| Generation of Mutant Pigs by Direct Pronuclear Microinjection of CRISPR/Cas9 Plasmid Vectors
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Author:
Date:
2017-06-05
[Abstract] A set of Cas9 and single guide CRISPR RNA expression vectors was constructed. Only a very simple procedure was needed to prepare specific single-guide RNA expression vectors with high target accuracy. Since the de novo zygotic transcription had been detected in mouse embryo at the 1-cell stage, the plasmid DNA vectors encoding Cas9 and GGTA1 gene specific single-guide RNAs were micro-injected into zygotic pronuclei to confirm such phenomenon in 1-cell pig embryo. Our results demonstrated that mutations caused by these CRISPR/Cas9 plasmids occurred before and at the 2-cell stage of pig embryos, indicating that besides the cytoplasmic microinjection of in vitro transcribed RNA, the pronuclear microinjection of CRISPR/Cas9 DNA vectors provided an efficient solution to ...
[摘要] 构建了一套Cas9和单引导CRISPR RNA表达载体。 需要一个非常简单的程序来制备具有高目标精度的特异性单向RNA表达载体。 由于在1细胞期的小鼠胚胎中已经检测到了合并的合子转录，所以将编码Cas9和GGTA1基因特异性单向RNA的质粒DNA载体微注射到合子原核中以确认 1细胞猪胚胎现象。 我们的研究结果表明，这些CRISPR / Cas9质粒引起的突变发生在猪胚胎的2细胞阶段之前和之后，表明除了体外转录的RNA的细胞质显微注射外，CRISPR / Cas9 DNA载体为产生基因敲除猪提供了有效的解决方案。
背景 由于最初发现了大肠杆菌基因组（Ishino）中的大肠杆菌基因组下游的32bp间隔的串联重复的高度保守的29个碱基对（bp）序列，等等，1987; Nakata等人，1989），在约50％至50bp的大小变化的短定期间隔重复序列的家族中发现约50％细菌和90％的古细菌（Makarova等人，2015）。根据它们的特征结构，Mojica（Mojica等人，2009）和Jansen（Jansen等人）引入了定期交织的短回文重复序列（CRISPR）的名称， ，2002），目前普遍使用。首先通过核苷酸序列比对（Jansen等人，2002）在CRISPR基因座的侧翼首先鉴定了一组CRISPR相关基因 cas1 ...
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| A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9
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Author:
Date:
2017-06-05
[Abstract] The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of ...
[摘要] 聚类规则间隔短回文重复（CRISPR）/ CRISPR相关蛋白9（Cas9）系统是使用最广泛的基因组编辑工具。一个常见的CRISPR / Cas9系统由两个组成部分组成：单导RNA（sgRNA）和Cas9。在特定靶序列引入双链断裂（DSB）需要两种成分。该系统的一个缺点是实验室中sgRNA的生产是费力的，因为它需要在体外转录反应和sgRNA纯化之间克隆sgRNA序列。通过sgRNA靶向Cas9活性的替代方案是用两种小RNA：CRISPR RNA（crRNA）和反式激活性crRNA（tracrRNA）进行靶向。这两种小RNA可以化学合成，这使得与sgRNA相比，这些RNA的产生不那么困难。 CRISPR / Cas9系统的另一个缺点是已经报告了脱靶效应。然而，已经开发了改进形式的Cas9以最小化离靶效应。例如，仅当两个引导RNA在规定的距离内结合相对的链时，切口酶型Cas9（nCas9）和FokI结构域融合的催化无活性的Cas9（FokI-dCas9; fCas9）才诱导DSB。在本协议中，我们描述了使用结合crRNA，tracrRNA和Cas9修饰形式的CRISPR / Cas9系统来生产突变小鼠的实验系统。该方法不仅有利于制备用于基因组编辑系统的试剂，而且可以降低脱靶效应的风险。
背景 ...
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