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