这项工作得到了加拿大卫生研究所（CIHR 365252）、克雷姆比尔基金会和安大略研究基金卓越研究第8轮（RE08-065）的项目赠款的支持。桑帕德·库马尔·洛加坦是加拿大癌症协会奖学金（BC-F-16#31919）的接受者。

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</ol>

作者没有什么可透露的。

CRISPR/Cas9基因组编辑已广泛应用于体外和体内研究，以研究基因功能和细胞过程。大多数体内研究都利用CRISPR/Cas9基因编辑的细胞移植到动物模型（异种移植或异种移植）。虽然这是研究癌症遗传学和细胞功能的有力工具，但它仍然缺乏原生组织微环境，并可能引起伤害和/或免疫反应。
为了克服这些挑战，几个小组已经率先在体内CRISPR方法产生几个，多路复用的自动鼠标模型在过去几年15，16，17，18。这些项目通常依靠腺相关病毒 （AAV） 向目标器官提供 sgRNA。AAV 允许生成非常高的滴定器，从而促进体内操作所需的高滴定病毒的生产。然而，AAV的使用严重限制了可以分析到大约40-50个基因的基因数量，因为AAV与扁豆病毒不同，不能稳定地整合到基因组DNA中，使得sgRNA库的读出不切实际。基于 AAV 的筛查需要使用目标站点的直接读取突变，例如，有针对性的捕获测序。但是，基于 PCR 的多路复用捕获测序将"可筛选"库中的可捕获目标数量限制在通常为数十个 15、16、17、18 的顺序。
与体内CRISPR屏幕中的AAV相比，此处描述的方法使用扁平性sgRNA。鉴于扁桃体构造集成到目标细胞的DNA中，sgRNA表示可以与任何传统的CRISPR屏幕19，20进行分析。因此，这种方法允许同时分析数百个基因，甚至可以无缝地扩展到全基因组的体内屏幕。例如，一个全基因组屏幕，具有78，000毫克RNA和30倍的覆盖率将需要约90个胚胎，如先前描述的类似的shRNA屏幕4。由于最常见的近亲繁殖小鼠菌株的垃圾大小约为8-12幼崽/幼崽/幼崽，经验丰富的外科医生可以很容易地在更小的时间内注射所有小鼠，因此这种全基因组筛查只需要大约10-12个水坝和手术。
体内屏幕的成功取决于高滴定病毒的产生。虽然扁豆结构，表达一个sgRNA的兴趣，Cas9和Cre（例如，pSECC）是一个方便和可行的一体式解决方案，他们有扁桃体辣椒（~10 kb的总尺寸）的包装限制，导致整体较低的病毒滴定。为了克服这一挑战，我们使用 R26-LSL-Cas9-GFP 小鼠和只含有 Cre-重组酶和 sgRNA 的扁豆病毒构造。由此产生的扁桃体结构长度仅为 7 kb，产生超过 108 PFU/mL 的病毒滴定器。
目标和特定的库可以在几天内快速生成，复杂的基因相互作用网络可以在几周内在体内进行研究。Cas9介导的突变可以在野生类型的小鼠中进行，以研究器官发育、平衡或疾病表型（癌症、炎症性皮肤疾病等），也可以很容易地与携带任何肿瘤突变或突变组合的小鼠结合，以模拟人类患者的遗传状况。此外，克隆方法可以通过在一体质粒中添加兼容的限制站点和 sgRNA 序列来克隆 CRISPRa 或 CRISPRi 库。值得注意的是，所有操纵基因功能的库不仅可用于小鼠模型，还可用于其他动物模型和器官培养物。该方法共同突出了实用性，并提供了一个样板，用于直接在体内CRISPR集成体细胞基因编辑和小鼠建模，以快速评估体内的基因功能。

后基因组时代癌症研究面临的挑战之一是挖掘因果基因突变的大量基因组数据，并识别基因网络中可以针对治疗的节点。虽然生物信息分析对实现这些目标大有帮助，但建立有效的体外和体内模型是破译生物系统和疾病状态的复杂性和促进药物开发的先决条件。虽然传统的转基因小鼠模型在体内癌症遗传学研究中被广泛使用，但其成本、时间和劳动密集型性质在很大程度上禁止了对现代基因组学解开的数百个假定癌症基因的系统分析。为了克服这一瓶颈，我们结合了先前建立的子宫内注射方法1，2的超声波引导与CRISPR/Cas9（聚集定期间歇短巴林德罗米重复）基因编辑技术3，同时诱导和研究数百个基因在皮肤和单只小鼠口腔的功能丧失突变。
此处描述的方法利用工程扁豆病毒的超声波引导注射到胚胎日E9.5活小鼠胚胎的羊水腔中。含有CRISPR/Cas9成分的扁桃体货物会转导单层表面等体，后者后来导致皮肤和口腔的上皮。皮肤由外表皮组成，其次是地下室膜和真皮。表皮是一种分层上皮，由内基层组成，与地下膜保持接触，具有增殖和干细胞能力。基底层产生上面的分化层，如旋转层、颗粒层和地层角膜层2、4。血统追踪研究表明，这种直接在体内CRISPR/Cas9方法基因操纵组织驻存干细胞在基底层，持续整个成年。由于扁豆病毒可以滴定以在克隆密度下转导E9.5表面细胞，因此这种方法可用于生成含有数千个离散基因克隆的马赛克小鼠。然后，下一代测序可用于以多路复用的方式分析CRISPR/Cas9介质基因消融在这些克隆中的影响。
我们最近使用这种方法评估了484个基因的功能，这些基因显示人类头部和颈部鳞状细胞癌（HNSCC）5的反复突变。HNSCC是一种毁灭性的癌症，死亡率高达40-50%，是全世界第6大最常见的癌症。HNSCC 出现在上气道或口腔的粘膜衬里中，与烟草和酒精消费或人类瘤病毒（ HPV ）感染有关。皮肤鳞状细胞癌（SCC）是皮肤肿瘤，是人类第二常见的癌症。角质SCC和HNSCC在病理学和分子学上非常相似，在TP53、PIK3CA、NOTCH1和HRAS8中出现改变的病例比例很高。虽然只有少数基因在高频下发生变异，但有数百个基因在低频下发生变异（+lt;5%），这种现象通常被称为长尾分布。由于大多数长尾基因缺乏生物或临床验证，我们利用这种体内CRISPR筛选技术，在p53、Pik3ca或Hras中具有敏感突变的肿瘤易发小鼠中模拟这些基因的功能丧失，并发现了几个与p53、Pik3ca或Hras合作的新型肿瘤抑制基因，以触发肿瘤发育5。
在这里，我们描述了一个详细的协议，以产生多路sgRNA扁平CRISPR sgRNA库，并在鼠标表面等体中执行CRISPR/Cas9基因敲除屏幕。值得注意的是，这种方法可以应用于整合其他基因操作技术，如CRISPR激活（CRISPRa）和CRISPR灭活（CRISPRi），或修改为针对小鼠的其他器官系统来研究基因功能。

<table><tbody><tr><td>0.45 micron filter</td><td>Sigma</td><td>S2HVU02RE</td><td></td></tr><tr><td>12k or 92k oligo chip</td><td>Customarray Inc. (Genscript)</td><td></td><td></td></tr><tr><td>15 cm cell culture plates</td><td>Corning</td><td></td><td></td></tr><tr><td>293FT</td><td>Invitrogen</td><td>R70007</td><td></td></tr><tr><td>293NT</td><td>Systems Biosciences</td><td>LV900A-1</td><td></td></tr><tr><td>Alkaline phosphatase</td><td>NEB</td><td>M0290L</td><td></td></tr><tr><td>Amplicillin</td><td>Fisher Scientific</td><td>BP1760-25</td><td></td></tr><tr><td>ATP</td><td>NEB</td><td>9804S</td><td></td></tr><tr><td>BsmBI</td><td>NEB</td><td>R0580L</td><td></td></tr><tr><td>Chromic gut suture</td><td>Covidien</td><td></td><td></td></tr><tr><td>Deep sequencing (Next-Seq or Hi-Seq)</td><td>Illumina</td><td></td><td></td></tr><tr><td>DNA-cleanup kit</td><td>Zymo Research</td><td>D4008</td><td></td></tr><tr><td>DNAesy Blood and Tissue DNA extraction kit</td><td>Qiagen</td><td>69506</td><td></td></tr><tr><td>Endura electrocompetent cells</td><td>Lucigen</td><td>60242-1</td><td></td></tr><tr><td>Glass Capillaries</td><td>Drummond</td><td>3-000-203-G/X</td><td></td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>High-Speed Centrifuge</td><td>Beckman Coulter</td><td>MLS-50</td><td></td></tr><tr><td>LB Agar</td><td>Wisent Technologies</td><td>800-011-LG</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P97</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M5904</td><td></td></tr><tr><td>Mini-prep plasmid Kit</td><td>Frogga Bio</td><td>PDH300</td><td></td></tr><tr><td>Mouse oxygen anaesthesia system</td><td>Visual Sonics</td><td></td><td></td></tr><tr><td>Nanoject II micromanipulator</td><td>Drummond</td><td></td><td></td></tr><tr><td>NEBuffer 3.1 (Buffer for BsmBI)</td><td>NEB</td><td>R0580L</td><td></td></tr><tr><td>Needle sharpener</td><td>Sutter Instrument</td><td>BV-10</td><td></td></tr><tr><td>Oligo cleanup kit</td><td>Zymo research</td><td>D4060</td><td></td></tr><tr><td>PAGE purified illumina sequencing primer</td><td>IDT DNA</td><td></td><td></td></tr><tr><td>PEI (polyethyleneimine)</td><td>Sigma</td><td>408727-100ML</td><td></td></tr><tr><td>Permoplast modeling clay</td><td></td><td></td><td></td></tr><tr><td>Petridish with central opening</td><td>Visual Sonics</td><td></td><td></td></tr><tr><td>pMD2.G</td><td>Addgene</td><td>12259</td><td></td></tr><tr><td>psPAX2</td><td>Addgene</td><td>12260</td><td></td></tr><tr><td>Q5 Polymerase 2x Master mix</td><td>NEB</td><td>M0494L</td><td></td></tr><tr><td>Qubit Fluorometric Quantification</td><td>Invitrogen</td><td>Q33327</td><td></td></tr><tr><td>Semicircular Silicone plug</td><td>Corning</td><td></td><td></td></tr><tr><td>Silicone membrane</td><td>Visual Sonics</td><td></td><td></td></tr><tr><td>T4 DNA ligase</td><td>NEB</td><td>M0202L</td><td></td></tr><tr><td>Ultra-centrifuge tubes</td><td>Beckman Coulter</td><td>344058</td><td></td></tr><tr><td>Vevo2000 ultrasound system</td><td>Visual Sonics</td><td></td><td></td></tr></tbody></table>

in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity

转基因小鼠模型 （GEMM） 在评估基因功能、模拟人类疾病以及作为临床前模型评估治疗途径方面发挥了重要作用。然而，它们的时间、劳动和成本密集型性质限制了它们对基因功能进行系统分析的效用。基因组编辑技术的最新进展克服了这些限制，并允许以多路复用和快速的方式在特定小鼠器官内快速生成特定的基因扰动。在这里，我们描述了一种基于CRISPR/Cas9的方法（聚集定期间歇短脑重复），在小鼠皮肤和口腔的上皮产生数千个基因敲除克隆，并提供一个协议，详细说明   在肿瘤抑制基因的直接体内CRISPR屏幕执行必要的步骤。这种方法可以应用于其他器官或其他CRISPR/Cas9技术，如CRISPR激活或CRISPR灭活，以研究基因在组织失落期间或在各种疾病环境中的生物功能。

