这项工作得到了中科院战略优先研究项目 (XDB02050007, XDA01010409), 国家高技术研发 & 开发计划 (863 计划; 2015AA020307), 国家自然科学基金 (自然科学基金资助 31522037, 31500825, 31571509,31522038), 中国青年千人才项目 (以 hy), 突破中国科学院工程, 上海市科技委员会项目 (16JC1420202), 中国科学技术部 (大部分; 2016YFA0100500)。

在上海生物科学研究院 (CAS) 的生物医学研究伦理学委员会批准了包括动物实验在内的所有程序。
1. 捐助者质粒的设计
<ol><li>
sgRNA 的选择
	<ol>
<li>使用在线 CRISPR 设计工具预测目标区域上的 sgRNAs11,12,13,14,15。对于Cdx2轨迹, 设计六个不同的 sgRNAs (Cdx2-sgRNA1~Cdx2sgRNA6), 其周围有更高级别和较低潜在目标 (图 1)16的 stop 密码子。</li>
		<li>进行线性化2µg 的 Cas9-CMV-EGFP 表达载体和 sgRNA BbsI 消化 (µL 1 BbsI 2 h 在37°c 在最后集中 1 U/µL 在容量20µL)。然后用1% 琼脂糖凝胶在1×泰缓冲器中纯化产品。</li>
		<li>将一对 sgRNA 寡核苷酸混合在10µL 的 1× T4 DNA 连接酶缓冲液中, 最终浓度为50µM. 用温度梯度从95°c 孵化到25°c, 温度变化率为5摄氏度/5 分钟 (95 摄氏度为5分钟) 孵育寡聚体溶液;, 然后90°c 为5分钟, 85 °c 5 分钟,等.), 这将退火的寡核苷酸。</li>
		<li>将退火产物的4µL、2µL 的线性化向量与1µL T4 dna 连接酶在µL 1× dna T4 缓冲区的10连接酶中, 然后结扎22摄氏度 1-2 h(图 1 B).</li>
	</ol>
</li>
	<li>
sgRNA 的测量核酸酶测定 注: sgRNA 试验用的靶向效率由测量员核酸酶化验 (也称为 T7 限制性 I (T7EI) 化验) 17 进行评估.选择 sgRNA 的高 DNA 解切效率和低距离之间的 sgRNA 切割点和停止密码子。<ol>
<li>染 Cas9-sgRNA-EGFP 表达载体到 N2a 细胞系中培养 DMEM 补充10% 胎牛血清, 1% 和1% 非必需氨基酸的转染套件 (见材料表)。在 5% CO2中孵育转染的细胞在37°c。</li>
		<li>在48小时的孵化后, 通过荧光活化细胞分类收集5000转染细胞 (GFP+), 并使用非转染细胞作为控制。</li>
		<li>消化收集的细胞在2-5 µL oflysis 缓冲 (0.1% 海卫 X-100, 0.1% 吐温 20, 100 µg/毫升蛋白酶 k) 在56摄氏度30分钟, 然后热灭活蛋白酶 k 在95°c 10 分钟。</li>
		<li>使用制造商的协议, 通过嵌套 PCR (表 1) 放大示例。PCR 产品的大小设置为 300-500 bp。<ol>
<li>将裂解产物的1µL 与 DNA 聚合酶和一对外引物混合, 识别 sgRNA 靶点周围的序列 (0.1 µM, 最终浓度) (表 1), 并在20µL 的体积内执行主 PCR。</li>
			<li>激活 DNA 聚合酶在95°c 5 分钟, 并执行主要 PCR 为30个周期在95°c 为三十年代, 60 °c 为三十年代和72°c 为二十四年代 (1 min/1 kb), 以最后的引伸在72°c 为5分钟。</li>
			<li>采用1µL 的初级 pcr 产物和一对嵌套内引物进行二次 pcr。</li>
		</ol>
</li>
		<li>在20µL 1× T7EI 反应缓冲器 (50 毫米氯化钠) 中变性和重新退火300-600 的纯化 PCR 产物, 10 毫米的三盐酸, 10 毫米氯化镁2, 1 毫米的有效率的 pH 值 7.9) 使用温度梯度从95°c 到25°c 以率5°c/5 分钟。</li>
		<li>在退火的 PCR 产物中加入1µL 的 T7EI 酶, 并在37摄氏度上消化2小时。然后在1×TAE 缓冲区中的2% 琼脂糖凝胶上运行消化产物120伏40分钟, 直到碎片分离 (参见材料表)。</li>
		<li>使用 ImageJ 确定切割和未切割 DNA 的带强度。使用以前报告的9 (图 1C) 中的方法计算 indel 频率。</li>
	</ol>
</li>
	<li>
捐赠载体的构建 注意: 为Cdx2基因生成 HMEJ 供体向量, 构造一个供方 DNA (800 bp HAL-p2A-mCherry-800 bp), 两端有 23 nt Cdx2-sgRNAs 目标序列 (图 1D和图 1E)。靶序列的 PAM 与同源臂的末端相邻。吉布森大会建议 HMEJ 捐赠者克隆。<ol>
<li>放大 800 bp 左同源臂 (HAL) 与前 primer-1F (包含 15-20 nt 重叠序列从向量, 23 nt Cdx2-sgRNA 目标序列, 约 20 nt 序列从 HAL) 和反向 primer-1R (包含 15-20 bp 重叠序列从p2A-mCherry 和大约 20 nt 序列从 HAL) 在0.1 µM 最后浓度使用小鼠基因组 DNA 在 200 ng/µL (图 1D,表 1)。</li>
		<li>放大 p2A-mCherry 插入片段与前 primer-2F (包含 15-20 nt 重叠序列从 HAL 和大约 20 nt 序列从插入片断) 和反向 primer-2R (包含 15-20 nt 重叠序列从哈和大约 20 nt 序列从插入片段) 在0.1 µM 最终浓度使用基因组 DNA 或质粒与记者序列在 100 ng/µL 或 30 ng/µL (图 1D,表 1)。</li>
		<li>放大 800 bp 右同源臂 (哈) 与前 primer-3F (包含 15-20 nt 重叠序列从向量, 23 nt Cdx2-sgRNA 目标序列, 约 20 nt 序列从哈) 和反向 primer-3R (包含 15-20 nt 重叠序列从p2A-mCherry 和大约 20 nt 序列从哈) 在0.1 µM 最后浓度使用小鼠基因组 DNA 在 200 ng/µL (图 1D,表 1)。</li>
		<li>根据制造商的说明 (表 1), 在1×TAE 缓冲器上运行所有的 pcr 产品, 并在1% 琼脂糖凝胶上使用凝胶萃取试剂盒纯化预期尺寸的 pcr 产品。</li>
		<li>用 KpnI 和 XbaI 消化50-100 的构造载体。混合2µL 的线性化向量在 30-40 ng/µL 与三 PCR 扩增片段 (1 µL 为每, 100-200 ng/µL) 在2x 吉布森混合。添加 H2O 可将最终卷调整为10µL. 孵育50°c 的混合物60分钟。</li>
		<li>根据制造商的指示, 用所有组装的产品转换合格的大肠杆菌细胞, 并提取质粒结构。通过 DNA 测序验证 HMEJ 的捐献者。</li>
	</ol>
</li>
</ol>2. 利用 HMEJ 方法对小鼠胚胎进行基因组编辑
<ol><li>
Cas9 mRNA 的生产
	<ol>
<li>对于 Cas9 mRNA 的制备, 通过使用表 1中列出的适当底漆对, 将 T7 启动程序序列添加到 Cas9 编码区域。在最终浓度为0.1 µM 和 20 Cas9 表达载体的 Cas9 中加入底漆, 1×高保真 DNA 聚合酶组合。用 H2O 将最终卷调整为50µL。</li>
