A Course-Based Undergraduate Research Experience for High-Throughput Reverse Genetic Studies in Arabidopsis Thaliana with CRISPR-Cas9
A Course-Based Undergraduate Research Experience for High-Throughput Reverse Genetic Studies in Arabidopsis Thaliana with CRISPR-Cas9
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DOI：
10.20944/preprints202008.0619.v1
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发表时间：
2020-08
期刊：
影响因子：
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通讯作者：
Alison M. Mills;V. Jagannatha;Alejandro Cortez;Michael A Guzmán;J. Burnette;Matthew A. Collin;
中科院分区：
文献类型：
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作者：
Alison M. Mills;V. Jagannatha;Alejandro Cortez;Michael A Guzmán;J. Burnette;Matthew A. Collin;
CRISPR-Cas9等基因编辑工具为动植物基因研究创造了前所未有的机会。我们设计了以课程为基础的本科生研究体验(CURE)，以培训入门的生物学学生了解基因编辑技术的概念和实施，并发展他们在数据管理和科学交流方面的软技能。我们提供了两个版本的课程，可以在五周内每周召开两次会议来实施。在远程学习版中，学生进行同源搜索，设计指导RNA和引子，并学习分子克隆的原理。此版本适用于获取实验室设备或面对面指导有限的情况，例如为应对新冠肺炎大流行而发生的关闭。在面对面的版本中，学生设计指导RNA，克隆CRISPR-Cas9结构，并对模式植物拟南芥进行遗传转化。治疗的高度平行性质使其有可能针对数十到数百个基因，这取决于可用的课程部分的数量。在敏化的突变背景中应用这种方法，可以对遗传抑制基因或增强子进行有针对性的反向遗传筛选。这门课程可以很容易地适应其他生物或使用基因编辑的项目。
Gene editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene editing technology as well as develop their soft skills in data management and scientific communication. We present two versions of the course that can be implemented with twice-weekly meetings over a five-week period. In the remote-learning version, students perform homology searches, design guide RNAs and primers, and learn the principles of molecular cloning. This version is appropriate when access to laboratory equipment or in-person instruction is limited, such as closures that have occurred in response to the Covid-19 pandemic. In the in-person version, students design guide RNAs, clone CRISPR-Cas9 constructs, and perform genetic transformation of the model plant Arabidopsis thaliana. The highly parallel nature of the CURE makes it possible to target dozens to hundreds of genes, depending on the number of course sections available. Applying this approach in a sensitized mutant background enables focused reverse genetic screens for genetic suppressors or enhancers. The course can be readily adapted to other organisms or projects that employ gene editing.