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下面的协议涉及哺乳动物细胞的工作; 对无菌技术/细胞培养的熟悉是意料之中的. 
 1. 单元格线和区域性条件 <ol> <li> 使500毫升培养基的培养116细胞: 麦考伊和 #39; s 5A 改良培养基补充10% 胎牛血清 (FBS), 2 毫米 l-谷氨酰胺和1% 青霉素 (这是完整的中). 
	注: 在电镀前, 在 T-75 或 T-175 烧瓶中生长116-19 细胞。当90% 汇合时, 每个 T-75 烧瓶将产生 8.4 x 10 6 单元格, 大约五 10 cm 板, 每个 T-175 将产生 18.4 x 10 6 单元格, 大约十五10厘米的板. </li>
</ol> 2。从烧瓶中提取单元格 <ol> <li> 吸去培养基, 用 Dulbecco 和 #39; 用磷酸盐缓冲的盐水, 不含钙和 magniesium (PBS) (10 毫升用于 T-75 或25毫升 T-175), 和抽吸. </li>
	<li> 使用2毫升吸管 (2 毫升为 T-175 或1毫升 T-75) 添加胰蛋白酶滴到烧瓶。将烧瓶放置在37和 #176 的恒温箱中; C 和 5% CO 2 为 5 min, 以允许单元格分离. </li>
	<li> 点击烧瓶, 以确保所有的细胞脱落, 然后用完全的介质淬火, 分散在烧瓶的整个表面 (8 毫升 T-175 或4毫升 T-75). </li>
	<li> 吸管向上和向下多次打破细胞团簇, 并将细胞转移到一个15毫升锥形管 </li>
	<li> 在将单元格向下旋转前, 取10和 #181; l 从 15 mL 圆锥, 并与10和 #181 相结合; 台盼蓝 l 计数细胞。在 125 x g 和16和 #176 旋转5分钟的细胞; C. </li>
</ol> 3。计算单元格 <ol> <li> 传输10和 #181; 与台盼蓝混合的细胞 L 例。计算外部的4网格 (每个网格包含16平方)。
	<ol> <li> 从每组十六角正方形中取平均单元格计数. </li>
		<li> 乘以 1万 (10 4 ). </li>
		<li> 乘以用于收获细胞的培养基的总体积, 以矫正从台盼蓝加法的稀释. 
		注: 公式格式计算重单元格的体积如下: <img alt="Equation 1" src="/files/ftp_upload/56195/56195eq1.jpg"/> <img alt="Equation 2" src="/files/ftp_upload/56195/56195eq2.jpg"/> </li> </ol>
	</li> </ol> 4。电镀单元格 <ol> <li> 对每个10厘米的单元格进行同步, 添加5毫升的完整介质和6和 #181; M aphidicholin (12 和 #181; L 2.5 毫米的股票在 200-证明乙醇). </li>
	<li> 传输100和 #181; L 悬浮细胞颗粒, 2.5 x 10 6 细胞, 每10厘米的板块和漩涡轻轻地混合. </li>
	<li> 在37和 #176 上孵育板; C 和 5% CO 2 16-24 h 以同步 G1/S 边框上的单元格. </li>
</ol> 5。从 Aphidicolin 同步中释放单元格 <ol> <li> 4 小时之前的目标, 吸介质, 用 pbs 冲洗, 吸入 pbs, 并添加5毫升的完整培养基 </li> <li> 将其放回孵化器在37和 #176; C 和 5% CO 2 4 h </li>
</ol> 6。RNA 络合 <ol> <li> 将突变的 eGFP 基因序列输入到张实验室和 #39 的在线生成器 25 (http://crispr), 并选择与目标站点紧密接近的 crispr 指南序列。从商业来源获取 CRISPR 指南序列. </li>
	<li> 存储 CRISPR RNA (crRNA), 反式活化 crRNA (tracrRNA) 和 Cas9 蛋白, 20 和 #176; C 和根据制造商的建议使用。
	<ol> <li> 将摩尔浓度的 RNA 混合到45和 #181; m. 增加6.75 和 #181; l 200 和 #181; 我的股票的 crRNA 和6.75 和 #181; l 200 和 #181; m 库存 tracrRNA 到1.5 毫升离心管。添加16.50 和 #181; i 缓冲区, 使最后的体积30和 #181; 我. </li>
	</ol> </li> <li> 在热块或 PCR 机上加热95和 #176; C 为5分钟. 
	注意: 热! </li>
	<li> 允许冷却到室温. 
	注: 如果使用 PCR 设备, 请将冷却设置为0.2 和 #176;
	<li> 对每个示例执行以下步骤。
	<ol> <li> 稀释2.22 和 #181; crRNA: 2.78 和 #181 的 tracrRNA 复合体; TE 缓冲器 (10 毫米三, ph 8.0 和0.1 毫米 EDTA; ph 8.0) 到最后容量5和 #181; l. </li>
		<li> 稀释1.67 和 #181; 从60和 #181 中 Cas9 蛋白; M 3.33 和 #181; l. 低中等至最终体积5和 #181. </li>