图1A 显示了寡核苷酸的设计，用于在单个 12k 或 92k 寡头芯片中以经济高效的方式对多个自定义 CRISPR 库进行多路复用。一旦选择 sgRNA（蓝色编码），寡核苷酸将设计为限制位点（橙色 BsmBI）和库特定的 PCR 引物对（绿色编码）。几个库可以通过使用引物对的独特组合来设计，用于在单个寡头芯片中进行多重组合。当 PCR 使用特定的 PCR 底漆对放大库时，始终只包含水负控制，并在 agarose 凝胶上运行所有反应。负控制车道不应有任何 100 bp 的波段，而库放大的车道应有一个 100 bp 波段。如果没有波段，请确保适当选择 PCR 引正器对。 图1B 显示了克隆库的质量控制步骤以及病毒的生产和浓缩过程。应注意保持sgRNA从克隆到转导的平等代表性。下一代PCR测序读取从质粒和扁豆病毒库转导细胞放大DNA应表现出高相关性。必须仔细分析任何偏差，以检查在扁豆病毒准备之前或之后是否丢失了表示。如果从质粒DNA读取的sgRNA不能显示sgRNA的等同表示，那么病毒制备和浓缩过程必须小心重复。如果在质粒DNA中发生等量sgRNA表示的丧失，则必须重复整个克隆过程，包括从寡头芯片中放大库的PCR，并缩短放大周期。CRISPR 图书馆筛选的成功在很大程度上取决于图书馆中现存在 sgRNA 的平等代表性。
图2 显示了为操纵怀孕小鼠的E9.5胚胎而设置的超声波引导微注射。整个设置被放置在生物安全级别II组柜下，以保持整个程序的无菌状态，并避免因外科手术而感染怀孕的老鼠。应小心避免在注射时对子宫角施加压力。针必须非常锋利，使子宫壁上的伤口和羊膜是最小的。