		<li>激活 DNA 聚合酶在95°c 5 分钟, 并执行 PCR 为36个周期在95°c 为三十年代, 60 °c 为三十年代和68°c 为4分钟 (1 min/1 kb), 以最后的引伸在68°c 为10分钟。</li>
		<li>纯化 T7-Cas9 PCR 产品的体外转录 (IVT), 然后转录 0.5-1 µg DNA 的 mRNA 转录试剂盒在37°c 8 小时的总容积20µL, 根据制造商的说明 (见材料表)。</li>
		<li>添加1µL 的 DNase 的混合物, 以删除 DNA 模板在37°c 15 分钟. 在37°c 添加一个聚 a 尾45分钟, 并通过 RNA 纯化试剂盒恢复 Cas9 mRNA, 根据制造商的说明 (请参阅材料表)。</li>
	</ol>
</li>
	<li>
sgRNA 生产
	<ol>
<li>生成 sgRNA 模板驱动的 T7 启动者与高保真 DNA 聚合酶如上所述。选择一个包含矢量的 sgRNA 脚手架作为模板。使用的引物列在表 1中。</li>
		<li>纯化 T7-sgRNA PCR 产品, 并使用 0.5-1 µg DNA 作为体外转录的模板, sgRNA 使用短 RNA 转录试剂盒, 在20µL 的总容积中, 在37°c 为6小时, 按制造商的说明 (参见材料表/c11>)。</li>
		<li>添加1µL 的 DNase 的混合物, 并继续孵化在37°c 15 分钟删除 DNA 模板。用 RNA 纯化试剂盒净化 sgRNAs (见材料表)。</li>
		<li>将 sgRNA 稀释至500µL RNase 水中, 并将样品保存在−80°c, 长达3月。 注: CRISPR ribonucleoproteins (RNPs) 是替代替代品, 具有更好的切割效率18,19,20。</li>
	</ol>
</li>
	<li>
胚胎采集、显微注射和体外培养
	<ol>
<li>Superovulate 雌 B6D2F1 (C57BL/6 x DBA2J) 小鼠 (7-8 周大), 孕母马血清促性腺激素 (PMSG), 其次为人绒毛膜促性腺激素 (hCG) 48 小时后。在 hCG 注射以后, 议院女性与 B6D2F1 男性过夜。</li>
		<li>牺牲女性由 CO2麻醉, 24 h 在 hCG 注射以后。在 M2 培养基中收集受精胚胎的输卵管 (每个雌性的 30-50 胚)。</li>
		<li>将受精的胚胎 (一天注射大约300颗鸡蛋) 放入 KSOM 培养基 (5.55 克/升氯化钠, 0.19 克/升氯化钾, 0.05 克/升2PO4, 0.05 克/升 MgSO47H2O, 0.04 克/升葡萄糖, 1.12 克/升乳酸钠, 2.1 克/升 NaHCO3, 0.02 克/l-丙酮酸钠, 0.25 克/升 CaCl2· 2H2O, 0.004 克/升 EDTA, 0.146 克/升谷氨酰胺, 1 克/升牛血清白蛋白), 在37摄氏度的孵化器与 5% CO2。</li>
		<li>混合 Cas9 mRNA (100 ng/µL)、sgRNA (50 ng/µL) 和 HMEJ 捐助向量 (100 ng/µL), 并添加 H2O 将最终卷调整为10µL. 把混合物放在冰上。</li>
		<li>拉毛细管针 (外径1.0 毫米, 内径0.78 毫米与灯丝) 使用微拉出器 (参数: 热, 74; 拉, 60; 速度, 80; 时间/延迟, 200; 压力, 300。请参阅材料表)。商业针头可以替代微注射。</li>
		<li>在含有5µg/毫升细胞松弛素 B 的 HEPES-CZB 介质液滴中, 将混合物的可能体积注入受精卵的胞浆中, 并使用具有恒定流设置的 microinjector(图 2 a) (参见 材料表)21。 注: 每组受精卵应在20-30 分钟内注射细胞松弛素 B 可提高注射后小鼠受精卵的存活能力。或者, 显微注射可以与压电系统一起操作, 如前所述22。</li>
		<li>培养注射受精卵在 KSOM 培养基在37°c 在 5% CO2之下直到胚泡阶段在3.5 天以后为荧光观察 (图 2B和 2C)。</li>
	</ol>
</li>
	<li>
小鼠胚胎移植及生成的研究
	<ol>
<li>输精管切除术雄性小鼠与注射剂在同一天发情的雌性小鼠交配。</li>
		<li>培养注射受精卵入2细胞阶段在37°c 在 5% CO2之下, 并且转移 25-30 2 细胞胚胎到 pseudopregnant 的输卵管在0.5 天后 coitum (dpc)。接受的母亲送幼崽在 19.5 dpc。</li>
	</ol>
</li>
	<li>
鼠标基因分型
	<ol>
<li>根据制造商的说明 (请参阅材料表), 使用 DNA 提取试剂盒从脚趾或尾部样品中提取小鼠基因组 dna。</li>
		<li>用紫外/可见光谱测量的 200-400 ng 基因组 DNA, 确定 5 ' 和 3 ' 的敲接事件, 作为模板进行 PCR ampliﬁcation。<ol>
<li>激活 DNA 聚合酶在95°c 5 分钟, 并执行 PCR 为38个周期在95°c 为三十年代, 60 °c 为三十年代和72°c 为1分钟 (1 min/1 kb), 以最后的引伸在72°c 为10分钟。对于 5 ' 连接, 使用前底漆在 HAL 的上游, 与反向一个在敲入片段 (p2A-mCherry)。至于 3 ' 结, 使用前底漆在敲入片段 (p2A-mCherry), 与反向一个在下游的哈 (表 1)。</li>
		</ol>
</li>
		<li>运行6µL 的 PCR 产品1% 琼脂糖凝胶在1×TAE 缓冲和检查预期的片段大小。然后通过 DNA 测序验证它们 (图 2D)。</li>
	</ol>
</li>
</ol>3. 肝细胞中基于 HMEJ 的体内基因组编辑
<ol><li>将收件人 C57BL/6J 鼠标 (8 周) 放在一个限制装置中, 并将尾部通过狭缝。</li>
	<li>在2毫升盐水溶液中混合 HMEJ 供体载体 (30 µg) 和 spCas9 表达载体 (30 µg)。对于控制实验, 在2毫升盐水溶液 (图 3A) 中挂起 HMEJ 供体向量 (30 µg)。</li>
	<li>用70% 乙醇清洁鼠标尾部。将针插入尾静脉, 并在5-7 秒内注入质粒 DNA 溶液. 取出针头, 从抑制装置中松开鼠标。</li>
	<li>在注射后5-9 天后通过 CO2麻醉来牺牲老鼠。灌注小鼠 transcardially 0.9% 生理盐水, 然后用蠕动泵4% 多聚甲醛, 并在4摄氏度一夜之间修复肝脏。</li>
	<li>在一夜之间用30% 蔗糖脱水组织, 直到它沉到管子的底部。</li>
	<li>切片的冷冻组织在厚度为10µm 的肝脏样本。</li>
	<li>冲洗三次在0.1 米磷酸盐缓冲 (PB) 和孵化他们的主要抗体: 兔抗 mCherry (稀释在 5%) 隔夜在4摄氏度。</li>
	<li>在 PB 中洗涤三次, 然后用二级抗体孵化: Cy3-AffiniPure 山羊抗兔 IgG, 在室温下在眼眶上进行2小时的免疫。</li>
	<li>Counterstain 部分与 DAPI 20 分钟, 并在玻璃幻灯片上安装甘油, 以进一步荧光观察 (图 3B)。</li>
</ol>

