CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans

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