X-chromosome target specificity diverged between dosage compensation mechanisms of two closely related Caenorhabditis species.

An evolutionary perspective enhances our understanding of biological mechanisms. Comparison of sex determination and X-chromosome dosage compensation mechanisms between the closely related nematode species Caenorhabditis briggsae (Cbr) and Caenorhabditis elegans (Cel) revealed that the genetic regulatory hierarchy controlling both processes is conserved, but the X-chromosome target specificity and mode of binding for the specialized condensin dosage compensation complex (DCC) controlling X expression have diverged. We identified two motifs within Cbr DCC recruitment sites that are highly enriched on X: 13 bp MEX and 30 bp MEX II. Mutating either MEX or MEX II in an endogenous recruitment site with multiple copies of one or both motifs reduced binding, but only removing all motifs eliminated binding in vivo. Hence, DCC binding to Cbr recruitment sites appears additive. In contrast, DCC binding to Cel recruitment sites is synergistic: mutating even one motif in vivo eliminated binding. Although all X-chromosome motifs share the sequence CAGGG, they have otherwise diverged so that a motif from one species cannot function in the other. Functional divergence was demonstrated in vivo and in vitro. A single nucleotide position in Cbr MEX can determine whether Cel DCC binds. This rapid divergence of DCC target specificity could have been an important factor in establishing reproductive isolation between nematode species and contrasts dramatically with the conservation of target specificity for X-chromosome dosage compensation across Drosophila species and for transcription factors controlling developmental processes such as body-plan specification from fruit flies to mice.

Rapid genome shrinkage in a self-fertile nematode reveals sperm competition proteins.

To reveal impacts of sexual mode on genome content, we compared chromosome-scale assemblies of the outcrossing nematode Caenorhabditis nigoni to its self-fertile sibling species, C. briggsaeC. nigoni's genome resembles that of outcrossing relatives but encodes 31% more protein-coding genes than C. briggsaeC. nigoni genes lacking C. briggsae orthologs were disproportionately small and male-biased in expression. These include the male secreted short (mss) gene family, which encodes sperm surface glycoproteins conserved only in outcrossing species. Sperm from mss-null males of outcrossing C. remanei failed to compete with wild-type sperm, despite normal fertility in noncompetitive mating. Restoring mss to C. briggsae males was sufficient to enhance sperm competitiveness. Thus, sex has a pervasive influence on genome content that can be used to identify sperm competition factors.

Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs and CRISPR/Cas9 to engineer insertions and deletions.

Exploitation of custom-designed nucleases to induce DNA double-strand breaks (DSBs) at genomic locations of choice has transformed our ability to edit genomes, regardless of their complexity. DSBs can trigger either error-prone repair pathways that induce random mutations at the break sites or precise homology-directed repair pathways that generate specific insertions or deletions guided by exogenously supplied DNA. Prior editing strategies using site-specific nucleases to modify the Caenorhabditis elegans genome achieved only the heritable disruption of endogenous loci through random mutagenesis by error-prone repair. Here we report highly effective strategies using TALE nucleases and RNA-guided CRISPR/Cas9 nucleases to induce error-prone repair and homology-directed repair to create heritable, precise insertion, deletion, or substitution of specific DNA sequences at targeted endogenous loci. Our robust strategies are effective across nematode species diverged by 300 million years, including necromenic nematodes (Pristionchus pacificus), male/female species (Caenorhabditis species 9), and hermaphroditic species (C. elegans). Thus, genome-editing tools now exist to transform nonmodel nematode species into genetically tractable model organisms. We demonstrate the utility of our broadly applicable genome-editing strategies by creating reagents generally useful to the nematode community and reagents specifically designed to explore the mechanism and evolution of X chromosome dosage compensation. By developing an efficient pipeline involving germline injection of nuclease mRNAs and single-stranded DNA templates, we engineered precise, heritable nucleotide changes both close to and far from DSBs to gain or lose genetic function, to tag proteins made from endogenous genes, and to excise entire loci through targeted FLP-FRT recombination.