	</ol> </li> <li> 混合5和 #181; Cas9 蛋白与5和 #181; 复合 RNA 的 l. </ol> 7。捕获用于目标 <ol> 的单元格 <li> 吸气培养基, 用 pbs 5 毫升冲洗, 吸出 pbs, 并在每10厘米的盘子中加入1毫升的5ml 胰蛋白酶。把盘子放在孵化箱里37和 #176; C 和 5% CO 2 为 5 min. </li>
	<li> 在10厘米的板上点击, 以确保所有的细胞被脱落, 然后通过分散在整个板块表面的4毫升的完全介质淬火. </li>
	<li> 吸管向上和向下多次, 以打破细胞团簇和转移到一个15毫升锥形管细胞. </li>
	<li> 在将单元格向下旋转前, 取10和 #181; 从15毫升锥形管中将其与10和 #181 相结合; 台盼蓝的 l 计数细胞。通过旋转5分钟的 125 x g 和室温下的细胞颗粒. </li>
	<li> 吸气培养基, 用5毫升 PBS 冲洗。在室温下, 在 125 x g 处旋转5分钟, 将细胞颗粒. </li>
</ol> 8。计算单元格 <ol> <li> 传输10和 #181; 与台盼蓝混合的细胞 L 例。计算外部的4网格 (每个网格包含十六平方)。
	<ol> <li> 从每组十六角正方形中取平均单元格计数. </li>
		<li> 乘以 1万 (10 4 ). </li>
		<li> 乘以用于收获细胞的培养基的总体积, 以矫正从台盼蓝加法的稀释. 
		注意: 下面是公式格式, 用于计算重单元格的体积。重新挂起所需数量的细胞在无血清麦考伊和 #39; s 5A 修改介质. 
		<img alt="Equation 3" src="/files/ftp_upload/56195/56195eq3.jpg"/> <img alt="Equation 4" src="/files/ftp_upload/56195/56195eq4.jpg"/> </li> </ol> </li> </ol> 9. 目标示例 <ol> <li> 传输100和 #181; L 细胞悬浮 (5 x 10 5 细胞) 从步骤8.1 到每个电穿孔4毫米空白试管。添加10和 #181; 从步骤6.7 到100和 #181 的 RNP 复杂 l, 单元格的 l 为 5 x 10 5 单元格密度。将寡 (2 和 #181; M) 添加到每个示例中. 
	注: 对于阳性对照, 添加1和 #181; eGFP 在1和 #181; g/和 #181; 表达质粒 <ol> <li> 将机架带到一台电穿孔机上, 轻轻地轻拍每个样品, 并将它们放在室内。Electroporate 在 250 V, LV;2脉冲, 1 秒;13毫秒;单极脉冲. </li>
	</ol> </li> <li> 将机架移回引擎盖。将每个样品转移到含有2毫升完整介质 i 的井6井板。在检查校正级别之前, 在37和 #176、C 和 5% CO 2 中孵育72小时. </li>
</ol> 10。基因编辑细胞与转染效率的分析 <ol> <li> 吸入培养基并用2毫升 PBS 洗涤细胞。抽吸 PBS, 添加500和 #181; 5ml 胰蛋白酶对6井板的每一个井。把盘子放在孵化箱里37和 #176; C 和 5% CO 2 为 5 min. </li>
	<li> 点击该板, 以确保所有的细胞脱落, 然后用1毫升完全介质淬火, 分散在整个表面的井. </li>
	<li> 在室温下将电池传递到1.5 毫升离心管和 5000 x g 的颗粒上. </li>
	<li> 吸气介质。在500和 #181 中重新悬浮细胞颗粒; (0.5% BSA、2毫米 EDTA、2和 #181; PBS 中的碘碘化物). </li>
	<li> 用流式细胞仪测量细胞荧光 (eGFP +)。
	<ol> <li> 将校正效率计算为每个样本中活细胞总数的总活 eGFP 阳性细胞的百分比, 如在维里维尔托雷斯 et al 30 中所述。
	</ol> </li> </ol> 11。DNA 序列分析 <ol> <li> Electroporate 在 5 x 10 5 和 #8197 的浓度中同步和释放的116-19 单元格; cells/100 和 #8197; #181; L, 与 RNP 复合体在 100 pmols 和72NT 寡在2.0 和 #181; m </li>
	<li> 将单元格转移到6井板, 并允许它们恢复72小时. </li>
	<li> 使用一个 488 nm (100 兆瓦) 激光 eGFP +/-, 如在维尔-托雷斯 et al 30 中所描述的那样, 将单元格单独分类为96井板。
	注: 并非所有水井都能成功生长. </li>
	<li> 展开6周以上的单元格, 并按步骤7中的说明进行收割. </li>
	<li> 从有生长的油井中分离出细胞 gDNA, 使用商业上可用的 DNA 隔离套件 (见 材料表), 通过 PCR (718 bp; 前引物5和 #39;ATGGTGAGCAAGGGCGAGGA-3 和 #39; 反转底漆5和 #39;-ACTTGTACAGCTCGTCCATGC-3 和 #39;). </li>
	<li> 对样品进行 DNA 测序分析. </li>
</ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+gene+editing+in+mammalian+cells+using+ssODNs+and+TALENs.">Combinatorial gene editing in mammalian cells using ssODNs and TALENs.</a> Sci Rep. 4 (ii), 3791 (2014).</li><li>Bialk, P., Rivera-Torres, N., Strouse, B., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Gene+Editing+Activity+Directed+by+Single-Stranded+Oligonucleotides+and+CRISPR/Cas9+Systems.">Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.</a> PloS One. 10 (6), e0129308 (2015).</li><li>Yu, C., 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=Small+Molecules+Enhance+CRISPR+Genome+Editing+in+Pluripotent+Stem+Cells.">Small Molecules Enhance CRISPR Genome Editing in Pluripotent Stem Cells.</a> Cell Stem Cell. 16 (2), 142-147 (2015).</li><li>Rivera-Torres, N., Banas, K., Bialk, P., Bloh, K. M., Kmiec, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+Mutagenesis+by+CRISPR/Cas9+Ribonucleoprotein+Gene+Editing+in+Cells+Targeted+for+Point+Mutation+Repair+Directed+by+Short+Single-Stranded+DNA+Oligonucleotides.">Insertional Mutagenesis by CRISPR/Cas9 Ribonucleoprotein Gene Editing in Cells Targeted for Point Mutation Repair Directed by Short Single-Stranded DNA Oligonucleotides.</a> PLoS One. 12 (1), e0169350 (2017).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAS9+transcriptional+activators+for+target+specificity+screening+and+paired+nickases+for+cooperative+genome+engineering.">CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering.</a> Nat Biotechnol. 31 (9), 833-838 (2013).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Ran, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+genome+editing+using+Staphylococcus+aureus+Cas9.">In vivo genome editing using Staphylococcus aureus Cas9.</a> Nature. 520 (7546), 186-191 (2015).</li><li>Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L., Corn, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+homology-directed+genome+editing+by+catalytically+active+and+inactive+CRISPR-Cas9+using+asymmetric+donor+DNA.">Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA.</a> Nature Biotechnol. 34 (3), 339-344 (2016).</li><li>Slaymaker, I. M., Gao, L., Zetsche, B., Scott, D. A., Yan, W. X., Zhang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rationally+engineered+Cas9+nucleases+with+improved+specificity.">Rationally engineered Cas9 nucleases with improved specificity.</a> Science. 351 (6268), 84-88 (2016).</li></ol>

