A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research.
A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research.
复制标题
DOI：
10.1161/atvbaha.116.304790
复制
发表时间：
2016-06
期刊：
影响因子：
--
通讯作者：
Lowenstein CJ
中科院分区：
文献类型：
--
作者：
Miano JM;Zhu QM;Lowenstein CJ
以前针对小鼠基因组进行生物信息序列的添加、减去或替换的努力需要复杂的载体设计和一系列艰难的步骤，只有少数实验室才能掌握。简单而廉价的成簇规则间隔短回文重复序列（CRISPR）方法现在已经取代了传统的基因组修饰方法，几乎任何实验室都可以快速组装试剂，用于开发新的心血管研究小鼠模型。在这里，我们简要回顾了CRISPR在原核生物中的历史，重点介绍了导致其在动物王国中进行基因组修饰的重大发现。CRISPR技术的核心组成部分进行了审查和更新。总结了双组分和三组分CRISPR编辑的实用指针，以及在小鼠中的许多应用，包括移码突变，增强子和非编码基因的缺失，蛋白质编码和基因调控序列的核苷酸取代，用于条件基因失活的loxP位点的掺入，以及表位标签整合。基因分型策略和遗传镶嵌和无意中的目标讨论的主题。最后，临床应用和伦理考虑得到解决，因为生物医学界热切地接受基因组编辑的这一惊人创新，以解决以前棘手的问题。
Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of labs could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any lab can quickly assemble reagents for developing new mouse models for cardiovascular research. Here we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for two-component and three-component CRISPR editing are summarized with a number of applications in mice including frameshift mutations, deletion of enhancers and non-coding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions.