CRISPR/Cas9-targeted mutagenesis in Caenorhabditis elegans

Abstract

The generation of genetic mutants in Caenorhabditis elegans has long relied on the selection of mutations in large-scale screens. Directed mutagenesis of specific loci in the genome would greatly speed up analysis of gene function. Here, we adapt the CRISPR/Cas9 system to generate mutations at specific sites in the C. elegans genome.

Keywords: CRISPR; Caenorhabditis elegans; Cas9; double-strand break; genome engineering.

Figure 1

Experimental design and germline Cas9 expression. (A) Cas9/sgRNA in complex with a target site. RuvC and HNH endonuclease domains together generate a double-strand break. In the sgRNA sequence, green bases are crRNA derived and red bases tracrRNA derived. (B) Schematic of the Cas9 expression vectors used in this study, placing Cas9 or Cas9::EGFP under control of the eft-3 promoter or the hsp-16.48 heat-shock promoter. Versions lacking EGFP are not shown. (C) Germline expression and nuclear localization of Cas9::EGFP expressed from the hsp-16.48 heat-shock promoter. Shown is a maximum-intensity projection of a Z-stack. Bar, 10 μm. (D) Diagrams and sequences of the U6::sgRNA and T7::sgRNA vectors. Gray background, promoter or downstream regions; orange background, sgRNA sequence downstream of the target recognition sequence; red background, BsaI recognition sites; boxed nucleotides, sequences left as 5′ overhang after BsaI digestion. (E) Example of cloning a target sequence into the U6::sgRNA vector. The 20-bp target site is outlined in blue and the PAM in yellow. The consensus sequences we used for target site selection are also indicated. Detailed materials and methods are available in File S1.

Figure 2

Genomic mutations generated by Cas9/sgRNA. Mutations are shown relative to the wild-type sequences. Three mutations could not be resolved by sequencing and may correspond to insertion of a repeated sequence. Blue indicates sgRNA target site, and yellow is the PAM motif.