作者没有任何披露。

基因编辑已经成为主流的科学学科, 主要是因为 CRISPR/Cas9 系统的出现。这一显着的途径, 促进了细菌细胞的免疫力, 已被改成一种分子工具, 使人类染色体的基因组改变。CRISPR/Cas9 的自然功能是通过引入双 dna 片段来禁用病毒 dna, 从而导致碎片30,31。这一活动导致入侵外源 DNA 的破坏, 并导致原核免疫, 抑制随后感染相同的病毒粒子。令人惊讶的是, 这个系统表现出肺炎的行为, 因为它可以重新激活特定的 CRISPR gRNA 由以前的感染事件创建。
CRISPR/Cas9 催化的基因干扰的效率和准确性已被用于许多真核基因系统, 其目的是禁用功能性基因32,33。当其基本水平降低时, 这个过程包括两个根本步骤。第一个是双的突破, 而第二个依赖于同源端连接 (NHEJ) 的内在过程来完成淘汰赛。在大多数情况下, 双断裂导致钝端, 脱离的染色体片段, 和酶参与 NHEJ 反应的染色体断裂和行动, 以与破碎的两端。这个过程有时会涉及在被切断的站点上的单个核苷酸的丢失。即使失去几个碱基也会导致 frameshift, 而功能表达停止。利用 CRISPR/Cas9 NHEJ 的自然过程来创造基因敲除, 彻底改变了真核基因;目标站点的 DNA 丢失是可预测的, 而且是意料之中的。
与基因剔除不同的是, 一些研究人员一直致力于将 CRISPR/Cas9 的活动重新定向到同源性修复的过程中。研究的目的是基因矫正。在这个策略中, CRISPR/Cas9 结合了一个供体 DNA 模板, 它提供了基因信息来修复先天的错误或将突变植入健康的基因。早期报告突出了 CRISPR/Cas9 的显著能力, 以催化同源定向修复 (直接或间接) 表明, 该过程以高度精确的方式发生24,34。我们的实验室一直在研究同源修复或基因校正使用单寡核苷酸, 试图定义的机制和监管电路围绕它15,17,18 ,23。我们主要关注基因编辑单点突变, 因为这是已知的最基本的基因突变, 对许多遗传性疾病负责。我们对基因编辑的基本认识使我们质疑这些反应中 CRISPR/Cas9 活性的准确性, 因为基因敲除的 CRISPR/Cas9 作用导致了 indels 的形成。我们使用一个明确的模型系统, 而不是一个不可尚未临床相关的基因, 以决定性的简化的方式研究 on-site 突变。通过使用一个简单的目标基因, 在基因的校正可以被测量在型别和表型水平, 我们推断, 在目标站点发生的异质性可以被辨认以可靠和健壮的方式。
我们的数据证实, 成熟的基因编辑系统, 包括一个突变的 eGFP 基因集成到 HCT116 细胞, 可以提供基础信息有关的产生的遗传病变和过程中的 on-site 突变。CRISPR/Cas9 和单寡核苷酸供体 DNA 分子串联工作可导致精确修复的点突变的 eGFP 基因。我们提出了一个新的模型来修复点突变, 一个分子通路, 其中的供体 DNA 作为复制模板修复的突变基地, 一个过程中, 我们称之为精确的30。目标人群, 而不是显示纠正表型, 展示了各种细胞含有异构和广泛范围的 DNA indels 围绕靶点。在大约一半的克隆与未被矫正的人口隔离, 删除突变在目标地点观察。由于以前没有报告表明, 单寡核苷酸作为单基因编辑工具可以诱导 indels 在目标网站, 我们得出结论, CRISPR/Cas9 活动是对这些突变负责。
在这篇手稿中, 我们提供了一个详细的方法, 使目标站点的遗传异质性能够以可靠和健壮的方式进行测量。尽管对异地诱变的分析和制图已经给予了极大的关注, 但在靶点产生的异质突变很可能会对基因编辑在临床领域的成功或失败产生更大的影响。可能需要额外的技术或改良的 Cas9 蛋白来提高哺乳动物细胞的同源性错误修复的精确度35。其中一些技术包括使用辅助寡核苷酸作为桥梁, 将染色体末端结合在一起, 避免 NHEJ 的破坏性作用。由于基因编辑活动的结果, 确定目标地点的异质性程度是而且必须是任何治疗干预方案的重要组成部分。

