没有与本报告有关的利益冲突。

所有动物关心和实验规程跟随了指南从国立卫生研究院和机构动物关心和使用委员会 (IACUC) 在密执安大学。在整个协议中使用无 RNase 的解决方案和吸管提示。按照制造商指南 (请参阅材料表), 清洁工作区、吸管、管子和离心机, RNase 净化解决方案。
1. sgRNA 目标选择
<ol><li>使用 web 浏览器打开网页: http://crispor.tefor.net8<ol>
<li>输入 ~ 60 基对 (bp) 的序列侧翼所需的编辑兴趣 (即30 bp 5 ' 和 3 ' 的预期编辑)。</li>
		<li>从下拉菜单中选择秀丽线虫基因组。</li>
		<li>从下拉菜单中选择 Protospacer 相邻的主题 (20 bp-NGG-SpCas9、SpCas9-HF1、eSPCas9 1.1)。</li>
		<li>单击 "提交"。在 "结果" 页中, 选择最接近编辑兴趣的排名最高的目标序列。如果在侧翼 60 bp 输入序列中没有合适的目标站点, 请将查询序列扩展到 100 bp (即50 bp 在所需编辑的任意一侧)。</li>
	</ol>
</li>
	<li>从可用源 (例如synthego) 获得一个20的目标 sgRNA, 其规格如下: 3 nmol;没有修改。</li>
	<li>在收到后, 离心机冻干 sgRNA 含管在 > 1.2万 x g 1 分钟室温。添加60µL 的核酸酶的管, 并轻轻地重新悬浮吹打向上和向下 15-20 次使用 P200 吸管。最后浓度为50µM。</li>
</ol>2. 同源导向修复模板设计
<ol><li>设计一个 ssODN HDR 模板, 其中包含: 1) 兴趣的变异, 2) 一个独特的框架内限制限制性站点, 3) NGG PAM 序列中的一个或两个 Gs 的无声突变, 4) 50 bp 5 ' 和 3 ' 同源武器侧翼的第一个和最后一个突变。(图 1C)9,10。<ol>
<li>如果 NGG PAM 序列的突变是不可能的, 在 sgRNA 目标识别序列中引入 5-6 个无声突变, 以防止 sgRNA 介导的 ssODN HDR 模板的分裂。</li>
	</ol>
</li>
	<li>从可用的源例如idtdna 获得一个具有以下参数的 "Ultramer DNA 寡核苷酸": 比例: 4 nmol;配方: 无;净化: 标准脱盐。</li>
	<li>在收到后, 离心机冻干 ssODN 含管在 > 1.2万 x g 1 分钟室温。使用吹打吸管, 轻轻地将 ssODN 重新挂起至100µM 的最后浓度 (核酸酶 ddH 2 O), P200 15-20 倍.</li>
</ol>3. 设计基因分型底漆
<ol><li>设计一个在基因组修改侧翼的引物集, 以生成一个〜 400-700 bp PCR 扩增子11。</li>
	<li>优化 PCR 循环条件以生成单个和特定的扩增子11。</li>
</ol>4. 准备注塑混合料
<ol><li>离心机表 1试剂的最大速度为2分钟4摄氏度。</li>
	<li>按照列出的顺序, 将表 1试剂添加到无核酸酶的 PCR 管中。</li>
	<li>用 P20 吸管轻轻吹打上下10次, 彻底混合。</li>
	<li>在室温下孵育注射混合剂10分钟。</li>
</ol>5. 注塑协议
<ol><li>加载注入微, 大约有一半的注入混合12。在注射针头堵塞或断裂时, 保存剩余的注射组合。</li>
	<li>中断微提示, 使 ~ 20-30 每平方产生一个渐进流动的解决方案 12. 注: 较大的直径提示对动物健康有负面影响, 降低了1子代产量。</li>
