Sigma Life Science再次推出基因组编辑工具CRISPR
- Sigma-Aldrich2013年9月3日 9:13 点击:3884
2013年8月27日 Sigma-Aldrich公司宣布推出Sigma CRISPRs,一款经济的哺乳动物基因组编辑工具,适合筛选和探索性研究。这是继锌指核酸酶(ZFN)以来,Sigma推出的第二款基因组编辑工具。
CRISPR指的是规律成簇的间隔短回文重复(Clustered regularly interspaced short palindromic repeats)。CRISPR/Cas是在大多数细菌和古细菌中发现的一种天然免疫系统,可用来对抗入侵的病毒及外源DNA。Sigma CRISPRs利用可定制的RNA-核酸酶复合物来实现哺乳动物基因组特定位点的编辑。
在细菌中,来自入侵病毒的短DNA序列(间隔区)被掺入CRISPR位点,充当之前感染的“记忆”。再次感染引发互补的成熟CRISPR RNA(crRNA)找到匹配的序列,为CRISPR相关(Cas)核酸酶在特定DNA序列切割双链提供了特异性。
Cas9是由crRNA和tracrRNA(反式激活crRNA)引导、切割特定DNA序列的核酸酶。引导RNA(gRNA)可设计成包含发夹结构,以模拟tracrRNA-crRNA复合物。结合特异性是根据gRNA和三核苷酸NGG序列(protospacer adjacent motif,PAM)。对于位点特异的编辑,CRISPR/Cas9系统至少需要Cas9核酸酶和gRNA。
Sigma CRISPRs以单个质粒载体提供,其中包含GFP,可快速富集编辑过的细胞。当然,您也可以利用专门的生物信息学工具在线定制(www.sigma.com/crisprs)。
Sigma独有的在线设计工具让用户可根据CRISPR/Cas靶定的要求,识别人、小鼠和大鼠外显子中的最佳靶位点。它能够自动应用CRISPR/Cas最佳设计实践,包括靶位点与基因组中其他位点至少要求3个碱基对不匹配,从而最大限度减少了脱靶效应。
“Sigma在2008年开发出CompoZr ZFN技术,如今ZFN已成为基因组编辑的金标准。我们利用这一专长创造出Sigma CRISPRs,它让研究人员以更快、更经济的方式筛选目的基因。在利用Sigma CRISPRs 鉴定出重要靶点之后,可通过超特异的CompoZr ZFNs 来验证生物学上相关的突变,以减少潜在的脱靶,”Sigma Life Science功能基因组学市场部经理Shawn Shafer博士谈道。
Sigma-Aldrich研发部门的首席研究员Greg Davis博士认为:“操控哺乳动物基因组的能力对生物学而言是革命性的。Sigma CRISPR/Cas技术作为一款早期、经济的基因编辑工具,为开发和使用带来巨大希望。”
Sigma Life Science Launches CRISPR/Cas Genome Editing Tools
Derived from bacterial and archaeal immune defenses, which guard against invading viruses and foreign DNA, Sigma CRISPRs utilize customizable RNA—nuclease complexes that allow introduction of genome edits at a desired site in mammalian genomes with high efficiency.
Sigma's exclusive online design tool allows users to identify the best target site(s) within human, mouse, and rat protein-coding exons that adhere to CRISPR/Cas targeting requirements. It is the only commercial tool that minimizes the possibility of off-target effects by automatically applying CRISPR/Cas best design practices, including a minimum requirement of three base-pair mismatches between the target site and other sites in the genome. Researchers can also request CRISPR/Cas targets derived from their own design preferences or they can engage Sigma's expert CompoZr® Zinc Finger Nuclease (ZFN) bioinformatics team to produce a custom design based on cutting-edge public and proprietary CRISPR guidelines.
"Sigma developed CompoZr ZFN technology in 2008, and now ZFNs are the gold standard in genome editing. We have used that expertise to create Sigma CRISPRs, which provide researchers a quicker and more affordable way to screen genes of interest. After important targets have been identified with Sigma CRISPRs, the biologically relevant mutations can be validated with ultra-specific CompoZr ZFNs to reduce potential off-targeting," said
"The ability to manipulate mammalian genomes is transformative for biology. The Sigma CRISPR/Cas technology holds significant promise for development and use as an early, inexpensive tool for targeted gene editing," said
For more information, visit www.sigma.com/crisprs.
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