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An Introduction to CRISPR-Mediated Genome Editing in Fungi

机译:CRISPR介导的真菌基因组编辑介绍

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摘要

Central dogma, transformation, and genome editing are key biological concepts for which junior scientists must gain experience during training. Here we present an exercise that introduces these concepts in a single practical laboratory exercise. Our exercise utilizes CRISPR/Cas9 genome editing to introduce a stop codon into ADE2. This edit leads to the buildup of an adenine precursor that dyes the edited cells red. As the repair template, guide RNA, and Cas9 are all encoded in our vector, transformation can be performed in 2 hours. Furthermore, since all components of the Cas9/CRISPR system are encoded by the vector, specialized equipment and reagents, such as a PCR machine or oligonucleotides, are not required to perform the experiments as designed. As such, these exercises provide an efficient cost-effective introduction to a wide variety of key molecular biology concepts and lay the foundation for more rigorous investigations in upper-level classes and independent research projects.
机译:中心教条,转化和基因组编辑是关键的生物学概念,初级科学家必须在培训过程中获得经验。在这里,我们介绍一个练习,在一个实际的实验室练习中介绍这些概念。我们的练习利用CRISPR / Cas9基因组编辑将终止密码子引入ADE2。该编辑导致腺嘌呤前体的积累,该腺嘌呤前体将编辑后的细胞染成红色。由于修复模板,指导RNA和Cas9均在我们的载体中编码,因此可以在2小时内完成转化。此外,由于Cas9 / CRISPR系统的所有组件均由载体编码,因此不需要专门的设备和试剂(例如PCR机或寡核苷酸)来进行设计的实验。因此,这些练习为各种关键的分子生物学概念提供了有效的成本有效的介绍,并为在上层课程和独立研究项目中进行更严格的研究奠定了基础。

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