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D., 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=DNA+targeting+specificity+of+RNA-guided+Cas9+nucleases.">DNA targeting specificity of RNA-guided Cas9 nucleases.</a> Nature Biotechnology. 31 (9), 827-832 (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>Quadros, R. M., 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=Easi-CRISPR:+a+robust+method+for+one-step+generation+of+mice+carrying+conditional+and+insertion+alleles+using+long+ssDNA+donors+and+CRISPR+ribonucleoproteins.">Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins.</a> Genome Biology. 18 (1), 92 (2017).</li><li>Park, K. E., 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=Targeted+Gene+Knockin+in+Porcine+Somatic+Cells+Using+CRISPR/Cas+Ribonucleoproteins.">Targeted Gene Knockin in Porcine Somatic Cells Using CRISPR/Cas Ribonucleoproteins.</a> International journal of molecular sciences. 217 (6), (2016).</li><li>Woo, J. W., 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=DNA-free+genome+editing+in+plants+with+preassembled+CRISPR-Cas9+ribonucleoproteins.">DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins.</a> Nature biotechnology. 33 (11), 1162-1164 (2015).</li><li>Harms, D. W., 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=Mouse+Genome+Editing+Using+the+CRISPR/Cas+System.">Mouse Genome Editing Using the CRISPR/Cas System.</a> Current protocols in human genetics. 83, 11-27 (2014).</li><li>Yang, H., Wang, H., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+genetically+modified+mice+using+CRISPR/Cas-mediated+genome+engineering.">Generating genetically modified mice using CRISPR/Cas-mediated genome engineering.</a> Nature protocols. 9 (8), 1956-1968 (2014).</li><li>Grompe, M., 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=Loss+of+Fumarylacetoacetate+Hydrolase+Is+Responsible+for+the+Neonatal+Hepatic-Dysfunction+Phenotype+of+Lethal+Albino+Mice.">Loss of Fumarylacetoacetate Hydrolase Is Responsible for the Neonatal Hepatic-Dysfunction Phenotype of Lethal Albino Mice.</a> Genes &amp; development. 7 (12), 2298-2307 (1993).</li><li>Paulk, N. K., 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=Adeno-associated+virus+gene+repair+corrects+a+mouse+model+of+hereditary+tyrosinemia+in+vivo.">Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo.</a> Hepatology. 51 (4), 1200-1208 (2010).</li></ol>

