Type II clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated (Cas) system is a novel genome-editing tool for targeted mutagenesis in cultured cells and whole organisms1,2,3. Recently, the CRISPR-Cas9 system has been applied in C. elegans using various delivery approaches including the transgene method and the direct injection of Cas9 mRNA/single guide RNA (sgRNA) or Cas9-sgRNA ribonucleoproteins4,5,6. In the present study, we have developed a CRISPR-Cas9-based genome-editing tool for C. elegans by delivering gene-specific guide RNA (gRNA) through bacterial feeding to achieve gene disruptions in a time- and labor-saving manner.

We monitored the production of heritable mutations by directly injecting in vitro-synthesized ts-gRNA and Cas9 mRNA into the gonads of C. elegans hermaphrodite. We successfully obtained heritable mutants and observed that the majority of mutant progeny were produced in a relatively narrow time window (8-16 h post gonad injection) (Supplementary information, Figure S1B, S1C and Table S1B). To generate ideal transgenic lines that express Cas9 in the germline, we optimized and reengineered the plasmids. We modified the flanking sequences of the cas9 gene as shown in Figure 1A. The newly optimized Cas9 was driven by the pie-1 promoter, which worked with the tbb-2 3′ UTR to effectively express exogenous cas9 or gfp in the germline11,12 (Supplementary information, Figure S1Da and S1Db). By the direct gonad microinjection of a high concentration of Ppie-1::Cas9 and U6::dpy-5ts-gRNA plasmid DNA into P0 hermaphrodites, we obtained 37 and 43 F1 worms in two separate experiments. In each experiment, 3 F1 worms produced F2 Dpy progeny (Supplementary information, Table S1C). Using a low concentration of Ppie-1::Cas9 and U6::dpy-5ts-gRNA plasmid DNA, two (out of 91) and one (out of 42) F1 animals produced F2 Dpy progeny. Thus, injection of high and low concentrations of DNA resulted in mean mutation rates of 7.54% and 2.29%, respectively (Supplementary information, Table S1C). All mutations were confirmed by sequencing the dpy-5 locus of the F2 Dpy worms (Supplementary information, Figure S1E and Table S1D).

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