Mandecki (1986) 的先驱研究表明, 使用寡核苷酸转化为细菌的质粒 DNA 的永久性变化为1, 而瓦德和瓦德 (1986) 在酵母中进行了类似的研究, 如2。此后不久, 谢尔曼和他的同事发表了一系列的论文, 其中单寡核苷酸被引入酵母细胞中, 以使基因的遗传变化3。在微生物细胞的开创性工作的基础上, Kmiec 和同事开始开发独特的单寡核苷酸, 指导单个基地修复哺乳动物细胞4。这个概念是基于生物化学数据的, 它表明, 承载 RNA 的分子比完全由 DNA 基础组成的基因更紧密地束缚在目标部位。虽然有许多报告的基因编辑成功使用嵌合体寡核苷酸5,6,7, 编辑水平保持高度可变的实验之间和跨不同的目标/细胞组合.
单链的供体 dna 更倾向于双供者的 dna, 因为它不太可能在基因组8中的随机站点上集成。然而, 外部介绍的单寡核苷酸容易迅速降解细胞核酸。为了防止寡核苷酸的降解, 已采用了几种策略。包含所需序列的酶保护的单 DNA 足以在 0.1-1% 范围6中获得编辑效率。改进和更一致的转染技术, 加上表型读数, 允许更一致的编辑由多个研究组8,9,10,11, 12,13。
在过去的10年中, 有一个重要的努力, 使目标细胞更适于基因编辑。一个策略采用了单元周期的调制14,15,16。当编辑频率在正常反应条件下与单寡核苷酸 (ssODNs) 引入到培养哺乳动物细胞徘徊在0.1% 和1% 之间时, 基因编辑的频率增加 3-到5倍, 当寡核苷酸在 S 期的过渡过程中被引入细胞。在一个单独的系列实验中, Brachman 和 Kmiec (2005) 表明, 当 2 "3" dideoxycytosine (ddC) 在细胞中孵育 24 h 之前, 在添加寡核苷酸之前获得了相对较高的精确基因编辑 (3-5%) (17.ddC 降低了 DNA 复制叉运动的速率, 这表明 ssODNs 在复制单元格中的基因编辑可能涉及将寡合并到活动复制的区域11,18。在一起, 这些研究导致了一个概念, 即捐赠人的 DNA 成为一个不断增长的复制叉, 作为基因编辑的真正作用机制的一部分, 独立于是否存在双断裂或不是19。因此, 通过 dna 配对和单同化途径, 对染色体内的点突变或 dna 片段的替换进行修正。
用小分子药物诱导生长细胞中的随机双 dna 断裂创造了一个环境, 当细胞试图修复损伤2021时, dna 复制事件就会停止。复制叉的这种暂时的减速使单编辑寡核苷酸更有效地穿透染色质结构, 提高了目标辅助功能15。通过 VP16、博莱霉素或喜树碱等药物的双 DNA 断裂的产生,22,23, 已被证明可以刺激基因编辑水平近10倍, 高达6-8%。然而, 在治疗环境中, 随机双 DNA 断裂是不可取的。
基因编辑与可编程的核酸和寡核苷酸在细胞分裂期间也被刺激24,25.当在同步的小贩116细胞中使用酸传递进行同源定向修复时, 在转录激活剂样效应核酸 (TALENs被使用与单寡核苷酸, 两个被介绍入复制的细胞人口26,27 。最近, Bialk 和同事表明, 修复单一基地突变的单寡核苷酸和阵列定期 interspaced 短复发重复 Cas9 (CRISPR/Cas9) 分子发生的更高的功效当单元格填充正在遍历 S 阶段28时。CRISPR 分子由 RNA 和功能组成, 用于识别目标 DNA 序列中被指定用于解理的区域。Cas9 是一种细菌酶, 其功能是在 nucleolytic 交换反应中将双 DNA 劈裂。因此, CRISPR 在基因组目标上定位复合体, Cas9 核酸执行双断裂。林et al.(2014) 还显示了细胞周期对实现高频率基因编辑的重要性, 使用改良的 CRISPR/Cas9 ribonulceoprotien (RNP) 复合介导的分娩系统在初生的成纤维细胞、人胚胎干细胞和其他单元格行24。因此, 基因编辑和细胞周期进展之间建立的单基因编辑的关系是适用于组合基因编辑使用可编程核酸和捐赠人的 DNA 模板。虽然基因编辑的组合方法已经汇集了各种各样的合作伙伴与捐赠人脱氧核糖核酸模板, 多数工作者在领域使用 CRISPR/Cas9 提供双断裂作用。这种选择是基于这种特殊的基因工具的易用性和灵活性, 它可以用来禁用基因功能或引进一个外国的 DNA 片段到一个特定的网站。与基因置换相比, 淘汰赛的产生在技术上更容易, 如果将基因的 "正确" 或正常拷贝纳入疾病现场, 必须精确地进行。一些研究人员正在识别和研究使用特定的药物和试剂, 使突变基地的纠正和正常的基因序列在适当的位置在高频率的插入29。
最近, 维瑞-托雷斯et al.30使用组合基因编辑, 利用专门设计的 CRISPR/Cas9 系统的双断裂活动和单寡核苷酸供体 DNA 模板提供的遗传信息, 修复点突变在一个单一副本的增强绿色荧光灯蛋白 (eGFP) 基因集成到 HCT116 细胞。作者利用这种良好模型细胞系来评价靶点周围的特异性。数据显示目标站点存在异质性, 特别是在不包含更正点突变的单元格中。在这篇手稿中, 我们详细地讨论了这些工作者用来研究由 CRISPR/Cas9 基因编辑产生的 on-site 突变异质性的方法。

