作者想感谢珍妮特Schermuly编制重组的T7 RNA聚合酶和诺曼Ebelt的协助编制图1。这项工作是由德意志研究联合会（DFG FOR1680）和马普学会的资金。

1。代的长前crRNA的底物，通过PCR <ol><li>设计PCR引物定位的CRISPR群集的间隔区。添加的T7 RNA聚合酶（T7RNAP）的启动子序列（5&#39;-taatacgactcactata-3&#39;）的正向引物和用于克隆的限制性位点的两种引物（例如，BamHⅠ和HindⅢ酶切的pUC19， 图2A到载体中的PCR产物） 。 注:T7RNAP需要适当的转录起始的一个胍残留物。 </li><li>放大您的预crRNA的PCR从基因组DNA序列的。 </li><li>分离PCR产物通过琼脂糖凝胶电泳，凝胶中提取所需的频带。用限制酶消化PCR产物，创建粘性末端（例如BamHI和HindIII， 图2A）。你的PCR产物用PCR纯化试剂盒纯化，以消除裂解的副产物。 </li><li>设置了连接反应，含有T4 DNA连接酶，T4 DNA连接酶缓冲液和切割的PCR产物和相应的粘性末端的脱磷酸化的线性pUC载体的摩尔比为3:1。在16°C过夜孵育混合物。变换连接混合物通过标准的协议和主管大肠杆菌 DH5α细胞，用蓝白斑筛选，找出成功的结扎。 </li><li>隔离质粒的白色菌落，用质粒制备试剂盒。确定阳性克隆质粒测序。另外，菌落PCR可能会被用于筛查。 </li></ol> 2。代中间预crRNA基板的，通过退火的DNA寡核苷酸<ol><li>设计正向和反向所需的CRISPR重复/间隔区序列的寡核苷酸。该寡核苷酸含有序列的T7 RNA聚合酶启动子，以及终端的限制性位点（例如，BamHⅠ和HindⅢ酶切的pUC19）。终止的寡核苷酸，以确保粘性末端形式后退火的（参见图2B </s仲&gt;）。 </li><li> 5&#39;-磷酸化1 nmol的每个寡核苷酸含有5微升T4多核苷酸激酶（PNK），2微升T4 PNK 10x缓冲液，2微升ATP（10毫摩尔）在一个单独的20μl反应。孵育各样品在37℃下进行1小时</li><li>杂交的两个磷酸化的寡核苷酸。结合1μl的前进的磷酸化寡核苷酸的混合物（从2.2），1微升的磷酸化的反向寡核苷酸混合物（从2.2），1微升T4 DNA连接酶10x缓冲液在10微升反应。孵育5分钟的样品在95℃C对加热块或在沸水中，关闭热源，并让该混合物冷却至室温（约2-3小时）。 注意:在这个关键的步骤中，在缓慢冷却过程有利于相比，每个单独的寡核苷酸形成的结构内的两个寡核苷酸退火。 </li><li>结扎4微升杂交组合，1μl的消化和去磷酸化的pUC载体（0.1微克/微升）用T4 DNA连接酶，T4 DNA连接酶10x缓冲液和20微升连接混合物中的10mM ATP。在16°C过夜孵育样品。 </li><li>结扎的质粒转化感受态大肠杆菌 DH5α细胞通过标准的协议和利用蓝白色的筛选。分离质粒和消化（筛选用于插入所需的大小）和随后的质粒测序确定阳性克隆。 </li></ol> 3。通过自定义的RNA寡核苷酸合成的短CAS RNA底物的代设计短期CAS RNA底物（例如，单一的重复序列， 图2C）和利用自定义的RNA寡核苷酸合成设施。 注:列入一个脱氧核糖核苷酸的RNA寡核苷酸（ 图2C）在指定的位置，可以用来查明RNA切割的部位。 4， 在体外T7 RNA聚合酶转录<ol><li>隔离质粒与设计的结构（1.9或2.7）。，使用maxiprep质粒纯化试剂盒。 </li><li>线性化的质粒用限制性内切酶切割的片段（例如HindⅢ位）的下游。确保完全消化。 注:如果发散定义的RNA转录物的3&#39;末端的需要的话，所设计的构造应包含一个附加的特定的限制位点为"运行"转录的HindⅢ序列的上游。 </li><li>纯化的线性化的质粒用酚:氯仿（1:1）提取和乙醇沉淀。恢复通过将沉淀重新悬浮于DEPC处理过的无菌水的核酸。 </li><li>设置的T7 RNA聚合酶体外流掉的转录混合物，该混合物含有40毫摩尔的Hepes / KOH（pH 8）中，22毫摩尔MgCl 2，5mM的二硫苏糖醇，1mM亚精胺，4mM的每个核苷三磷酸（ATP，CTP，GTP，UTP ），40〜100微克/毫升的消化质粒和0.1毫克/毫升于DEPC处理过的水的T7 RNA聚合酶。孵育在37℃下进行3小时</li><li>分析得到的RNA转录变性8M尿素的12％聚丙烯酰胺凝胶（ 图3A）。