CRISPR-Cas9-Guided Genome Engineering in Caenorhabditis elegans.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Kim, H-M; Colaiácovo, MP

Published Date

  • December 2019

Published In

Volume / Issue

  • 129 / 1

Start / End Page

  • e106 -

PubMed ID

  • 31763794

Pubmed Central ID

  • PMC6905509

Electronic International Standard Serial Number (EISSN)

  • 1934-3647

International Standard Serial Number (ISSN)

  • 1934-3639

Digital Object Identifier (DOI)

  • 10.1002/cpmb.106

Language

  • eng