作者没有什么可透露的。

HMEJ 供体质粒的构建最关键的步骤是: (1) sgRNA 的选择, 其 DNA 解切效率高, sgRNA 切割部位与停止密码子之间的距离小, (2) HMEJ 供体的适当构造。CRISPR/Cas9-mediated 对两种转基因供体载体 (包含 sgRNA 靶点和800的 bp 同源臂) 和靶向基因组在体内进行高效和精确靶向整合是必要的.采用 HMEJ 法生成敲鼠的最关键步骤是: (1) 制备高质量的 Cas9 mrna 和 sgRNA (Cas9 mrna 和 sgRNA 中不存在变性), (2) 制备高质量 HMEJ 供体质粒。质粒对胚胎发育无毒性作用。
最近, 还报告了一种基于 NHEJ 的方法, 用于有效的体内基因组编辑8。然而, 如前几份报告8所述, 不同类型的 indel 突变通常是在结点引起的, 因此很难实现精确的集成。在这里, 我们上面描述的基于 HMEJ 的策略显示了精确的目标集成, 几乎没有任何 indel 突变。因此, 一个基于 HMEJ 的策略可以是一个理想的平台, 用正确的方法替换突变序列 (如点突变), 而不适用于基于 NHEJ 的算法。
嵌合体是胚胎基因编辑的一个主要问题。在早期胚胎阶段注射 Cas9 蛋白代替 mRNA, 可以在没有嵌合体的情况下, 在一个细胞阶段实现转基因撞击。对于临床应用, 将 CRISPR/Cas9 系统送到成人组织中仍然具有挑战性。
基于 HMEJ 的基因组编辑有许多未来的潜在用途。它可以用来产生基因改良的动物模型。考虑到它在胚胎中的高敲效率, 这种方法可以显著减少产生基因改良动物模型所需的动物数量, 特别是开辟了生成非人类灵长类遗传模型的可能性。基于 HMEJ 的基因组编辑可以沿袭在成人组织中追踪单个细胞类型, 这对于动物模型特别有用, 因为缺乏可用的动物模型, 如非人类灵长类。它可以用于靶向基因治疗: HMEJ 策略最有吸引力的应用是基因治疗的临床使用。在本研究中, 我们修正了遗传性 tyrosinemia I. 型小鼠的出突变, 并通过流体动力学注入指示载体. 然而, CRISPR/Cas9 系统进入成人组织仍然是临床使用的主要技术挑战, 因为水动力注射不太可能在患者中执行。目前, 在将这种 HMEJ 的方法转化为临床前, 迫切需要进一步完善配送策略。