<table>
	<tbody>
		<tr>
			<td>McCoy&#8217;s 5A Modified medium</td>
			<td>ATCC</td>
			<td>30-2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>ATCC</td>
			<td>30-2214</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>ATCC</td>
			<td>30-2300</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>ATCC</td>
			<td>30-2200</td>
			<td>No Calcium or magnesium</td>
		</tr>
		<tr>
			<td>Trypsin EDTA Solution</td>
			<td>ATCC</td>
			<td>30-2101</td>
			<td></td>
		</tr>
		<tr>
			<td>Aphidicolin 1mg</td>
			<td>Thermo Fisher</td>
			<td>AC611970010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R CRISPR crRNA, 10 nmol</td>
			<td>IDT</td>
			<td></td>
			<td>No catalog number since its made to your specific gene target.</td>
		</tr>
		<tr>
			<td>CRISPR-Cas9 tracrRNA, 20 nmol</td>
			<td>IDT</td>
			<td>1072533</td>
			<td></td>
		</tr>
		<tr>
			<td>S.p. Cas9 Nuclease 3NLS, 500 &#181;g</td>
			<td>IDT</td>
			<td>1074182</td>
			<td></td>
		</tr>
		<tr>
			<td>IDTE Buffer</td>
			<td>IDT</td>
			<td>11-01-03-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeus Electroporation Cuvettes 0.4 Cm</td>
			<td>VWR</td>
			<td>10497-474</td>
			<td></td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporation Systems</td>
			<td>BioRad</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin 
			lyophilized powder, crystallized, &#8805;98.0% (GE)</td>
			<td>Sigma</td>
			<td>05470-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 M), pH 8.0</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide - 1.0 mg/mL Solution in Water</td>
			<td>ThermoFisher Scientific</td>
			<td>P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy Blood &#38; Tissue Kit (250)</td>
			<td>Qiagen</td>
			<td>69581</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well Standard Line Multiwell Cell Culture Plates</td>
			<td>VWR</td>
			<td>10062-892</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dishes</td>
			<td>Fisher</td>
			<td>12-565-90</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes (15 mL) (racked)</td>
			<td>Thermo Fisher</td>
			<td>AM12500</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Thermo Fisher</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td></td>
		</tr>
	</tbody>
</table>

a standard methodology to examine on-site mutagenicity as a function of point mutation repair catalyzed by crispr/cas9 and ssodn in human cells

组合基因编辑使用 CRISPR/Cas9 和单寡核苷酸是一种有效的策略, 纠正单基点突变, 这往往是对各种人类遗传性疾病负责。使用一个完善的细胞模型系统, 单突变 eGFP 基因的点突变集成到 HCT116 单元已修复使用这种组合方法。对纠正和未矫正的细胞的分析揭示了基因编辑的精确度和基因病变的发展, 当 indels 是建立在未矫正的细胞中的 DNA 序列周围的目标站点。在这里, 具体的方法用于分析这种组合方法的基因编辑点突变, 再加上详细的实验策略, 以测量 indel 形成在目标站点, 概述。该协议概述了一个基础的方法和工作流程的调查, 旨在开发 CRISPR/Cas9-based 基因编辑的人类治疗。这项工作的结论是, on-site 突变发生在点突变修复过程中的 CRISPR/Cas9 活性的结果。这项工作建立了一个标准化的方法, 以确定的程度突变, 这应该是一个重要的和关键的方面, 任何方法的临床实施。

