 和 5.5-7.5 单位生产的产品中, 仍有平均过长的碎片。大量的酶 (50 单位) 导致了10分钟后输入 DNA 的过度降解。因此, 选择了中间金额 (10 单位)。该文摘被放大以产生足够的消化片段, 以便随后的纯化和克隆 (图 2B)。虽然建议盲目选择 dna 片段的大小和只依赖于 dna 阶梯的方向, 以尽量减少暴露的 dna 片段的紫外线, 凝胶可以事后染色的质量控制的消化和切割。图 2B显示了一页凝胶的典型示例, 其中20和 30 bp 之间的 DNA 片段已被切除。凝胶纯化 MNase 片段被加载到20% 页凝胶, 以检查成功的大小选择和纯化的 MNase 消化片段 (图 2C)。该协议在手边是兼容的使用自定义的链接器序列, 允许克隆的 MNase 消化片段成 gRNA 表达载体的选择。这里, gRNA-PLKO9被用作主干。用标准 PCR 方法从 gRNA 表达载体中扩增连接。图 2D描述了一个有代表性的放大链接器序列的示例, 没有附加、不正确或没有模板增。接下来, 链接器增被消化与限制性酶, 以确保连接被结扎到 MNase 消化片段的正确方向。图 2D显示了 5 "和 3" 连接的琼脂糖凝胶, 分别与后 III 和 SacII 进行消化, 表明连接对预测的637和 295 bp 完全消化。凝胶图像的右手部分记录了被消化的链接器片段的切除。经过凝胶提取消化连接, 该协议的下一步是结扎连接到最终修复的 MNase 消化片段。由于链式序列是用 unphosphorylated 引物 PCR 生成的, 不应发生连接的自结扎。只有最终修复的 MNase 消化的 DNA 片段提供了结扎所需的磷酸基团。在尼克翻译后, 结扎产品被 PCR 放大。为了避免过量的 PCR 放大偏倚, 可能扭曲了 gRNA 序列在图书馆的表现, 放大的限制到少于20周期的总和。PCR 后, 在琼脂糖凝胶上, 放大产物难以形象化。因此, 为了检测产品 (但不用于库的准备工作), 将使用32周期的单独控制 PCRs。该控制 PCR 的结果显示在图 2E中。这允许优化结扎反应, 并确保反应是缺乏 PCR 文物, 有时发生在 "无碎片控制" (NFC)。图 2E显示了所需的增 (5 ' 链接器 + DNA 片段 + 3 ' 链接, 长度: 869 bp) 在结扎反应的放大后使用摩尔 (1:1) 的比例之间的片段和连结序列。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56264/56264fig1.jpg"> 图 1: 准备 gRNA 库的建议时间线.CORALINA 提供了一个简单而经济高效的策略, 从任何生物体的不同 DNA 来源中生成全面的 gRNA 库。在一个工作周内, 手头的议定书可以完成。连接器生成可以与 DNA 端修复并行执行。electrocompetent 细菌的制备需要两天时间, 包括一夜的生长步骤, 因此应在装配反应建立之前开始。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56264/56264fig1large.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/56264/56264fig2.jpg"> 图 2: 协议期间的关键步骤.( A) 控制的 BAC DNA 消化可以生成不同大小的碎片。这里显示的是 MNase 消化的优化。用不同剂量的 MNase 对纯化的 BAC dna 进行了10分钟的 MNase 10 U 生成所需长度 (20-30 bp) 的 dna 片段。(B) 使用聚丙烯酰胺凝胶切除的20和 30 bp 片段的大小选择。纯化 BAC DNA 治疗 10 U MNase 10 分钟。图像在切除后被记录下来。(C) 凝胶纯化片段的质量控制。经过凝胶纯化, 1/第六的纯化 MNase 片段被加载到20% 页凝胶检查成功的大小选择和纯化。(D) 对连接的组装和限制酶消化的链接器序列进行放大, 以确保定向克隆。分别使用酶和SacII放大并剪切了 5 "和 3" 连接。没有模板控制 (NTC) 被纳入控制 PCR 文物和 DNA 污染。左: 分析样品应用;右: 制备样品应用。图像被记录后, 凝胶切除。(E) 成功结扎连接到 DNA 片段的方法可以通过 pcr (pcr) 周期的增加 (32) 进行分析, 并通过执行不具有 H2O (ntc) 的模板控件或使用前一个尼克翻译步骤中的 ntc 进行控制作为输入 (NTC NT))。重要的是要包括一个无片段控制 (NFC), 这是一个从结扎和尼克翻译反应的放大, MNase 片段被省略。只有 MNase 片段与连结剂 DNA 结合的样品才能产生预期的增 (869 bp)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56264/56264fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about A Universal Protocol for Large-scale gRNA Library Production from any DNA Source at JoVE.com