	<li>如果可能的话, 在两种性腺武器中注射 10-15 只幼虫 (12)。如果不是, 注射入一个性腺胳膊是充足的。</li>
	<li>允许注射动物 (P0) 在 OP50-seeded 35 毫米线虫生长培养基 (NGM) 板上的室温下恢复 1-2 小时。随后, 单一注入 P0动物到单独 OP50-seeded 35 毫米 NGM 板使用白金线蠕虫挑选12。</li>
</ol>6. 屏幕 P0板和单 mCherry (+) F1s
<ol><li>注射后两天, 识别含有 F1子代的 P0板, 使用荧光显微镜 (图 1D, 天 2-3) 表示咽 mCherry。选择0板 , 其中包含最 mCherry ( + ) F1动物。</li>
	<li>从三选择的 P0板块, 单 8-12 mCherry (+) f1动物总计24-36 个 mCherry (+) f1s 使用蠕虫拾取 (图 1D, 天 2-3)。 注: mCherry (-) 动物也可以被单独从这些 P0板块, 但识别正确编辑的动物的可能性要低得多 (图 2A)。</li>
	<li>允许单个 F1s 自受精, 在室温下产卵 1-2 天。</li>
</ol>7. 单蠕虫 PCR 和基因分型
<ol><li>在产卵 1-2 天后, 使用蠕虫拾取 (表 2,图 1D, 天 4-5) 将 F1转换为单独的 PCR 带管帽, 其中含有7µL 的蠕虫裂解缓冲器。</li>
	<li>离心 PCR 管以最大速度在室温下1分钟将动物带到管底, 并在-80 摄氏度冷冻管1小时。 注意: 蠕虫可以无限期地存储在-80 摄氏度。</li>
	<li>溶解冷冻蠕虫在一个 thermocycler 使用以下程序:60 °c 为60分钟, 95 °c 为15分钟, 4 °c 举行。</li>
	<li>设置 PCR mastermix, 如表 3所示。</li>
	<li>将 pcr mastermix 的21µL 添加到干净的 pcr 管中, 然后从步骤3中添加4µL 的蠕虫裂解。混合使用吸管和运行 PCR 程序后, 制造商的指导方针 (见材料表)。</li>
	<li>根据制造商的说明 (请参阅材料表), 使用 DNA 清洁浓缩试剂盒纯化 PCR 反应。洗脱 DNA 通过增加10µL 水的旋转柱。</li>
	<li>设置限制酶 mastermix, 如表 4所示。</li>
	<li>添加10µL 的限制酶 mastermix 的每一个清洁 PCR 反应和孵化 1-2 小时在37摄氏度13。</li>
	<li>使用1x 三乙酸乙酯-EDTA (泰) 缓冲器14, 在1.5% 琼脂糖凝胶上分离消化 PCR 产物, 运行于 120 V。</li>
</ol>8. 被编辑的动物的识别和序列验证
<ol><li>检查酶消化样品是否存在表明可能被编辑的动物14 (图 1D, 天 4-5) 的带。 注: 加载一个 N2 控制, 以确定潜在的 indel 突变, 可以观察到的变化, 在带大小和/或意外和复杂的限制酶消化带状模式。</li>
	<li>在鉴定可能的被编辑的动物之后, 使用剩余的3µL 蠕虫裂解并且重复 PCR 反应。不清除或限制酶消化 PCR 反应后, 程序完成。在1% 琼脂糖凝胶上加载 PCR 反应, 用凝胶萃取套件15分离和提取带 (参见材料表)。</li>
	<li>桑格序列16凝胶提取 DNA 使用 F1 底漆识别正确编辑的动物 (图 1A) 注: 杂合子读将包含覆盖的峰值由于存在的野生类型和工程的等位基因。</li>
	<li>从经验证的异型编辑动物中获得纯合动物, 单 8-12 F2动物并执行步骤 7-8。</li>
</ol>

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我们感谢乞求实验室的成员对这篇手稿的批判性阅读。菌株由由 NIH 研究基础设施项目办公室 (P40 OD010440) 资助的秀丽遗传学中心提供。在乞讨实验室的研究得到了 NIH (R01 NS094678) 和肌肉营养不良协会 (MDA382300) 提供的资助, 以 A.A.B。

CRISPR-Cas9 系统是精确修改模型生物体基因组的有力而有效的工具。在这里, 我们演示了嵌合体 sgRNAs20与 ssODN HDR 模板结合在一起, 使得在C. 线虫中高效地生成基因组点突变。重要的是, 我们表明, RNP 交付产生高编辑效率时, 荧光作为一个联合选择标记, 突出的易用性和可靠性的技术。
C. 线虫基因组工程的大多数 CRISPR-Cas9 方法都依赖于特定的遗传背?...

最近的技术进步已经从根本上改变并加速了精确地设计基因组的能力。特别是, 依靠 RNA 引导的限制性 Cas9 在目标序列附近诱导双链断裂 (CRISPR-Cas9) 的系统, 已被广泛地用于精确地设计大多数模型生物体的基因组, 用于生物医学研究1,2,3,4。重要的是, 即使在诸如线虫5这样的困难物种中, CRISPR-Cas9 的使用也没有锁定基因组编辑。无论物种, 产生点突变与基于 CRISPR-Cas9 的基因组编辑系统依赖于三核心组件: 1) Cas9 限制性, 2) 一个单一的指南 RNA (sgRNA), 指示 Cas9 限制性到目标序列, 3) 用户设计同源定向修复 (HDR) 模板, 其中包含所需的感兴趣的编辑2。
有几种方法可用于将靶向 sgRNA 和 Cas9 核酸酶引入细胞, 包括质粒、RNA 和基于病毒的传递方法 6.最近, 在 CRISPR-Cas9-based 基因组编辑7中, 直接交付预复合 sgRNA-Cas9 Ribonucleoproteins (RNPs) 已成为一个强大而有效的工具。预复杂 CRISPR-Cas9 RNPs 的直接传递有几个明显的优点, 即: 1) RNPs 绕过细胞转录和翻译的需要, 2) RNPs 迅速清除, 这可能会增加特异性通过减少可用时间离靶解理和 3) RNPs 不包含外来的 DNA/RNA 元素, 通过随机积分绕过非本机序列引入宿主基因组。在一起, 这些属性可能会提供一个短暂的 CRISPR 的目标编辑, 同时尽量减少目标效果。
我们描述了一种简单有效的协议, 用于在C. 线虫中引入特定于站点的基因组变化。该协议包括目标 sgRNA 和单链寡核苷酸 (ssODN) HDR 模板设计, sgRNA-Cas9 RNP 络合和交付, 以及一个基因分型策略, 以明确识别正确编辑的动物。使用此策略, 不仅可以恢复所需的站点特定更改, 而且还可以恢复其他非特定的 indel 突变。因此, 我们的策略允许使用单一的策略生成一个二等位序列, 在这个方法中, 可以在 F1生成中生成单位、双位和 indel 突变体。

<table border="0" cellpadding="0" cellspacing="0" width="1144">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">CRISPRevolution sgRNA&#160; EZ Kit</td>
			<td style="width:216px;">Synthego Inc.</td>
			<td style="width:344px;"></td>
			<td style="width:227px;">chimeric sgRNA</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free TE</td>
			<td>Synthego Inc.</td>
			<td>provided with the sgRNA kit EZ kit</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nuclease-free water</td>
			<td>Synthego Inc.</td>
			<td>provided with the sgRNA kit EZ kit</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">4 nmole Ultramer DNA Oligo</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>ssODN HDR template</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alt-R S.p. HiFi Cas9 Nuclease 3NLS</td>
			<td>Integrated DNA Technologies</td>
			<td>1078728</td>
			<td>Cas9 protein</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">pCFJ90&#160;</td>
			<td>Addgene</td>
			<td>19327</td>
			<td>Pmyo-2::mCherry Marker Plasmid</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KCl</td>
			<td>Sigma</td>
			<td>P5405</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNA Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>D4004</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Q5 Hot Start High-Fidelity 2X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0494L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Proteinase K</td>
			<td>Sigma</td>
			<td>P2308</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass Borosilicate Glass Micropipettes</td>
			<td>Sutter Instruments</td>
			<td>BF100-78-10</td>
			<td>OD: 1.0mm. ID: 0.78mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma Hydrochloride</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MgCl2</td>
			<td>Sigma</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">NP-40</td>
			<td>Sigma</td>
			<td>74385</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tween-20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">RNaseZap Decontamination Solution</td>
			<td>Fisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rapid and facile pipeline for generating genomic point mutants in c. elegans using crispr/cas9 ribonucleoproteins