图4 显示了PCR的下一代测序读数，从质粒和扁豆病毒库中放大DNA，并从4个有代表性的肿瘤中读取。与质粒池或受感染细胞中的 sgRNA 演示相比，针对 Adam10 和 Ripk4 的 sgRNA 指南在肿瘤样本（三角形和钻石）中具有丰富的内容。 亚当10 和 Ripk4 功能作为肿瘤抑制剂5。通过为每个样本分配唯一的条形码和协议中概述的深序，可以多路复用几百个肿瘤。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61693/61693fig01.jpg"> 图1:克隆有针对性的CRISPR库。（A） 示意图代表 12 k 或 92 k 自定义寡头芯片的寡核苷酸设计。bsmBI（或其他兼容的）限制站点（橙色编码和下划线）在 sgRNA 的每一侧添加。箭头指示 BsmBI 切割站点。对于每个库，都添加了一对独特的 PCR 入门（绿色、棕色和紫色编码），以便使用池式芯片中的 PCR 进行具体放大。几个自定义库可以多路复用多达 12k 或 92k 寡头。（B） 显示来自CRISPR库的质粒DNA与来自同一扁平库的细胞的DNA的sgRNA表示的图形。每个点代表一个指南。充分代表在转导后保持，并具有一定的相关性。 <a href="https://www.jove.com/files/ftp_upload/61693/61693fig01large.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/61693/61693fig02v2.jpg"> 图2:超声波引导微投影设置的图片。（A） 携带E9.5胚胎的老鼠被麻醉并放置在加热平台上，子宫暴露在充满PBS的改性培养皿室中（由四个建模粘土稳定）。蓝色半圆形橡胶支撑着含有胚胎的子宫，微注射器的注射针位于右侧。超声波扫描头安装在顶部，将实时图像中继到后面的监视器上，针头可见。（B） E9.5胚胎的特写超声图像。伦蒂维拉尔图书馆被注射针头（见右侧）到羊水腔。 <a href="https://www.jove.com/files/ftp_upload/61693/61693fig02large.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/61693/61693fig03.jpg"> 图3:成功转导小鼠表面上皮。与克里伦蒂病毒（入口:相应的白光图像）一起转化的新生新记者LSL-纸屑小鼠全身、口腔、舌头和味觉的荧光图像。比例栏 = 500 μm <a href="https://www.jove.com/files/ftp_upload/61693/61693fig03large.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/61693/61693fig04.jpg"> 图4:肿瘤样本的深度测序。图显示来自CRISPR库的sgRNA表示质粒DNA与来自同一扁平库的细胞的DNA，以及来自4个具有代表性肿瘤的读数。红色和绿色圆表示在库和转导细胞中读取的 Adam10 和 Ripk4 sgRNA 的数量，而红色三角表示 Adam10 sgRNA 的读数，绿色钻石表示分别显示 1000 倍丰度的单独 HNSCC 小鼠在肿瘤中识别的 Ripk4 sgRNA 的读数。 <a href="https://www.jove.com/files/ftp_upload/61693/61693fig04large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>PCR1 转发引号</td>
			<td>加格克卡特卡特克</td>
		</tr>
<tr>
<td>PCR1 反向引号</td>
			<td>卡卡格特加</td>
		</tr>
</tbody></table>表1:PCR1反应的引言。 用于放大与扁豆病毒转化的细胞基因组DNA的sgRNA盒周围区域的前向和反向引引子。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>步</td>
			<td>温度</td>
			<td>时间</td>
			<td> </td>
		</tr>
<tr>
<td>1</td>
			<td>98°C</td>
			<td>30 秒</td>
			<td></td>
		</tr>
<tr>
<td>2</td>
			<td>98°C</td>
			<td>10 秒</td>
			<td></td>
		</tr>
<tr>
<td>3</td>
			<td>66°C</td>
			<td>30 秒</td>
			<td></td>
		</tr>
<tr>
<td>4</td>
			<td>72°C</td>
			<td>15 秒</td>
			<td>15 个周期（步骤 2-4）</td>
		</tr>
<tr>
<td>5</td>
			<td>72°C</td>
			<td>2 分钟</td>
			<td></td>
		</tr>
<tr>
<td>6</td>
			<td>4 °C</td>
			<td>拿</td>
			<td></td>
		</tr>
</tbody></table>表2:PCR1周期参数。 用于放大与扁豆病毒转化的细胞基因组DNA的sgRNA盒周围区域的PCR条件。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>501 FW</td>
			<td colspan="3">阿塔加塔克加卡塔卡塔克 阿克特克特克特加特加加卡奇格</td>
		</tr>
<tr>
<td>701房地</td>
			<td colspan="3">卡加加特格特加加特加特加加特格特 中加塔克塔塔克塔克塔克塔</td>
		</tr>
<tr>
<td colspan="4">*下划线基表示用于多路复用的光照（D501-510用于转发，D701-712用于反向）条形码位置。</td>
		</tr>
<tr>
<td colspan="4">*红色基座表示与扁平CRISPR质粒上的目标站点绑定的序列。此区域可根据使用的扁桃体向量进行修改。</td>
		</tr>
</tbody></table>表3:PCR2反应的条形码引言。 用于条形码的引物放大每个肿瘤样本（直接来自基因组DNA或使用 PCR1 的产品作为模板）。下划线区域表示可用于每个样本的独特条形码区域，以便在深测序反应中进行多路复用。红色基对表示这些引金绑定的扁平结构中的目标区域。可根据所使用的扁桃体构造类型对此目标区域进行修改。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>步</td>
			<td>温度</td>
			<td>时间</td>
			<td> </td>
		</tr>
<tr>
<td>1</td>
			<td>98°C</td>
			<td>30 秒</td>
			<td></td>
		</tr>
<tr>
<td>2</td>
			<td>98°C</td>
			<td>10 秒</td>
			<td></td>
		</tr>
<tr>
<td>3</td>
			<td>68 °C</td>
			<td>30 秒</td>
			<td></td>
		</tr>
<tr>
<td>4</td>
			<td>72°C</td>
			<td>15 秒</td>
			<td>10 个周期（步骤 2-4）</td>
		</tr>
<tr>
<td>5</td>
			<td>72°C</td>
			<td>2 分钟</td>
			<td></td>
		</tr>
<tr>
<td>6</td>
			<td>4 °C</td>
			<td>拿</td>
			<td></td>
		</tr>
</tbody></table>表4:PCR2循环参数。 用于条形码的 PCR 条件放大每个肿瘤样本（直接来自基因组 DNA 或使用 PCR1 的产品作为模板）。

Watch this Scientific Journal Video about In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity at JoVE.com

