The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been successfully used for genome editing in a variety of organisms. Here, we report the use of dual sgRNA-guided Cas9 nuclease to generate knockout mutants of protein coding genes, noncoding genes, and repetitive sequences in C. elegans. Co-injection of C. elegans with dual sgRNAs results in the removal of the interval between two sgRNAs and the loss-of-function phenotype of targeted genes. We sought to determine how large an interval can be eliminated and found that at least a 24 kb chromosome segment can be deleted using this dual sgRNA/Cas9 strategy. The deletion of large chromosome segments facilitates mutant screening by PCR and agarose electrophoresis. Thus, the use of the CRISPR/Cas9 system in combination with dual sgRNAs provides a powerful platform with which to easily generate gene knockout mutants in C. elegans. Our data also suggest that encoding multiple sgRNA sequences into a single CRISPR array to simultaneously edit several sites within the genome may cause the off-target deletion of chromosome sequences.

Small-RNA-mediated chromatin modifications have been widely studied in plants and S. pombe. However, direct evidence of small-RNA-guided sequence-specific chromatin alterations is scarce in animals. In C. elegans, the nuclear RNAi defective (Nrde) pathway functions to transport siRNA from the cytoplasm to the nucleus, modulate transcription elongation, induce histone H3 lysine 9 (H3K9) trimethylation, and mediate transgenerational inheritance of RNAi. Here, we show that both exogenous RNAi and NRDE-bound endogenous 22G RNAs can direct sequence-specific histone H3 lysine 27 (H3K27) trimethylation at targeted loci through the Nrde pathway. The resulting H3K27me3 status can be inherited by progeny for multiple generations. piRNAs and WAGO-1-associated siRNAs induce H3K27 methylation as well. Interestingly, CSR-1-associated endogenous siRNAs fail to trigger H3K27 methylation, whereas exogenous provision of dsRNAs can induce H3K27 methylation at the CSR-1-targeted loci via the Nrde pathway. We further observed distinct genetic requirements of H3K9 and H3K27 trimethylation. Whereas set-25 and met-2 are required for K9 methylation, mes-2 is required for K27 methylation. The depletion of mes-2 leads to a nuclear RNAi defective phenotype. These results indicate that dsRNA-triggered chromatin modification is a sequence-specific response that engages the Nrde pathway in C. elegans.

Ribosome biogenesis is a multistep process, during which mistakes can occur at any step of pre-rRNA processing, modification, and ribosome assembly. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that down-regulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNAs, we conducted both forward and reverse genetic screens to search for more suppressor of siRNA () mutants. We isolated a number of genes that are broadly conserved from yeast to humans and are involved in pre-rRNA modification and processing. Among them, SUSI-2(ceRRP8) is homologous to human RRP8 and engages in m1A methylation of the 26S rRNA. C27F2.4(ceBUD23) is an m7G-methyltransferase of the 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A-methyltransferase of the 18S rRNA. Mutation of these genes led to a deficiency in modification of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasmic-to-nuclear and cytoplasmic-to-nucleolar translocations of the Argonaute protein NRDE-3. The rRNA processing deficiency also resulted in accumulation of risiRNAs. We also isolated SUSI-3(RIOK-1), which is similar to human RIOK1, that cleaves the 20S rRNA to 18S. We further utilized RNAi and CRISPR-Cas9 technologies to perform candidate-based reverse genetic screens and identified additional pre-rRNA processing factors that suppressed risiRNA production. Therefore, we concluded that erroneous rRNAs can trigger risiRNA generation and subsequently, turn on the nuclear RNAi-mediated gene silencing pathway to inhibit pre-rRNA expression, which may provide a quality control mechanism to maintain homeostasis of rRNAs.

PIWI-interacting RNAs (piRNAs) play significant roles in suppressing transposons, maintaining genome integrity, and defending against viral infections. How piRNA source loci are efficiently transcribed is poorly understood. Here, we show that in Caenorhabditis elegans, transcription of piRNA clusters depends on the chromatin microenvironment and a chromodomain-containing protein, UAD-2. piRNA clusters form distinct focus in germline nuclei. We conducted a forward genetic screening and identified UAD-2 that is required for piRNA focus formation. In the absence of histone 3 lysine 27 methylation or proper chromatin-remodeling status, UAD-2 is depleted from the piRNA focus. UAD-2 recruits the upstream sequence transcription complex (USTC), which binds the Ruby motif to piRNA promoters and promotes piRNA generation. Vice versa, the USTC complex is required for UAD-2 to associate with the piRNA focus. Thus, transcription of heterochromatic small RNA source loci relies on coordinated recruitment of both the readers of histone marks and the core transcriptional machinery to DNA.