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/56844/crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated">Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells</a>. The phrases&#160;&#34;surveyor assay&#34;&#160;and&#160;&#34;Surveyor&#160;Nuclease&#34; have&#160;been updated to &#34;T7E1 assay&#34;&#160; to &#34; T7&#160;endonuclease I&#34; respectively.&#160;
Step 1.2 in&#160;the Protocol has been updated from:
<ol class="jove_content" start="2">
	<li>Surveyor nuclease&#160;assay of sgRNA 
	NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by surveyor nuclease assay (also known as T7 endonuclease I (T7EI) assay)17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon.</li>
</ol>
to:
<ol class="jove_content" start="2">
	<li>T7 endonuclease assay of sgRNA 
	NOTE: The targeting efficiency of the sgRNA used for the knock-in experiment is evaluated by T7 endonuclease (T7EI)&#160;assay17. Select the sgRNA with high DNA cleavage efficiency and a low distance between the sgRNA cutting site and the stop codon.</li>
</ol>
Figure 1 in the Representative Results has been updated&#160;from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56844/56844fig1.jpg" /> 
Figure 1: HMEJ-mediated targeted integration in vitro. 
(A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2a&#160;cells. GFP+ cells were sorted at day 3 for surveyor assay. (C) Surveyor assay for Cdx2 targeting: 6 different sgRNAs were designed for surveyor assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.&#160;<a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56844/56844fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56844/56844fig1v3.jpg" /> 
Figure 1: HMEJ-mediated targeted integration in vitro. 
(A) Experimental scheme for selection of sgRNAs: Six different sgRNAs (Cdx2-sgRNA1~Cdx2-sgRNA6) around the stop codon of the Cdx2 locus with a higher rank and off-target potential were chosen based on online CRISPR design tool. The protospacer adjacent motif (PAM) sequence is in red. (B) Experimental design: The Cas9-CMV-GFP expression plasmids expressing sgRNA, Cas9, and GFP were introduced into N2a&#160;cells. GFP+ cells were sorted at day 3 for T7EI&#160;assay. (C) T7EI assay for Cdx2 targeting: 6 different sgRNAs were designed for T7EI assay. Normal N2a cell genomic DNA serves as control. *, the sgRNA used for Cdx2-2A-mCherry knock-in experiment. (D) Schematic overview of construction of HMEJ donors using Gibson assembly. (E) Schematic overview of HMEJ-mediated gene targeting strategy at Cdx2 locus. HAL/HAR, left/right homology arm; triangles, sgRNA target sites; OF/OR, outer forward/reverse primer; IF/IR, inner forward/reverse primer. Figure modified from previous report10.&#160;<a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56844/56844fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for: <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/56844/crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated">Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells</a>. The phrases&#160;&#34;surveyor assay&#34;&#160;and&#160;&#34;Surveyor&#160;Nuclease&#34; have&#160;been updated to &#34;T7E1 assay&#34;&#160; to &#34; T7&#160;endonuclease I&#34; respectively.&#160;

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

精确的, 有针对性的基因组编辑通常需要生产转基因动物模型和临床治疗。为有效的靶向基因组编辑制定了各种策略, 如锌指核酸酶 (ZFN)、转录激活剂样效应核酸 (TALENs) 和 CRISPR/Cas9 系统。这些策略在基因组中创建目标 dna 双链断裂, 并利用内在的 dna 修复系统, 如同源重组 (HR)1,2, microhomology 介导的最终加入 (MMEJ)3,4,5和非同源端连接 (NHEJ)6,7,8 , 以诱导目标集成的转基因1,9。基于 HR 的策略是目前最常用的基因组编辑方法, 它在细胞系中非常有效, 但由于其在晚期 S/G2 阶段的受限发生而无法轻易进入非分裂细胞。因此, 基于 HR 的策略不适用于体内基因组编辑。最近, NHEJ 的策略被开发为有效的基因敲入在小鼠组织8。然而, 基于 NHEJ 的方法通常会在连接处引入 indels, 使得很难生成精确的基因组编辑, 尤其是在试图构建帧内融合基因8时。基于 MMEJ 的目标整合能够精确地进行基因组编辑。但是, 在以前的报告5中, 它只适度地提高了目标集成效率。因此, 在广泛的治疗应用程序3中迫切需要提高精确目标集成在体内的效率。
在最近发布的工作中, 我们演示了一种基于同源介导的端接 (HMEJ) 策略, 它在所有报告的策略中都显示了目标集成效率最高的体外和体内10。在这里, 我们描述了建立 HMEJ 系统的一个协议, 并构建了针对感兴趣的基因的单导 RNA (sgRNA) 向量, 以及窝藏 sgRNA 目标点的施主载体和800同源臂的 bp (图 1).在本协议中, 我们还描述了生成 DNA 敲除小鼠的详细步骤以及在组织中体内的目标集成的简要步骤。此外, 基于 HMEJ 的策略的概念验证研究表明, 它有能力纠正 "突变" 的变异并抢救/肝功能衰竭小鼠, 进一步揭示了其治疗潜力.