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

在这里，我们描述了一种快速和直接的体内CRISPR/Cas9筛查方法，使用超声波引导子宫胚胎扁平注射，同时评估几个基因在皮肤和免疫能力小鼠口腔的功能。

在Vivo CRISPR/Cas9筛查中同时评估小鼠皮肤和口腔中的基因功能

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

in-vivo-crisprcas9-screening-to-simultaneously-evaluate-gene-function

Nanyang Technological University

Research

JoVE Journal

Genetics

6.4K 次观看.  Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital. 在这里，我们描述了一种快速和直接的体内CRISPR/Cas9筛查方法，使用超声波引导子宫胚胎扁平注射，同时评估几个基因在皮肤和免疫能力小鼠口腔的功能。

视频: In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

用荧光PCR毛细管电泳技术在基因型CRISPR / Cas9介导的敲除突变体的高通量形式

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

科研

遗传学

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to generate RNA-guided nucleases, such as CRISPR-associated (Cas) 9 for site-specific eukaryotic genome editing. Genome engineering by Cas9 is used to efficiently, easily and robustly modify endogenous genes in many biomedically-relevant mammalian cell lines and organisms. Here we show an example of how to utilize the CRISPR/Cas9 methodology to understand the biological function of specific genetic mutations. We model calreticulin (CALR) mutations in murine interleukin-3 (mIL-3) dependent pro-B (Ba/F3) cells by delivery of single guide RNAs (sgRNAs) targeting the endogenous Calr locus in the specific region where insertion and/or deletion (indel) CALR mutations occur in patients with myeloproliferative neoplasms (MPN), a type of blood cancer. The sgRNAs create double strand breaks (DSBs) in the targeted region that are repaired by non-homologous end joining (NHEJ) to give indels of various sizes. We then employ the standard Ba/F3 cellular transformation assay to understand the effect of physiological level expression of Calr mutations on hematopoietic cellular transformation. This approach can be applied to other genes to study their biological function in various mammalian cell lines.

Targeted gene editing using CRISPR/Cas9 has greatly facilitated the understanding of the biological functions of genes. Here, we utilize the CRISPR/Cas9 methodology to model calreticulin mutations in cytokine-dependent hematopoietic cells in order to study their oncogenic activity.

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

癌症研究

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

小鼠卵母细胞在减数分裂过程中对人类基因功能的评估

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian cells, this technology represents a novel means to perform genome-wide genetic screens for functional genomics studies. Libraries of guide RNAs (sgRNA) targeting all open reading frames permit the facile generation of thousands of genetic perturbations in a single pool of cells that can be screened for specific phenotypes to implicate gene function and cellular processes in an unbiased and systematic way. CRISPR-Cas screens provide researchers with a simple, efficient, and inexpensive method to uncover the genetic blueprints for cellular phenotypes. Furthermore, differential analysis of screens performed in various cell lines and from different cancer types can identify genes that are contextually essential in tumor cells, revealing potential targets for specific anticancer therapies. Performing genome-wide screens in human cells can be daunting, as this involves the handling of tens of millions of cells and requires analysis of large sets of data. The details of these screens, such as cell line characterization, CRISPR library considerations, and understanding the limitations and capabilities of CRISPR technology during analysis, are often overlooked. Provided here is a detailed protocol for the successful performance of pooled genome-wide CRISPR-Cas9 based screens.

CRISPR-Cas9 technology provides an efficient method to precisely edit the mammalian genome in any cell type and represents a novel means to perform genome-wide genetic screens. A detailed protocol discussing the steps required for the successful performance of pooled genome-wide CRISPR-Cas9 screens is provided here.

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

通过显微注射卵母细胞生成转基因小鼠

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

发育生物学

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

CRISPR/Cas9核糖核酸蛋白在急性骨髓性白血病细胞中的RUNX1电子消光器的转录作用研究

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Cell Surface Receptor Identification Using Genome-Scale CRISPR/Cas9 Genetic Screens

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and functioning of multicellular organisms. Detecting these interactions remains technically challenging, however. This manuscript describes a systematic genome-scale CRISPR/Cas9 knockout genetic screening approach that reveals cellular pathways required for specific cell surface recognition events. This assay utilizes recombinant proteins produced in a mammalian protein expression system as avid binding probes to identify interaction partners in a cell-based genetic screen. This method can be used to identify the genes necessary for cell surface interactions detected by recombinant binding probes corresponding to the ectodomains of membrane-embedded receptors. Importantly, given the genome-scale nature of this approach, it also has the advantage of not only identifying the direct receptor but also the cellular components that are required for the presentation of the receptor at the cell surface, thereby providing valuable insights into the biology of the receptor.

