CRISPR‐Cas9‐Guided Genome Engineering in Caenorhabditis elegans

Abstract

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

Recent Advances in the Genetic, Anatomical, and Environmental Regulation of the C. elegans Germ Line Progenitor Zone

“…Because C. elegans is optically clear, imaging fluorescently labeled living animals is possible. CRISPR/Cas9-mediated genome editing works extraordinarily well in C. elegans [ 34 , 35 , 36 , 37 , 38 , 39 , 40 ], allowing for the endogenous tagging of proteins, as well as the stable transgenic marking of cell types. Fluorescently tagging endogenous proteins is the key to seeing fine structures that may be disrupted by the overexpression of transgenes, though these protein fusions must always be validated for functionality [ 10 ].…”

Quo Vadis Caenorhabditis elegans Metabolomics—A Review of Current Methods and Applications to Explore Metabolism in the Nematode

“…Via fourier-transformation (FT) the NMR spectra are generated, consisting of signal intensities of NMR active compounds depending on their resonance frequency. The most important nuclei at the analysis of biomolecules are 1 H, 13 C, 15 N, 19 F and 31 P.…”

“…Turnover rates were determined in different mutants and identified fat-5, fat-6 and fat-8 as membrane maintenance regulators. Furthermore, 15 N tracers were used to follow the phospholipid turnover in more detail. This analysis revealed that fat-7 also influences the turnover of lipids towards slower metabolism [139].…”

“…This leads to degradation of the homologous messenger RNA (mRNA) [ 5 , 9 , 10 ]. Also, clustered regularly interspersed short palindromic repeats (CRISPR)–Cas9 has been applied in C. elegans for genome engineering, which has been reviewed elsewhere [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ].…”

Chromosome‐level genome reference and genome editing of the tea geometrid

“…The CRISPR/Cas9 system has been utilized to edit genomes in a number of eukaryotic organisms (Ablain et al, 2015;Karageorgi et al, 2019;Kim & Colaiácovo, 2019;Suzuki et al, 2016). To implement genetic manipulation in the geometrid, we developed the CRISPR/Cas9-based genome editing system in E. grisescens.…”