<table><tbody><tr><td>pX330</td><td>Addgene</td><td>42230</td><td></td></tr><tr><td>pAAV vector</td><td>Addgene</td><td>37083</td><td></td></tr><tr><td>pX260</td><td>Addgene</td><td>42229</td><td></td></tr><tr><td>AAV_Efs_hSpCas9_NLS_FLAG-SV40</td><td>Addgene</td><td>97307</td><td>AAV vector for encoding a human codon-optimized SpCas9 driven by EFs promoter</td></tr><tr><td>AAV_Actb HMEJ donor_U6_sgRNA_EF1a_GFP_polyA</td><td>Addgene</td><td>97308</td><td>HMEJ donor for fusing a p2A-mCherry reporter to mouse Actb. EGFP driven by EF1a promoter and U6-driven sgRNAs targeting Actb. AAV backbone.</td></tr><tr><td>AAV_Cdx2 HMEJ donor</td><td>Addgene</td><td>97319</td><td>HMEJ donor for fusing a p2A-mCherry reporter to mouse Cdx2.&amp;nbsp;</td></tr><tr><td>Lipofectamine 3000 Transfection Reagent</td><td>Life Technology</td><td>L3000015</td><td></td></tr><tr><td>Nuclease-Free Water</td><td>Life Technologies</td><td>AM9930</td><td></td></tr><tr><td>Bbs I</td><td>New England Biolabs</td><td>R0539S</td><td></td></tr><tr><td>NEB Buffer 2</td><td>New England Biolabs</td><td>B7002S</td><td></td></tr><tr><td>T7 endonuclease I</td><td>New England Biolabs</td><td>M0302L</td><td></td></tr><tr><td>NEBuilder HiFi DNA Assembly Master Mix</td><td>New England Biolabs</td><td>E2621L</td><td></td></tr><tr><td>Plasmid EndoFree-Midi Kit</td><td>Qiagen</td><td>12143</td><td></td></tr><tr><td>MMESSAGE MMACHINE T7 ULTRA</td><td>Life Technologies</td><td>AM1345</td><td></td></tr><tr><td>MEGACLEAR KIT 20 RXNS</td><td>Life Technologies</td><td>AM1908</td><td></td></tr><tr><td>MEGASHORTSCRIPT T7 KIT 25 RXNS</td><td>Life Technologies</td><td>AM1354</td><td></td></tr><tr><td>Flaming/Brown Micropipette Puller</td><td>Sutter Instrument</td><td>P-97</td><td>&amp;nbsp;Micropipette Puller (parameters: heat, 74; pull, 60; velocity, 80; time/delay, 200; pressure, 300)</td></tr><tr><td>Borosilicate glass</td><td>Sutter Instrument</td><td>B100-78-10</td><td>type of capillaries (outer diameter 1.0 mm, inner diameter 0.78 mm with filament)&amp;nbsp;</td></tr><tr><td>FemtoJet microinjector</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Freezing microtome</td><td>Leica</td><td>CM1950-Cryostat</td><td>thickness&amp;nbsp;of&amp;nbsp;40&amp;nbsp;&amp;mu;m&amp;nbsp;for&amp;nbsp;brain,&amp;nbsp;10 &amp;mu;m&amp;nbsp;for&amp;nbsp;liver</td></tr><tr><td>Rabbit anti-mCherry</td><td>GeneTex</td><td></td><td></td></tr><tr><td>Cy3-AffiniPure Goat Anti-Rabbit IgG</td><td>Jackson Immunoresearch</td><td></td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>11965092</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>10099141</td><td></td></tr><tr><td>NEAA</td><td>Gibco</td><td>11140050</td><td></td></tr><tr><td>Pen,Strep,Glutamine</td><td>Gibco</td><td>10378016</td><td></td></tr><tr><td>Gel Extraction Kit</td><td>Omega</td><td>D2500-02</td><td></td></tr><tr><td>FACS</td><td>BD AriaII</td><td></td><td></td></tr><tr><td>PMSG</td><td>Ningbo Sansheng Medicine</td><td>S141004</td><td></td></tr><tr><td>HCG</td><td>Ningbo Sansheng Medicine</td><td>B141002</td><td></td></tr><tr><td>Cytochalasin B</td><td>Sigma</td><td>CAT#C6762</td><td></td></tr><tr><td>KSOM+AA with D-Glucose and Phenol Red</td><td>Millipore</td><td>CAT#MR-106-D</td><td></td></tr><tr><td>M2 Medium with Phenol Red</td><td>Millipore</td><td>CAT#MR-015-D</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td></td><td></td></tr></tbody></table>

crispr/cas9-mediated targeted integration in vivo using a homology-mediated end joining-based strategy

CRISPR/Cas9 系统作为一种有前景的基因组编辑平台, 具有很好的遗传操作能力, 特别是转基因靶向整合。然而, 由于同源重组 (HR) 的效率低下, 以及非同源端连接 (NHEJ) 的各种 indel 突变, 在无分裂细胞中,体内基因组编辑仍然是一个巨大的挑战。在这里, 我们描述了一个基于同源介导的端接 (HMEJ) 的 CRISPR/Cas9 系统, 用于有效的体内精确目标集成。在该系统中, 靶向基因组和包含同源臂 (约 800 bp) 的单导 RNA (sgRNA) 靶序列的施主载体被 CRISPR/Cas9。这种基于 HMEJ 的策略在小鼠受精卵以及肝细胞体内实现了有效的转基因集成。此外, 以 HMEJ 为基础的策略提供了一种有效的方法来矫正肝细胞中的 fumarylacetoacetate 水解酶 () 突变, 并抢救缺陷致肝功能衰竭小鼠. 结合起来, 专注于目标整合, 这种基于 HMEJ 的战略为各种应用提供了一个有前景的工具, 包括基因改良动物模型的生成和靶向基因治疗。