聚簇定期穿插回文重复 (CRISPR)-CRISPR 相关蛋白 9 (Cas9) 原核自适应免疫防御系统已被联合选择作为一个强大的工具, 精确的真核基因组工程。在这里, 我们提出了一个快速和简单的方法, 使用嵌合体单导 rna (sgRNA) 和 CRISPR-Cas9 Ribonucleoproteins (RNPs), 以有效和精确地产生基因组点突变的C. 线虫。我们描述了一个 sgRNA 目标选择的管道, 同源定向修复 (HDR) 模板设计, CRISPR-Cas9-RNP 络合和交付, 以及一个基因分型策略, 使正确编辑的动物的健壮和快速识别。我们的方法不仅允许简单的生成和识别所需的基因突变动物, 而且还有助于在大约 4-5 天内检测其他复杂的 indel 等位基因, 效率高, 筛选工作量减少。

人类超氧化物歧化酶 1 (SOD1) 的突变占家庭肌萎缩侧索硬化症的 10-20%, 这是一种破坏性的神经退行性疾病, 总是导致瘫痪和死亡17。人类 SOD-1 是一个进化保守的蛋白质共享55% 的身份和70% 相似性与线虫SOD-1 蛋白 (图 1B)。为了演示 CRISPR-Cas9 RNP 方法的简便性、可行性和有效性, 我们针对sod-1 线虫基因引入了蠕虫基因组?...

Watch this Scientific Journal Video about A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins at JoVE.com

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

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

在这里, 我们提出了一个方法, 以工程的基因组的C. 线虫使用 CRISPR-Cas9 ribonucleoproteins 和同源依赖修复模板。

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

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Research

JoVE Journal

Genetics

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

7.5K 次观看.  University of Michigan. 在这里, 我们提出了一个方法, 以工程的基因组的C. 线虫使用 CRISPR-Cas9 ribonucleoproteins 和同源依赖修复模板。

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

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

科研

遗传学

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 ...

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 ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the African clawed frog, Xenopus, a longstanding model organism for biomedical research. Traditional breeding schemes for creating homozygous mutant lines with CRISPR/Cas9-targeted mutagenesis have several time-consuming and laborious steps. To facilitate the creation of mutant embryos, particularly to overcome the obstacles associated with knocking out genes that are essential for embryogenesis, a new method called leapfrogging was developed. This technique leverages the robustness of Xenopus embryos to &#34;cut and paste&#34; embryological methods. Leapfrogging utilizes the transfer of primordial germ cells (PGCs) from efficiently-mutagenized donor embryos into PGC-ablated wildtype siblings. This method allows for the efficient mutation of essential genes by creating chimeric animals with wildtype somatic cells that carry a mutant germline. When two F0 animals carrying &#34;leapfrog transplants&#34; (i.e., mutant germ cells) are intercrossed, they produce homozygous, or compound heterozygous, null F1 embryos, thus saving a full generation time to obtain phenotypic data. Leapfrogging also provides a new approach for analyzing maternal effect genes, which are refractory to F0 phenotypic analysis following CRISPR/Cas9 mutagenesis. This manuscript details the method of leapfrogging, with special emphasis on how to successfully perform PGC transplantation.

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Genes essential for survival pose technical hurdles for creating mutant lines. Leapfrogging circumvents lethality by combining genome editing with primordial germ cell transplantation to create wild-type animals carrying germline mutations. Leapfrogging also permits the efficient generation of homozygous null mutants in the F1 generation. Here, the transplantation step is demonstrated.

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

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 ...

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 ...

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. ...

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 ...