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

生成大型 gRNA 库的方法应简单、高效且具有成本效益。我们描述了一种基于目标 DNA 酶消化的 gRNA 库的生成协议。这种方法, CORALINA (全面 gRNA 图书馆一代通过控制核酸活动) 提出了一个替代昂贵的定制寡核苷酸合成。

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

a-universal-protocol-for-large-scale-grna-library-production-from-any

Nanyang Technological University

Research

JoVE Journal

Genetics

11.0K 次观看.  Ludwig-Maximilian-Universität, BioMedical Center. 生成大型 gRNA 库的方法应简单、高效且具有成本效益。我们描述了一种基于目标 DNA 酶消化的 gRNA 库的生成协议。这种方法, CORALINA (全面 gRNA 图书馆一代通过控制核酸活动) 提出了一个替代昂贵的定制寡核苷酸合成。

视频: A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

科研

遗传学

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

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 ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

A Customizable Protocol for String Assembly gRNA Cloning (STAgR)

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic engineering became applicable to a large range of systems. Moreover, many transcriptional and epigenomic engineering approaches are now generally feasible for the first time. One reason for the broad applicability of CRISPR lies in its bipartite nature. Small gRNAs determine the genomic targets of the complex, variants of the protein Cas9, and the local molecular consequences. However, many CRISPR approaches depend on the simultaneous delivery of multiple gRNAs into individual cells. Here, we present a customizable protocol for string assembly gRNA cloning (STAgR), a method that allows the simple, fast and efficient generation of multiplexed gRNA expression vectors in a single cloning step. STAgR is cost-effective, since (in this protocol) the individual targeting sequences are introduced by short overhang primers while the long DNA templates of the gRNA expression cassettes can be re-used multiple times. Moreover, STAgR allows single step incorporation of a large number of gRNAs, as well as combinations of different gRNA variants, vectors and promoters.

A Customizable Protocol for String Assembly gRNA Cloning STAgR

Here, we present string assembly gRNA cloning (STAgR), a method to easily multiplex gRNA vectors for CRISPR/Cas9 approaches. STAgR makes gRNA multiplexing simple, efficient and customizable.

字符串装配 grna 克隆 (stacr) 的可自定义协议

The bacterial CRISPR/Cas9 system has substantially increased methodological options for life scientists. Due to its utilization, genetic and genomic ...

Evaluation of a Universal Nested Reverse Transcription Polymerase Chain Reaction for the Detection of Lyssaviruses

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription polymerase chain reaction (nested RT-PCR) was developed to detect the conserved region of the nucleoprotein (N) gene of lyssaviruses. The method applies reverse transcription (RT) using viral RNA as template and oligo (dT)15 and random hexamers as primers to synthesize the viral complementary DNA (cDNA). Then, the viral cDNA is used as a template to amplify an 845 bp N gene fragment in first-round PCR using outer primers, followed by second-round nested PCR to amplify the final 371 bp fragment using inner primers. This method can detect different genetic clades of rabies viruses (RABV). The validation, using 9,624 brain specimens from eight domestic animal species in 10 years of clinical rabies diagnoses and surveillance in China, showed that the method has 100% sensitivity and 99.97% specificity in comparison with the direct fluorescent antibody test (FAT), the gold standard method recommended by the World Health Organization (WHO) and the World Organization for Animal Health (OIE). In addition, the method could also specifically amplify the targeted N gene fragment of 15 other approved and two novel lyssavirus species in the 10th Report of the International Committee on Taxonomy of Viruses (ICTV) as evaluated by a mimic detection of synthesized N gene plasmids of all lyssaviruses. The method provides a convenient alternative to FAT for rabies diagnosis and has been approved as a National Standard (GB/T36789-2018) of China.

A pan-lyssavirus nested reverse transcription polymerase chain reaction has been developed to detect specifically all known lyssaviruses. Validation using rabies brain samples of different animal species showed that this method has a sensitivity and specificity equivalent to the gold standard fluorescent antibody test and could be used for routine rabies diagnosis.

一种通用嵌套逆转录酶链反应检测裂解病毒的评价

To detect rabies virus and other member species of the genus Lyssavirus within the family Rhabdoviridae, the pan-lyssavirus nested reverse transcription ...

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.