在小鼠胚胎中基于 HMEJ 的基因组编辑:为了定义小鼠受精卵中基于 HMEJ 的方法的敲除效率, 我们将 Cas9 mRNA、sgRNA 靶向Cdx2基因和 HMEJ 捐献者放入鼠标受精卵中, 该基因被设计用来将 p2A-mCherry 报告器中的一个子项与 Cdx2 的最后一个密码子结合起来. 基因 (图 2A)。注射的受精卵发展成囊胚在文化中。为了评价 mCherry 荧光的有效性, 我们用荧光显微镜对其进行了分析, 发现12.9% 的囊胚接受 HMEJ 捐献者是阳性的 mCherry, 这在滋养外胚层中严格表达 (图2B、2C)。通过对 PCR 阳性小鼠进行排序, 我们还发现所有检测到的集成事件都是 5 ' 和 3 ' 连接点的精确帧内集成 (图 2D)。
基于 HMEJ 的基因组编辑在成人组织和 HMEJ 介导的基因治疗:为了研究 HMEJ 基因组的编辑是否可以应用于成人组织, 我们在Actb基因的停止密码子前插入 mCherry 盒, 传感ActbHMEJ 构造 C57/B6J 小鼠肝的尾静脉流体力学注入 (图 3A)。经过7天的注射, 我们发现近半数的转染肝细胞表达 mCherry 染色在肝脏部分 (图 3B)。
为了探讨使用 HMEJ 的基因治疗策略的可能性, 我们使用了 fumarylacetoacetate 水解酶 () 缺陷小鼠.tyrosinemia 鼠标是一种成熟的遗传性类型 I (HTI) 鼠标模型, 它在基因的外显子5中放置一个插入片段, 导致以下序列23中的 frameshift 突变. 为了维护 trifluoromethylbenzoyl /小鼠, 我们用酪氨酸分解通路上游的抑制剂, 2-(2-硝基-4-)-13-cyclohexanedione (NTBC) 24 处理了.在这里, 我们出发去看看 MMEJ 和 HMEJ 介导的基因矫正是否能拯救/鼠标中的. 突变. 我们水力注入 Cas9 构造和-MMEJ 或 HMEJ 构造, 旨在将5到14的子显式 cDNA 插入遗传基因的内含子 4, 到. - 小鼠肝脏 ( 图 3C). 注射后一周, NTBC 被撤回以诱发肝脏损伤 (图 3C)。在 NTBC 退出后, HMEJ 小鼠接收的纠正肝细胞比基于 MMEJ 的方法显示更有效的扩散 (图 3 D ).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56844/56844fig1.jpg"> 图 1: HMEJ 介导的目标集成体外. (A) 选择 sgRNAs 的实验方案: 六个不同的 sgRNAs (Cdx2-sgRNA1~Cdx2sgRNA6) 基于在线 Cdx2 设计选择了具有较高秩和非目标电位的CRISPR轨迹的终止密码子。工具.protospacer 相邻的母题 (PAM) 序列为红色。(B) 实验设计: 将 sgRNA、Cas9 和 GFP 表达的 Cas9-CMV-GFP 表达质粒引入 N2a 细胞。GFP+单元格在3天被排序为验船师化验。(C) 对Cdx2目标进行测量的检测: 6 种不同的 sgRNAs 是为测量师设计的。正常 N2a 细胞基因组 DNA 作为控制。*, 用于Cdx2的 sgRNA 2 mCherry 敲入实验。(D) 使用吉布森组件构造 HMEJ 捐助者的示意图概述。(E) HMEJ 介导的基因靶向策略在Cdx2轨迹中的示意图概述。哈尔, 左/右同源臂;三角形, sgRNA 目标站点;/或, 外向前/反向底漆;IF/IR, 内前/反向底漆。从上一个报表10修改的图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56844/56844fig1large.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/56844/56844fig2.jpg"> 图 2: 通过 HMEJ 介导的目标集成对小鼠胚胎进行基因组编辑 (A) 微注射实验方案: 将 Cas9 mRNA (100 ng/µL)、sgRNA (50 ng/µL) 和供体质粒 (100 ng/µL) 的混合物注射到小鼠受精卵中。(B) 由 HMEJ 策略编辑的小鼠胚胎的代表荧光图像。酒吧, 20 µm. (C) 按 mCherry+囊胚百分比表示的敲出效率。数字在每个酒吧之上, 总囊胚计数。(D) 在Cdx2轨迹上进行基因编辑的小鼠序列分析。PCR 产品从 5 ' 和 3 ' 结点被放大了测序。上部, 同源臂;紫色, p2A;红色, mCherry;或哈尔, 右或左同源的手臂。虚线标记为清晰而省略的区域。从上一个报表10修改的图。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56844/56844fig2large.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/56844/56844fig3.jpg"> 图 3: HMEJ 介导的目标集成在体内. (A) 水动力尾静脉注射示意图概述。通过水动力尾静脉注入的方式, 表达了供体序列和 sgRNA 的质粒, 以及表达 spCas9 的质粒。(B) 代表肝细胞免疫荧光图像。肝切片采集7天后注射。缩放条, 50 µm, 转染的细胞。(C) MMEJ 或 HMEJ 介导的基因置换策略的质粒, 旨在将5到14的外显子的发 cDNA 插入到发基因的内含子4中, 并通过水动力注射将其传递到了该方法. NTBC on: NTBC - 小鼠保存在水上;NTBC: 撤退 NTBC 水 (NTBC 撤退的第一天被定义了0天, 是注射之后的第七天)。(D) MMEJ 的免疫组化染色, 其肝脏部分由 ./-小鼠注射而成, 其为 HMEJ 质粒。缩放条, 100 µm 从以前的报告修改过的图5,10。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56844/56844fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy at JoVE.com