通过Mono Q阴离子交换色谱法13可被纯化的RNA转录的RNA成分的沉淀和再悬浮于DEPC处理的无菌水，通过乙醇沉淀回收。以供将来使用，存储的RNA在-80℃下</li></ol> 5。 Cas6核酸内切酶分析<ol><li>建立一个 运行20微升T7 RNA聚合酶体外转录混合物（见4.4），它包含一个减少量为2mM ATP，并辅以2.5微升的α-[32 P]-ATP（10毫居里/毫升，5000次/毫摩尔）。通过变性8 M尿素12％聚丙烯酰胺凝胶的凝胶提取，纯化反应产物。可视化带的放射自显影。 </li><li>生产和净化目的的核证机关的蛋白质。在这个例子中，CAS6 产气荚膜梭菌嗜热通过热沉淀和Ni-NTA柱层析纯化。 </li><li>设置核酸内切酶测定反应（例如， 梭状芽孢杆菌嗜热 Cas6，反应混合物含有20毫摩尔的Hepes（KOH PH8），250 mM KCl中，2毫摩尔的MgCl 2，1mM DTT的，12000每分钟RNA底物和1μM酶液于37℃下30分钟）。 </li><li>装入5μl的反应混合物（+ 10微升RNA载样缓冲液含95％甲酰胺），8 M尿素12％聚丙烯酰胺凝胶上。可视化的裂解产物电泳后，用放射自显影。 </li></ol> 6。代表性的成果在图3A示出一个例子的RNA底物CAS核酸内切酶活性的分析。分析体外转录反应中加入100μl的5微升的等分试样进行加载。请注意，在RNA的生产效率的不同的结构之间的变化。有些因素吨帽子被观察到的影响后得到的RNA的量是（i）的初始序列+1 G的转录起始所需的，（ⅱ）的可能性转录时RNA结构的形成，及（iii）的限制位点的选择用于生成运行乳沟位置。 RNA核酸内切酶活性的调查，需要两个高度纯化的重组CAS蛋白（ 图3B）和适当的阴性对照。在理想的情况下，这种阴性对照样品相差尽可能少从调查的CAS核酸内切酶反应。这可以通过以下来实现的RNA用无CAS表达式（及之后的相同的纯化步骤）的反应缓冲液和细胞裂解液中的孵育。一个理想的阴性对照是在建议的裂解位点的脱氧核糖核苷酸的添加。在图3C中，5&#39;端标记的重复序列的裂解的梭状芽胞杆菌嗜热 Cas6所示。下<html>相同的条件下，这种重复是不基板了当一个脱氧核糖核苷酸位-9引入。此方法还提供了有关的裂解位点的信息。最后，一​​个长的内部标记的预crRNA裂解Cas6观察两个裂解片段。 <img alt="图1" src="/files/ftp_upload/4277/4277fig1.jpg" /> 图1。CRISPR / CAS活动的总体示意图。概述如下到CRISPR群集（适应），由Cas6核酸内切酶的转录和加工成小crRNAs CRISPR阵列的，摄取的crRNAs成梯级络合物和干扰的的病毒DNA的序列（protospacer）的插入重复上之间的互补crRNA和protospacer的病毒攻击。 Protospacer相邻图案（PAM）标记的的病毒protospacer序列。 IGURE"SRC ="/ files/ftp_upload/4277/4277fig2.jpg"/&gt; 图2。RNA底物的CAS蛋白的生成。该计划产生的工作流（A）长前crRNA的基板，（B）中间的前crRNA基板和（C）的简称CAS RNA底物，为前crRNA生产。例嗜热梭状芽孢杆菌的的CRISPR阵列的序列。 <a href="http://www.jove.com/files/ftp_upload/4277/4277fig2large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图3" src="/files/ftp_upload/4277/4277fig3.jpg" /> 图3。Cas6核酸内切酶的测定。 A）甲苯胺蓝染色聚丙烯酰胺凝胶的自定义设计的RNA寡核苷酸（250皮摩尔），并在体外 RNA转录体（5微升的一个典型的100μl反应）。 B.）SDS-PAGE凝胶的Cas6准备（80皮摩尔） 产气荚膜梭菌嗜热后热沉淀，在50℃1小时，Ni-NTA柱色谱法。 C.）检测的的信使Cas6活动5&#39;末端重复序列标记和预crRNA的体外转录。 -9位的dNTP的引入废除Cas6裂解短的CAS的RNA底物的（S， 图2C）。 A 5&#39;终端8个核苷酸的标记也产生为crRNA成熟从长期预crRNA基板（L， 图2A）。变性8 M尿素12％聚丙烯酰胺凝胶上分离的条带，并通过放射自显影可视化。

