Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

Abstract

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 followed by 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 efficiently repaired 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.

Antimicrobial effectors in the nematode Caenorhabditis elegans : an outgroup to the Arthropoda

“…A common approach for this relies on RNAi, which may, however, not always work with high efficiency, especially if genes are expressed in specific cell types such as neurons [78,79]. Another approach is to use knock-out mutants, which are already available for a large number of C. elegans genes [80] and which can be produced through application of CRISPR/Cas technology [81,82]. On the other hand, additional information on antimicrobial functions can be obtained through analysis of synthesized proteins and peptides.…”

Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans

“…1 D and E). Because of their close linkage, we used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated genome editing (33) to introduce mutations in dsh-2 and mig-5 in animals carrying the dsh-1(ok1445) allele. Loss-of-function dsh-2 and mig-5 alleles with small deletions that caused frameshifts were identified by genotyping (Fig.…”

Oligonucleotide-based targeted gene editing in C. elegans via the CRISPR/Cas9 system

“…Compared with the zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), which have been used for genome editing [1], the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR-associated (Cas) system has emerged as a new powerful tool for genome modifications. It has recently been adopted for genome editing in human cell lines [2][3][4], mouse [5], zebrafish [6], C. elegans [7][8][9][10][11][12], and plants [13].…”

“…The DSDBs can be repaired either through non-homologous end joining (NHEJ), which leads to generation of random deletions, insertions, or both (InDels) [2-5, 7-9, 11, 13], or through homologous recombination (HR), which could generate specific and precise nucleotide or sequence replacements [3,5,9,10,12] when a plasmid or a single-stranded oligonucleotide (oligo) template is also present. The use of oligonucleotides as donor templates, which can be rapidly synthesized through commercial sources, to achieve Cas9-mediated knock-ins has not yet been reported in C. elegans.…”