Engineering the Caenorhabditis elegans Genome Using Cas9-Triggered Homologous Recombination

By D. Dickinson, Jordan D. Ward, D. Reiner, B. Goldstein. Nature Methods, 2013

925 citations recorded by the source corpus.

Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

Subfields: Aging, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/c7e89afaab0e7a7541deacff1141e75ee5da2cf7

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Citations in the corpus

High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes (2015, background): “Second, the efficiency of HDR typically is low, requiring the screening of large number of animals or the use of selection markers that are integrated alongside the desired edits (Dickinson et al. 2013, 2015).”
sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites (2014, methodology): “The CRISPR-Cas9 system has been successfully used in gene targeting of different species, including the monkey, human induced pluripotent stem cells, the mouse, the rat, the zebrafish and the fly [9–22].”
Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins (2014, background): “Since then, RGENs have been successfully used to modify genomes in various species including model organisms (Cho et al. 2013b; Dickinson et al. 2013; Friedland et al. 2013; Gratz et al. 2013; Li et al. 2013a,c; Wang et al. 2013; Sung et al. 2014) and plants (Li et al. 2013b; Nekrasov et al. 2013;…”
Reprogramming homing endonuclease specificity through computational design and directed evolution (2013, methodology): “For example, the CRISPR/Cas9 family of nucleases is becoming a popular catalyst for genome engineering (6,49–51), and the methods presented in this work could be used to increase the speciﬁcity of Cas9 (52–54) or expand the targeting potential of this class of enzymes, currently limited by the PAM…”
Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells (2014, positive): “The simplicity of targeting any locus with a single protein and a programmable sgRNA has quickly led to widespread use of Cas9 11,12 in applications such as genome editing 7,8,13–16 , disease gene repair 17,18 and knock-in of specific tags 8,19 .”
Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA (2014, result): “However , when we genetically fused Cas9 to a CPP that consists of four Gly, nine Arg, and four Leu (Matsui et al. 2003; Ding et al. 2010), it was difficult to obtain purified protein in suitable quantities, which is in line with results with ZFNs (Gaj et al. 2012) and TALENs (Liu et al. 2014)…”