CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans.
CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans.
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DOI：
10.1002/cpmb.106
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发表时间：
2019-12-01
影响因子：
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通讯作者：
Colaiacovo, Monica P
中科院分区：
文献类型：
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作者：
Kim, Hyun-Min;Colaiacovo, Monica P
CRISPR-Cas（成簇规则间隔短回文重复序列-CRISPR相关蛋白）系统已成功用于多种生物体（包括线虫秀丽隐杆线虫）的高效、靶向基因组编辑。最近的研究开发了多种 CRISPR-Cas9 方法，通过两种主要的 DNA 双链断裂修复途径：非同源末端连接和同源重组来增强基因组工程。在这里，我们描述了 Cas9 介导的线虫基因组编辑以及单引导 RNA (sgRNA) 和修复模板克隆（规范的无标记和盒式选择方法）的协议，以及将 Cas9、sgRNA 和修复模板 DNA 传递到种系中所需的注射方法。 © 2019 by John Wiley & Sons, Inc. 基本方案 1：引导 RNA 制备 替代方案 1：使用融合 PCR 进行 sgRNA 克隆 基本方案 2：准备同源重组修复模板 替代方案 2：为盒式选择方法制备修复模板供体 基本方案 3：注射动物 基本方案 4：使用无标记方法筛选转基因蠕虫 替代方案 3：使用无标记方法筛选转基因蠕虫盒式磁带选择方法。
The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein) system is being used successfully for efficient and targeted genome editing in various organisms, including the nematode Caenorhabditis elegans. Recent studies have developed a variety of CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: nonhomologous end joining and homologous recombination. Here, we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning (canonical marker-free and cassette selection methods), as well as injection methods required for delivering Cas9, sgRNAs, and repair template DNA into the germline. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Guide RNA preparation Alternate Protocol 1: sgRNA cloning using fusion PCR Basic Protocol 2: Preparation of a repair template for homologous recombination Alternate Protocol 2: Preparation of repair template donors for the cassette selection method Basic Protocol 3: Injecting animals Basic Protocol 4: Screening transgenic worms with marker-free method Alternate Protocol 3: Screening transgenic worms with cassette selection method.