Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination

Abstract

Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR-Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

Figure 1.

CRISPR–Cas targeting in C. elegans. (A) Vectors optimized to express Cas9 and sgRNA in the C. elegans germline. NLS, nuclear localization signal. (B) Targeting sequences used for different genes, together with the 3′ PAM sequence.

Figure 2.

Efficiency of CRISPR–Cas-induced mutations. (A–D) Mutations induced by CRISPR–Cas9 using targeting sequences for ben-1 (A and B), unc-4 (C) and unc-5 (D). For ben-1a, only a subset of the mutations we obtained is shown. (E) Efficiency of CRISPR-induced mutagenesis at different loci.

Figure 3.

CRISPR-targeted knock-ins. Schematic showing targeted knock-in of an HygR cassette at the ben-1 locus. The double-strand break induced at ben-1 by CRISPR–Cas9 is repaired using the transgene containing the HygR cassette, leading to gene conversion.