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没有利益冲突的声明。

所提出的方法使产生的CAS核酸内切酶底物不同尺寸范围不同序列设计的自由。用于产生合成的RNA寡核苷酸基板的最直接的方法是有限的短RNA的设计，由于增加了成本和技术的局限性，在创建更长的RNA的低聚物。自定义的RNA合成的实际和经济的最大在于虽然成功RNA合成已被报道用于超过100个核苷酸长度的未修饰RNA的低聚物，在40个核苷酸以下。然而，任何给定的序列可以合成和有针对性地引入经修饰的核苷酸（如脱氧核糖核苷酸），可以被用来分析详细的裂解位点。停止前的cRNA应通过体外转录产生。 含有T7 RNA聚合酶启动子的寡核苷酸退火允许生产的中间长度预crRN为。例行和经济上的合成的DNA寡核苷酸的最大长度仅仅是150个碱基以上，代表最大的合成预crRNAs通过此方法。几个退火，形成粘性末端的DNA寡核苷酸对相互的组装可以延长这个最大的长度，但必须在克隆的构造越来越多的挑战。这种方法的主要优点是生成crRNA预构造（及因此CAS基板）核酸内切酶的能力与任何所需的顺序。这允许测试合成crRNA设计。 最后，可以得到更大的预crRNAs从PCR amplificates整个基因组的CRISPR元素或其馏分。改建的体外转录的模板质粒可以通过位点定向诱变引入用于生成预crRNA变种。这些结构可以被用来分析全球信使核糖核酸图案内的整个前crRNA。 通过T7 RNA聚合酶体外转录的未修饰RNA，用于生产的先驱工作的基础上转移RNA 14，雀麦花叶病毒RNA 15。 T7 RNA聚合酶启动子的共识由识别域（-17至-5）和一个起始域（-4至6）在一个基本的鸟苷1 16的转录起始。设备的持仓+1的下游可以改变，它允许为几乎任何所需的RNA序列的转录。所提出的方法在体外决胜转录模板用于产生允许的完整的预crRNAs的匹配的基因组的的CRISPR区域的或合成的的预crRNA变种在体外合成。转录产生的具有5&#39;-末端三磷酸本除非浸过水的GMP的转录反应，得到5&#39;单磷酸末端14。这种总站的需要，如果是被标记的成绩单用T4多核苷酸激酶和γ-[32 P]-ATP。的Cas6和Cas6样酶的裂解活性生成的crRNA包含5&#39;-羟基和2&#39;，3&#39;-环磷酸末端。然后，这些RNA进行分析，以识别的级联复杂的。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>南极磷酸酶</td><td> NEB </td><td> M0289L </td><td></td></tr><tr><td> 在的体外转录级超纯水三磷酸核苷</td><td>耶拿生物科学</td><td> NU-1010 - NU-1013 </td><td></td></tr><tr><td>液相色谱，ktapurifier 100; </td><td> GE医疗集团</td><td></td><td></td></tr><tr><td> DNA寡核苷酸</td><td> MWG操纵子</td><td></td><td></td></tr><tr><td> RNA寡核苷酸</td><td> MWG操纵子</td><td></td><td></td></tr><tr><td>凝胶提取试剂盒</td><td> QIAGEN </td><td> 28704 </td><td></td></tr><tr><td> PCR纯化试剂盒</td><td> QIAGEN </td><td> 28104 </td><td></td></tr><tr><td> Spin Miniprep试剂盒</td><td> QIAGEN </td><td> 27106 </td><td></td></tr><tr><td>质粒试剂盒</td><td> QIAGEN </td><td> 12163 </td><td></td></tr><tr><td> BamHI位</td><td> NEB </td><td> R0136L </td><td></td></tr><tr><td> HindIII位</td><td> NEB </td><td> R0104L </td><td></td></tr><tr><td> T4 DNA连接酶</td><td> NEB </td><td> M0202S </td><td></td></tr><tr><td> T4多聚核苷酸激酶</td><td> Ambion公司</td><td> AM2310 </td><td></td></tr><tr><td> T7 RNA聚合酶</td><td>自己编写</td><td></td><td></td></tr><tr><td>脱氧解决方案组合</td><td> NEB </td><td> N0447L </td><td></td></tr><tr><td>迷你的PROTEAN利电泳系统</td><td> Bio-Rad公司</td><td> 165-8001 </td><td></td></tr><tr><td>风暴840扫描仪</td><td&gt; GE医疗</td><td> 163723 </td><td></td></tr><tr><td>存储Phosphorscreen </td><td>分子动力学</td><td> 63-0034-81 </td><td></td></tr><tr><td>单Q 5/50 GL </td><td> GE医疗集团</td><td> 17-5166-01 </td><td></td></tr><tr><td> phusion高-Fidelity DNA聚合酶</td><td> NEB </td><td> M0530L </td><td>使用GC缓冲液</td></tr><tr><td>甲苯胺蓝</td><td>西格玛</td><td> T3260 </td><td></td></tr><tr><td> pUC19中</td><td> NEB </td><td> N3041S </td><td></td></tr><tr><td> γ-[32 P]-ATP，α-[32 P]-ATP </td><td>哈特曼分析</td><td> SRP，SRP-401-307 </td><td></td></tr></tbody></table>

substrate generation for endonucleases of crispr/cas systems

病毒和它们的原核宿主的相互作用塑造细菌和古细菌生活的演变。原核生物开发了多种策略逃避病毒攻击，包括限制修饰，流产感染和CRISPR / CAS系统。这些发现在许多细菌和古细菌的适应性免疫系统组成的 C上光ŕegularly，我 nterspaced 短p alindromicŕEPEAT（CRISPR）序列和的 C RISPR数sociated（CAS）的基因（图1）1-3。 CAS蛋白质和重复套不同的定义至少有三大不同类型的CRISPR / CAS系统4。通用的蛋白质CAS1，CAS2，提出要参与病毒DNA的摄取将生成一个新的间隔件元件之间延伸CRISPR簇5的5&#39;末端的两个重复。整个群集被转录到的前体crRNA的含有所有的隔板和重复序列，并随后通过酶处理成更小的crRNAs 6-8多样化Cas6系列。这些crRNAs组成的间隔区序列的5&#39;末端（8个核苷酸）和3&#39;末端标记的重复序列9来自两侧。一个重复感染的病毒现在被封锁的蛋白复合物（级联）作为新的crRNA会被定向到病毒的DNA，将其识别为通过互补碱基10。最后，CRISPR / CAS 1型系统，核酸CAS3会破坏检测到入侵者的DNA 11,12。 这些过程定义CRISPR / CAS的适应性免疫系统的原核生物，并开了一个迷人的研究领域所涉及的CAS蛋白的研究。许多CA蛋白的功能仍然是难以捉摸和明显的多样性的CRISPR / CAS系统的原因仍然被照亮。通过详细的计算分析，大多数CA蛋白的潜在活动进行了预测。一个主要部分的CAS蛋白或建议作为核酸内切酶4。 这里，我们提出的方法，产生crRNAs和前体的cRNA CAS号核糖核酸内切酶的研究。不同的核酸内切酶的测定需要，可以直接合成的RNA寡核苷酸或不再crRNA和预crRNA的序列，该序列通过在体外 T7 RNA聚合酶转录运行产生的任一短的重复序列。这种方法允许将用于生成内部标记的核酸内切酶基板和的合成或突变crRNAs的创建的放射性核苷酸。 cas6核酸内切酶活性是利用成熟预crRNAs成crRNAs与5&#39;-hydr氧基和2&#39;，3&#39;-环磷酸末端。