我们使用一个模型系统来研究哺乳动物细胞的基因编辑, 这依赖于在 HCT116 细胞中整合的 eGFP 基因内的点突变的修正。重要的是要注意, 这是一个单基因;因此, 可以对 DNA 的改变或突变进行较不复杂的观点。图 1A显示突变的 eGFP 基因序列与目标基地, 第三个基地的停止密码标签, 突出以红色。72基寡核苷酸, 这是部分互补的 non-transcribed 链的 eGFP 基因 (72NT), 并旨在诱导基础交换从一个 G 到一个 C, 也说明。此外, 在图 1A中还描述了一个 CRISPR, 指定为 2C, 以 5 "到 3" 方向指示的 protospacer 序列。为了进行这种基因编辑反应, 我们使用了一个核糖 (RNP) 组成的 CRISPR (cr) rna 和 tracr (tr) rna 耦合纯化 Cas9 蛋白 (图 1B), 而不是使用哺乳动物表达载体组成的 Cas9 基因和适当的具体指南 RNA 序列。
当专门设计的 RNP 粒子通过电穿孔将单寡核苷酸传递给 HCT116 细胞时, 基因编辑, 通过修复 eGFP 中的单碱突变来证明, 在 72 h 的孵化后使用流式.功能修复是可见的绿色荧光的出现在目标细胞, 细胞, 也可以从整个人口分离, 因为这种荧光排序。如图 2所示, 渐进剂量响应可以看作是 RNP 和72NT 增量的协调水平。在图中显示的72NT 的分子比、picomoles RNP 和 micromolar 浓度, 是基于基因编辑反应的最佳剂量, 这依赖于 Cas9 和特定的引导 RNA 从转染表达的引入向量.图 2中的插图显示了在没有 RNP 粒子的情况下进行的基因编辑反应。在这里, 大约1% 的目标细胞被纠正时, 10 倍高浓度的72NT 寡核苷酸用于单基因编辑反应。
为了确定目标部位是否存在遗传异质性--so-called on-site 诱变效应--我们决定研究基因编辑活动在单个细胞中的结果。虽然比检查总体人口要费力得多, 但当检测到无性扩展细胞的基因组时, 可以确定基因足迹或病变的真正量度。我们重复了图 2中描述的实验, 这次只使用了 100 pmol 的 RNP 复合体和2.0 µmol 的72NT 寡核苷酸。如上图所示, 116 细胞与 aphidicholine 同步24小时, 并在 G1/S 边境被捕。4小时后, 细胞被释放, 并通过电穿孔引入基因编辑工具。72小时后, 对细胞进行了分析, 并将其单独归类为96井板 (实验过程在图 3中说明)。用流式细胞仪对96孔板的单个井进行克隆扩增, 以显示绿色荧光细胞。重要的是, 缺乏 eGFP 表达的细胞也被隔离, 并在相同的条件下以类似的方式进行分类。
经过14天的生长, 大多数的个体克隆已经扩展到足以使 DNA 分离。因此, 选择了 eGFP 阳性样本的16无性系, 并通过 DNA 测序分析了靶点周围的遗传完整性。利用桑格测序法, 利用序列可视化软件对野生等位基因的序列进行比较, 以此来对种群内等位基因序列的相关信息进行组合.RNP 复合体的切口位置由一个黑色箭头表示, 位于绿条 (2C crRNA) 上。如图 4A所示, 所有 16 eGFP 阳性单元格都包含目标站点的预测的核苷酸交换。转换后的 C 残滓以红色突出显示, 并且在 eGFP 正序下提供了反映精确变化的峰值剖面。在类似的方式, 15 非克隆菌株, 充分扩展, 以使 DNA 提取和测序, 分析了异质性的目标地点。据预测, 在大约一半的样本中, 没有观察到 DNA 基础交换。这反映在目标站点的 G 残滓的维护中, 如图 4B所示。在这些实验中检查的克隆扩张的其余部分显示了一个异质的缺失突变种群, 因此缺乏绿色荧光。删除大小从一个基地到19基地不等。重要的是要注意, 我们只检查了15样本的 eGFP 阳性细胞, 虽然我们认为这是相当代表的类型的遗传损害留下的 CRISPR/Cas9 活动, 有一种可能性, 其他类型或形式的 indels可能存在于目标人群中。
同时,图 4中显示的结果确认了 eGFP 目标系统中的表型读数。转换的 G 至 C 核苷酸使绿色荧光的出现在纠正 HCT116 细胞。在为本实验隔离的克隆或先前的实验中, 没有观察到目标站点周围的基替换27,28。这些数据还表明, 通过点突变修复而无法进行基因编辑的细胞仍未被修正, 但在某些情况下, 没有被改变, 在目标部位周围存在一系列的遗传异质性。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56195/56195fig1.jpg"> 
图1。(A) 突变 eGFP 基因基因编辑的模型系统.野生和突变的 eGFP 基因与靶密码子的适当片段, 位于序列的中心, 分别显示为绿色和红色。靶向交换的核苷酸是粗体和下划线。蓝色突出显示的基地代表 2C CRISPR protospacer 序列, 橙色基地突出了 PAM 网站。在这些实验中使用的寡核苷酸长度为72个碱基, 在三终端基上承载硫修饰的连接;72的目标 non-transcribed (NT) 链 (72NT)。(B) CRISPR/Cas9核糖装配反应。crRNA 提供目标特异性 (20 基地, 红色部分) 对应的 2C protospacer 序列和一个互动领域 (蓝色) 与 tracrRNA (绿色)。crRNA 和 tracrRNA 在摩尔浓度下退火。添加 Cas9 蛋白 (灰色) 以完成 RNP 组件。引导 rna (gRNAs) 直接和激活 Cas9 切, 然后切割目标 DNA。图的下部分显示了2C 种子序列和 tracrRNA 序列。这一数字是从 al., N. et 。(2017).<a href="//ecsource.jove.com/files/ftp_upload/56195/56195fig1large.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/56195/56195fig2.jpg"> 
图2。基因编辑是剂量依赖性时, 由 RNP 和 ssODN 的指示.同步和释放的小贩116-19 细胞是 electroporated 与 24-120 pmol CRISPR/Cas9 RNP 和 0.6-3.0 µM 的72mer。经过72小时的恢复期后, 利用流式细胞仪测量基因编辑活性。基因编辑被显示为校正效率 (%), 由可行的 eGFP 阳性细胞的数量除以种群中的存活细胞总数决定。误差线是由三组数据点产生的, 使用标准误差的基本计算, 在三多个单独的实验中产生。嵌入: 单代理基因编辑。在相同条件下, 单寡核苷酸 (72NT) 在无 RNP 复合体的作用下, 基因编辑活动被作为增加浓度的函数。这一数字是从 al., N. et 。(2017).<a href="//ecsource.jove.com/files/ftp_upload/56195/56195fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56195/56195fig3.jpg"> 
图3。分离单细胞无性系的实验策略.显示 eGFP 表达的细胞是阳性的, 并以流式单细胞细胞为单体进入单个井进行克隆扩张。细胞缺乏 eGFP 表达是孤立和排序, 在相同的方式和扩展在相同的条件。然后, DNA 被分离, eGFP 基因被放大, 并受桑格测序, 以分析围绕目标部位的基因编辑活动。这一数字是从 al., N. et 。(2017).<a href="//ecsource.jove.com/files/ftp_upload/56195/56195fig3large.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/56195/56195fig4.jpg"> 
