Iyer Jyoti, DeVaul Nicole, Hansen Tyler, Nebenfuehr Ben
Department of Chemistry and Biochemistry, The University of Tulsa, Tulsa, OK, USA.
The Department of Anatomy and Cell Biology, The George Washington University School of Medicine and Health Sciences, Tulsa, OK, USA.
Methods Mol Biol. 2019;1874:431-457. doi: 10.1007/978-1-4939-8831-0_25.
In this chapter, we describe the procedure for generating genetically modified Caenorhabditis elegans using microinjection via the Cas9-mediated Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing technique. Specifically, we describe the detailed method of performing CRISPR editing by microinjection using the Cloning-free Co-CRISPR method described by the Seydoux lab. This microinjection protocol can also be used for CRISPR editing with protocols from other labs as well as for a variety of other editing techniques including Mos1-mediated single-copy transgene insertions (MosSCI), transcriptional activator-like nucleases (TALENs), and zinc-finger nucleases (ZFNs). Further, this microinjection protocol can also be used for injecting plasmid DNA to generate heritable extrachromosomal arrays for gene expression and mosaic analysis, performing RNAi by injection and delivering RNA, dyes or other molecules into the C. elegans germline.
在本章中,我们描述了通过Cas9介导的成簇规律间隔短回文重复序列(CRISPR)编辑技术,利用显微注射法生成转基因秀丽隐杆线虫的程序。具体而言,我们描述了使用Seydoux实验室所描述的无克隆共CRISPR方法,通过显微注射进行CRISPR编辑的详细方法。该显微注射方案也可用于采用其他实验室方案的CRISPR编辑,以及多种其他编辑技术,包括Mos1介导的单拷贝转基因插入(MosSCI)、转录激活样效应核酸酶(TALENs)和锌指核酸酶(ZFNs)。此外,该显微注射方案还可用于注射质粒DNA以生成用于基因表达和嵌合体分析的可遗传的染色体外阵列,通过注射进行RNA干扰,并将RNA、染料或其他分子递送至秀丽隐杆线虫的生殖系中。