Watch this Scientific Journal Video about Substrate Generation for Endonucleases of CRISPR/Cas Systems at JoVE.com

Substrate Generation for Endonucleases of CRISPR/Cas Systems

CRISPR / CAS系统介导的适应性免疫中的细菌和古细菌。提出了许多CA蛋白作为核糖核酸内切酶作用于不同长度的crRNA前体。在这里，我们说明了三种不同的方法，CAS核酸内切酶活性的生化分析以产生前crRNA的底物。

CRISPR / CAS系统内切酶的底物生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

substrate-generation-for-endonucleases-of-crisprcas-systems

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JoVE Journal

Biology

27.2K 次观看.  Max-Planck-Institute for Terrestrial Microbiology. CRISPR / CAS系统介导的适应性免疫中的细菌和古细菌。提出了许多CA蛋白作为核糖核酸内切酶作用于不同长度的crRNA前体。在这里，我们说明了三种不同的方法，CAS核酸内切酶活性的生化分析以产生前crRNA的底物。

视频: Substrate Generation for Endonucleases of CRISPR/Cas Systems

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

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 ...

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 ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

crispr 介导的人类真菌病原体白色念珠菌基因组编辑

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...

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 ...

使用 CRISPRmass 的高通量 UAS-cDNA/ORF 质粒文库构建

CRISPR-Based Modular Assembly for High-Throughput Construction of a UAS-cDNA/ORF Plasmid Library

Functional genomics screening offers a powerful approach to probe gene function and relies on the construction of genome-wide plasmid libraries. Conventional approaches for plasmid library construction are time-consuming and laborious. Therefore, we recently developed a simple and efficient method, CRISPR-based modular assembly (CRISPRmass), for high-throughput construction of a genome-wide upstream activating sequence-complementary DNA/open reading frame (UAS-cDNA/ORF) plasmid library. Here, we present a protocol for CRISPRmass, taking as an example the construction of a GAL4/UAS-based UAS-cDNA/ORF plasmid library. The protocol includes massively parallel two-step test tube reactions followed by bacterial transformation. The first step is to linearize the existing complementary DNA (cDNA) or open reading frame (ORF) cDNA or ORF library plasmids by cutting the shared upstream vector sequences adjacent to the 5&#39; end of cDNAs or ORFs using CRISPR/Cas9 together with single guide RNA (sgRNA), and the second step is to insert a UAS module into the linearized cDNA or ORF plasmids using a single step reaction. CRISPRmass allows the simple, fast, efficient, and cost-effective construction of various plasmid libraries. The UAS-cDNA/ORF plasmid library can be utilized for gain-of-function screening in cultured cells and for constructing a genome-wide transgenic UAS-cDNA/ORF library in Drosophila.&#160;

作者聚焦:使用 CRISPRmass 简化全基因组质粒文库构建

We present a protocol for CRISPR-based modular assembly (CRISPRmass), a method for high-throughput construction of UAS-cDNA/ORF plasmid library in Drosophila using publicly available cDNA/ORF resources. CRISPRmass can be applied to editing various plasmid libraries.