Prior Harriet, MacConnachie Lauren, Martinez Jose L, Nicholl Georgina C B, Beg Asim A
Department of Pharmacology, University of Michigan.
Department of Pharmacology, University of Michigan;
J Vis Exp. 2018 Apr 30(134):57518. doi: 10.3791/57518.
The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.
成簇规律间隔短回文重复序列（CRISPR）-CRISPR相关蛋白9（Cas9）原核适应性免疫防御系统已被用作一种强大的工具，用于精确的真核基因组工程。在此，我们展示了一种使用嵌合单导向RNA（sgRNA）和CRISPR-Cas9核糖核蛋白（RNP）在秀丽隐杆线虫中高效精确地产生基因组点突变的快速简便方法。我们描述了一个用于sgRNA靶点选择、同源定向修复（HDR）模板设计、CRISPR-Cas9-RNP复合及递送的流程，以及一种基因分型策略，该策略能够强大且快速地鉴定出正确编辑的动物。我们的方法不仅允许轻松产生和鉴定所需的基因组点突变动物，还能在大约4至5天内高效且减少筛选工作量地检测其他复杂的插入缺失等位基因。
