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><tbody><tr><td>CRISPRevolution sgRNA&amp;nbsp; EZ Kit</td><td>Synthego Inc.</td><td></td><td>chimeric sgRNA</td></tr><tr><td>Nuclease-free TE</td><td>Synthego Inc.</td><td>provided with the sgRNA kit EZ kit</td><td></td></tr><tr><td>Nuclease-free water</td><td>Synthego Inc.</td><td>provided with the sgRNA kit EZ kit</td><td></td></tr><tr><td>4 nmole Ultramer DNA Oligo</td><td>Integrated DNA Technologies</td><td></td><td>ssODN HDR template</td></tr><tr><td>Alt-R S.p. HiFi Cas9 Nuclease 3NLS</td><td>Integrated DNA Technologies</td><td>1078728</td><td>Cas9 protein</td></tr><tr><td>pCFJ90&amp;nbsp;</td><td>Addgene</td><td>19327</td><td>Pmyo-2::mCherry Marker Plasmid</td></tr><tr><td>KCl</td><td>Sigma</td><td>P5405</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034</td><td></td></tr><tr><td>DNA Clean &amp;amp; Concentrator</td><td>Zymo Research</td><td>D4004</td><td></td></tr><tr><td>Zymoclean Gel DNA Recovery Kit</td><td>Zymo Research</td><td>D4002</td><td></td></tr><tr><td>Q5 Hot Start High-Fidelity 2X Master Mix</td><td>New England Biolabs</td><td>M0494L</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P2308</td><td></td></tr><tr><td>Glass Borosilicate Glass Micropipettes</td><td>Sutter Instruments</td><td>BF100-78-10</td><td>OD: 1.0mm. ID: 0.78mm</td></tr><tr><td>Trizma Hydrochloride</td><td>Sigma</td><td>T5941</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>M2393</td><td></td></tr><tr><td>NP-40</td><td>Sigma</td><td>74385</td><td></td></tr><tr><td>Tween-20</td><td>Fisher Scientific</td><td>BP337-100</td><td></td></tr><tr><td>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 ...

a-rapid-facile-pipeline-for-generating-genomic-point-mutants-c

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

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

科研

发育生物学

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 ...

遗传学

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

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

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

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

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

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 ...

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 ...

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 ...

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been harnessed by researchers to study important biologically relevant problems. The unparalleled power of the CRISPR/Cas genome editing method allows researchers to precisely edit any locus of their choosing, thereby facilitating an increased understanding of gene function. Several methods for editing the C. elegans genome by CRISPR/Cas9 have been described previously. Here, we discuss and demonstrate a method which utilizes in vitro assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. Specifically, in this article, we go through the step-by-step process of introducing premature stop codons into the C. elegans rbm-3.2 gene by homology-directed repair using this method of CRISPR/Cas9 editing. This relatively simple editing method can be used to study the function of any gene of interest and allows for the generation of homozygous-edited C. elegans by CRISPR/Cas9 editing&#160;in less than two weeks.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

The goal of this protocol is to enable seamless CRISPR/Cas9 editing of the C. elegans genome using assembled ribonucleoprotein complexes and the dpy-10 co-CRISPR marker for screening. This protocol can be used to make a variety of genetic modifications in C. elegans including insertions, deletions, gene replacements and codon substitutions.

CRISPR/Cas9 使用dpy-10联合 CRISPR 筛选标记和组装的核糖核蛋白复合物编辑C. elegans rbm-3.2基因。

The bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated protein (Cas) system has been ...

Construction of Homozygous Mutants of Migratory Locust Using CRISPR/Cas9 Technology

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an important research model for insect metamorphosis. The CRISPR/Cas9 system can accurately locate at a specific DNA locus and cleave within the target site, efficiently introducing double-strand breaks to induce target gene knockout or integrate new gene fragments into the specific locus. CRISPR/Cas9-mediated genome editing is a powerful tool for addressing questions encountered in locust research as well as a promising technology for locust control. This study provides a systematic protocol for CRISPR/Cas9-mediated gene knockout with the complex of Cas9 protein and single guide RNAs (sgRNAs) in migratory locusts. The selection of target sites and design of sgRNA are described in detail, followed by in vitro synthesis and verification of the sgRNAs. Subsequent procedures include egg raft collection and tanned-egg separation to achieve successful microinjection with low mortality rate, egg culture,&#160;preliminary estimation of the mutation rate, locust breeding as well as detection, preservation, and passage of the mutants to ensure population stability of the edited locusts. This method can be used as a reference for CRISPR/Cas9 based gene editing applications in migratory locusts as well as in other insects.

This study provides a systematically optimized procedure of CRISPR/Cas9 ribonuclease-based construction of homozygous locust mutants as well as a detailed method for cryopreservation and resuscitation of the locust eggs.

基于CRISPR/Cas9技术构建迁徙蝗虫纯合突变体

The migratory locust, Locusta migratoria, is not only one of the worldwide plague locusts that caused huge economic losses to human beings but also an ...

生物学

Single Worm PCR: A Method to Extract and Amplify Genomic DNA

Source: Prior, H., et al. <a href="https://www.jove.com/video/57518/a-rapid-facile-pipeline-for-generating-genomic-point-mutants-c">A Rapid and Facile Pipeline for Generating Genomic Point Mutants in&#160;C. elegans&#160;Using CRISPR/Cas9 Ribonucleoproteins.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes a technique to rapidly screen a single nematode for a genetic marker by PCR screening. In the example protocol, we screen for CRISPR-based genome editing.