SUMMARY Organisms exhibit a fascinating array of gene-silencing pathways, which have evolved in part, to confront invasive nucleic acids such as transposons and viruses. A key question raised by the existence of these pathways is how do they distinguish “self” from “non-self” nucleic acids? Evidence exists for a number of mechanisms that might facilitate detection of foreign sequences including mechanisms that sense copy-number, unpaired DNA, or aberrant RNA (e.g. dsRNA). Here we describe an RNA-induced epigenetic silencing pathway, RNAe, that permanently silences single-copy transgenes. We show that the Piwi Argonaute PRG-1 and its genomically encoded piRNA cofactors initiate RNAe, while maintenance depends on chromatin factors and the WAGO Argonaute pathway. Our findings support a model in which PRG-1 scans for foreign sequences, while two other Argonaute pathways serve as epigenetic memories of “self” and “non-self” RNAs. These findings suggest how organisms may utilize RNAi-related mechanisms not only to recognize and silence foreign genes, but also to keep inventory of all genes expressed in the germ-line.

Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans. The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols. Here we report efficient and straightforward CRISPR-Cas9 genome-editing methods for C. elegans, including a Co-CRISPR strategy that facilitates detection of genome-editing events. We describe methods for detecting homologous recombination (HR) events, including direct screening methods as well as new selection/counterselection strategies. Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.

SUMMARY Organisms can develop adaptive sequence-specific immunity by re-expressing pathogen-specific small RNAs that guide gene silencing. For example, the C. elegans PIWI-Argonaute/piRNA pathway recruits RNA-dependent RNA polymerase RdRP to foreign sequences to amplify a trans-generational small RNA-induced epigenetic silencing signal (termed RNAe). Here we provide evidence that in addition to an adaptive memory of silenced sequences, C. elegans can also develop an opposing adaptive memory of expressed/self mRNAs. We refer to this mechanism, which can prevent or reverse RNAe as RNA-induced epigenetic gene activation (RNAa). We show that CSR-1, which engages RdRP-amplified small RNAs complementary to germline-expressed mRNAs, is required for RNAa. We show that a transgene with RNAa activity also exhibits accumulation of cognate CSR-1 small RNAs. Our findings suggest that C. elegans adaptively acquires and maintains a trans-generational CSR-1 memory that recognizes and protects self mRNAs, allowing piRNAs to recognize foreign sequences innately, without need for prior exposure.