This manuscript describes a genome-scale cell-based screening approach to identify extracellular receptor-ligand interactions.

使用基因组-规模CRISPR/Cas9基因屏幕进行细胞表面受体识别

Intercellular communication mediated by direct interactions between membrane-embedded cell surface receptors is crucial for the normal development and ...

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors of the diseases. To validate the contribution of a dysfunction of the mutated genes to disease development, the generation of animal models carrying the mutations is one obvious approach. While germline genetically engineered mouse models (GEMMs) are popular biological tools and exhibit reproducible results, it is restricted by time and costs. Meanwhile, non-germline GEMMs often enable exploring gene function in a more feasible manner. Since some brain diseases (e.g., brain tumors) appear to result from somatic but not germline mutations, non-germline chimeric mouse models, in which normal and abnormal cells coexist, could be helpful for disease-relevant analysis. In this study, we report a method for the induction of CRISPR-mediated somatic mutations in the cerebellum. Specifically, we utilized conditional knock-in mice, in which Cas9 and GFP are chronically activated by the CAG (CMV enhancer/chicken &#223;-actin) promoter after Cre-mediated recombination of the genome. The self-designed single-guide RNAs (sgRNAs) and the Cre recombinase sequence, both encoded in a single plasmid construct, were delivered into cerebellar stem/progenitor cells at an embryonic stage using in utero electroporation. Consequently, transfected cells and their daughter cells were labeled with green fluorescent protein (GFP), thus facilitating further phenotypic analyses. Hence, this method is not only showing electroporation-based gene delivery into embryonic cerebellar cells but also proposing a novel quantitative approach to assess CRISPR-mediated loss-of-function phenotypes.

CRISPR-mediated Loss of Function Analysis in Cerebellar Granule Cells Using In Utero Electroporation-based Gene Transfer

Conventional loss-of-function studies of genes using knockout animals have often been costly and time-consuming. Electroporation-based CRISPR-mediated somatic mutagenesis is a powerful tool to understand gene functions in vivo. Here, we report a method to analyze knockout phenotypes in proliferating cells of the cerebellum.

CRISPR 介导的小脑颗粒细胞在宫内电穿孔基因转移中的功能分析损失

Brain malformation is often caused by genetic mutations. Deciphering the mutations in patient-derived tissues has identified potential causative factors ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

受精卵显微注射用于在小鼠中产生基因盒敲入和絮状等位基因

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

Evaluation of Abnormal Growth-related Genes of Hematopoietic Stem and Progenitor Cells by Combining CRISPR/Cas9 Technology with Cell Counting

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. However, the accumulation of cancerous mutations in hematopoietic stem and progenitor cells (HSPCs) can block normal differentiation, induce aberrant proliferation, and ultimately lead to leukemogenesis. To identify and/or evaluate the cancerous mutations, we integrated CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to investigate a model gene Trp53, that is essential for abnormal proliferative ability of HSPCs. Specifically, bone marrow cells enriched for HSPCs from Cas9 mice were harvested and then subjected to viral transfection with single-guide RNAs (sgRNAs) targeting one or several candidate genes to introduce genetic alterations in HSPCs. Then, a CCK-8 assay was performed to investigate the proliferative capacity of transfected HSPCs. The sgRNA targeting efficiency was confirmed by a Tracking of Indels by Decomposition assay. These identified genes may play crucial roles in leukemogenesis and could serve as potential therapeutic targets.

Here, we present a protocol that integrates CRISPR/Cas9 technology with the Cell Counting Kit-8 (CCK-8) assay to identify candidate genes that are crucial for the abnormal proliferative capacity of hematopoietic stem and progenitor cells.

将 CRISPR/Cas9 技术与细胞计数相结合评估造血干细胞和祖细胞生长相关基因的异常

Hematopoietic stem cells possess the ability for long-term self-renewal and the potential to differentiate into various types of mature blood cells. ...