图4。(a) 位分析 eGFP 阳性细胞作为克隆种群扩展.无性分离和扩展的 eGFP 阳性样本 (十六克隆) 的分析在该网站周围的目标基地和 DNA 从每个, 收获, 纯化, 放大, 并测序。位分析是使用桑格测序, 组装使用序列可视化软件, 并与序列的野生等位基因, 这是说明在顶部的数字。RNP 复合体的切口位置表示为位于绿条 (2C crRNA) 上的一个黑色小箭头。(B)位分析 eGFP 阴性细胞扩展为克隆种群.十五个样本, 从未被矫正的种群中克隆出来, 在目标核苷酸周围的地点随机选择并分析 indel 形成。如上所述, 位分析是使用桑格测序和组装使用序列可视化软件。再一次, 在图的顶部的野生等位基因的序列, 连同 RNP 的切口站点被提出。这一数字是从 al., N. et 。(2017).<a href="//ecsource.jove.com/files/ftp_upload/56195/56195fig4large.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 Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells at JoVE.com

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

该协议概述了 CRISPR/Cas9-based 基因编辑系统的工作流程, 用于修复哺乳动物细胞的点突变。在这里, 我们使用一个组合的方法来基因编辑和详细的后续实验策略, 测量 indel 形成在目标 site-in 本质, 分析现场诱变。

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

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

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7.7K 次观看.  Christiana Care Health Services. 该协议概述了 CRISPR/Cas9-based 基因编辑系统的工作流程, 用于修复哺乳动物细胞的点突变。在这里, 我们使用一个组合的方法来基因编辑和详细的后续实验策略, 测量 indel 形成在目标 site-in 本质, 分析现场诱变。

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

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

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

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

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

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

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

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遗传学

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable markers.

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

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

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

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

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

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

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

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

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

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

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

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

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

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

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

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

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

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

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

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

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

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

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

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

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

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

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

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

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

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

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

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

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

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

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

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

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

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

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...