Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C. elegans.

Single-guide RNAs can target exogenous CRISPR-Cas proteins to unique DNA locations, enabling genetic tools that are efficient, specific and scalable. Here we show that short synthetic guide Piwi-interacting RNAs (piRNAs) (21-nucleotide sg-piRNAs) expressed from extrachromosomal transgenes can, analogously, reprogram the endogenous piRNA pathway for gene-specific silencing in the hermaphrodite germline, sperm and embryos of Caenorhabditis elegans. piRNA-mediated interference ('piRNAi') is more efficient than RNAi and can be multiplexed, and auxin-mediated degradation of the piRNA-specific Argonaute PRG-1 allows conditional gene silencing. Target-specific silencing results in decreased messenger RNA levels, amplification of secondary small interfering RNAs and repressive chromatin modifications. Short (300 base pairs) piRNAi transgenes amplified from arrayed oligonucleotide pools also induce silencing, potentially making piRNAi highly scalable. We show that piRNAi can induce transgenerational epigenetic silencing of two endogenous genes (him-5 and him-8). Silencing is inherited for four to six generations after target-specific sg-piRNAs are lost, whereas depleting PRG-1 leads to essentially permanent epigenetic silencing.

Engineering rules that minimize germline silencing of transgenes in simple extrachromosomal arrays in C. elegans.

Transgenes are prone to progressive silencing due to their structure, copy number, and genomic location. In C. elegans, repressive mechanisms are particularly strong in the germline with almost fully penetrant transgene silencing in simple extrachromosomal arrays and frequent silencing of single-copy transgene insertions. A class of non-coding DNA, Periodic An/Tn Clusters (PATCs) can prevent transgene-silencing in repressive chromatin or from small interfering RNAs (piRNAs). Here, we describe design rules (codon-optimization, intron and PATC inclusion, elevated temperature (25 °C), and vector backbone removal) for efficient germline expression from arrays in wildtype animals. We generate web-based tools to analyze PATCs and reagents for the convenient assembly of PATC-rich transgenes. An extensive collection of silencing resistant fluorescent proteins (e.g., gfp, mCherry, and tagBFP) can be used for dissecting germline regulatory elements and a set of enhanced enzymes (Mos1 transposase, Cas9, Cre, and Flp recombinases) enable efficient genetic engineering in C. elegans.

Necessity and Contingency in Developmental Genetic Screens: EGF, Wnt, and Semaphorin Pathways in Vulval Induction of the Nematode Oscheius tipulae.

Genetic screens in the nematode Caenorhabditis elegans identified the EGF/Ras and Notch pathways as central for vulval precursor cell fate patterning. Schematically, the anchor cell secretes EGF, inducing the P6.p cell to a primary (1°) vulval fate; P6.p in turn induces its neighbors to a secondary (2°) fate through Delta-Notch signaling and represses Ras signaling. In the nematode Oscheius tipulae, the anchor cell successively induces 2° then 1° vulval fates. Here, we report on the molecular identification of mutations affecting vulval induction in O. tipulae A single Induction Vulvaless mutation was found, which we identify as a cis-regulatory deletion in a tissue-specific enhancer of the O. tipulae lin-3 homolog, confirmed by clustered regularly interspaced short palindromic repeats/Cas9 mutation. In contrast to this predictable Vulvaless mutation, mutations resulting in an excess of 2° fates unexpectedly correspond to the plexin/semaphorin pathway. Hyperinduction of P4.p and P8.p in these mutants likely results from mispositioning of these cells due to a lack of contact inhibition. The third signaling pathway found by forward genetics in O. tipulae is the Wnt pathway; a decrease in Wnt pathway activity results in loss of vulval precursor competence and induction, and 1° fate miscentering on P5.p. Our results suggest that the EGF and Wnt pathways have qualitatively similar activities in vulval induction in C. elegans and O. tipulae, albeit with quantitative differences in the effects of mutation. Thus, the derived induction process in C. elegans with an early induction of the 1° fate appeared during evolution, after the recruitment of the EGF pathway for vulval induction.

Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis.

Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.