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

聚簇定期 interspaced 短回文重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统为基因工程提供了一个有前途的工具, 并开辟了转基因目标整合的可能性。我们描述了一种基于同源介导的端接 (HMEJ) 策略, 用于有效的 DNA 靶向集成体内和使用 CRISPR/Cas9 的靶向基因治疗。

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 ...

crisprcas9-mediated-targeted-integration-vivo-using-homology-mediated

Nanyang Technological University

Research

JoVE Journal

Genetics

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

14.7K 次观看.  Chinese Academy of Sciences. 聚簇定期 interspaced 短回文重复/CRISPR 相关蛋白 9 (CRISPR/Cas9) 系统为基因工程提供了一个有前途的工具, 并开辟了转基因目标整合的可能性。我们描述了一种基于同源介导的端接 (HMEJ) 策略, 用于有效的 DNA 靶向集成体内和使用 CRISPR/Cas9 的靶向基因治疗。

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

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 ...

科研

遗传学

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

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

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

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

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

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 ...

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

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 ...

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 ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been successfully repurposed for genome engineering in many different living organisms. Most commonly, the wildtype CRISPR associated 9 (Cas9) or Cas12a endonuclease is used to cleave specific sites in the genome, after which the DNA double-stranded break is repaired via the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway depending on whether a donor template is absent or present respectively. To date, CRISPR systems from different bacterial species have been shown to be capable of performing genome editing in mammalian cells. However, despite the apparent simplicity of the technology, multiple design parameters need to be considered, which often leave users perplexed about how best to carry out their genome editing experiments. Here, we describe a complete workflow from experimental design to identification of cell clones that carry desired DNA modifications, with the goal of facilitating successful execution of genome editing experiments in mammalian cell lines. We highlight key considerations for users to take note of, including the choice of CRISPR system, the spacer length, and the design of a single-stranded oligodeoxynucleotide (ssODN) donor template. We envision that this workflow will be useful for gene knockout studies, disease modeling efforts, or the generation of reporter cell lines.

CRISPR-Cas is a powerful technology to engineer the complex genomes of plants and animals. Here, we detail a protocol to efficiently edit the human genome using different Cas endonucleases. We highlight important considerations and design parameters to optimize editing efficiency.

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

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 ...

<meta charset="utf8"></head><body>使用 CIRCLE-Seq 检测脱靶 CRISPR-Cas9 切割事件

CIRCLE-Seq for Interrogation of Off-Target Gene Editing

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over other in vitro methods, including minimal sequencing depth requirements, low background, and high enrichment for Cas9-cleaved gDNA. These advantages enhance sensitivity in identifying both intended and unintended cleavage events. This study provides a comprehensive, step-by-step procedure for examining the off-target activity of CRISPR-Cas9 using CIRCLE-seq. As an example, this protocol is validated by mapping genome-wide unintended cleavage sites of CRISPR-Cas9 during the modification of the AAVS1 locus. The entire CIRCLE-seq process can be completed in two weeks, allowing sufficient time for cell growth, DNA purification, library preparation, and Illumina sequencing. The input of sequencing data into the CIRCLE-seq pipeline facilitates streamlined interpretation and analysis of cleavage sites.

<meta charset="utf8"></head><body>CIRCLE-Seq 用于研究脱靶基因编辑

A significant barrier to technologies like CRISPR is the off-target events that can disrupt vital genes. 'Circularization for In Vitro Reporting of Cleavage Effects by Sequencing' (CIRCLE-seq) is a technique designed to identify unintended cleavage sites. This method maps the genome-wide activity of CRISPR-Cas9 with high sensitivity and without bias.

CIRCLE-Seq 用于研究脱靶基因编辑

Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of ...

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

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此视频中的章节

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

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

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

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

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

CRISPR/Cas9 诱导人多潜能干细胞内源蛋白标记的应用

利用 Crispr-ca 在哺乳动物细胞系中进行基因组编辑

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

CIRCLE-Seq 用于研究脱靶基因编辑

CIRCLE-Seq 用于研究脱靶基因编辑