Characterizing a standardized BioPart for BAG-specific expression in C. elegans.

Biological parts (BioParts) are modular and standardized DNA sequences that encode biological functions and contribute to the efficient biological engineering of complex systems. Here, we characterize a short BioPart (P flp-17 , 300 bp) for bright multicopy and single-copy BAG-specific expression starting from the gastrula stage in hermaphrodite and male C. elegans . We have generated standardized P flp-17 cloning vectors for BAG-specific gfp and mScarlet expression compatible with extra-chromosomal arrays and for single-copy transgene insertion. The short P flp-17 promoter is easy to generate by gene synthesis and has been incorporated into our online transgene design tool (www.wormbuilder.org/transgenebuilder ) .

Characterizing a standardized BioPart for PVQ-specific expression in C. elegans.

Synthetic biology relies on standardized biological parts (BioParts), and we aim to identify cell-specific promoters for every class of neuron in C. elegans . Here, we characterize a short BioPart (P nlp-17 , 300 bp) for PVQ-specific expression. P nlp-17 ::mScarlet showed bright, persistent, and specific expression in hermaphrodite and male PVQ neurons from multicopy arrays and single-copy insertions starting from the comma stage. We generated standardized P nlp-17 cloning vectors with gfp and mScarlet compatible with single-copy or array expression for PVQ-specific transgene expression or identification. To facilitate gene synthesis, we have incorporated P nlp-17 as a standard BioPart in our online transgene design tool (www.wormbuilder.org/transgenebuilder).

Characterizing short germline-specific promoters with a range of expression levels in C. elegans.

A core tenet of synthetic biology is that well-characterized regulatory elements are essential for engineering biological systems. Here, we characterize the specificity and expression levels of 18 short (254 to 880 bp) candidate germline promoters using a single-copy gfp reporter assay in C. elegans . Six promoters resulted in ubiquitous expression, three did not drive detectable expression, and nine were germline-specific. Several promoters drove stronger germline expression than the commonly-used mex-5 promoter. The promoters range across expression levels and facilitate, for example, low expression of toxic transgenes or high expression of gene editing enzymes, and their compactness facilitates gene synthesis.

Acute and inherited piRNA-mediated silencing in a rde-3 ribonucleotidyltransferase mutant.

We recently developed a piRNA-based silencing assay (piRNAi) to study small-RNA mediated epigenetic silencing: acute gene silencing is induced by synthetic piRNAs expressed from extra-chromosomal array and transgenerational inheritance can be quantified after array loss. The assay allows inheritance assays by injecting piRNAs directly into mutant animals and targeting endogenous genes ( e.g. , him-5 and him-8 ) with obvious phenotypes (increased male frequency). Here we demonstrate the piRNAi assay by quantifying acute and inherited silencing in the ribonucleotidyltransferase rde-3 (ne3370) mutant. In the absence of rde-3, acute silencing was reduced but still detectable, whereas inherited silencing was abolished.

Target-dependent suppression of siRNA production modulates the levels of endogenous siRNAs in the Caenorhabditis elegans germline.

Despite the prominent role of endo-siRNAs in transposon silencing, their expression is not limited to these 'nonself' DNA elements. Transcripts of protein-coding genes ('self' DNA) in some cases also produce endo-siRNAs in yeast, plants and animals. How cells distinguish these two populations of siRNAs to prevent unwanted silencing of active genes in animals is not well understood. To address this question, we inserted various self-gene or gfp fragments into an LTR retrotransposon that produces abundant siRNAs and examined the propensity of these gene fragments to produce ectopic siRNAs in the Caenorhabditis elegans germline. We found that fragments of germline genes are generally protected from production of ectopic siRNAs. This phenomenon, which we termed 'target-directed suppression of siRNA production' (or siRNA suppression), is dependent on the germline expression of target mRNA and requires germline P-granule components. We found that siRNA suppression can also occur in naturally produced endo-siRNAs. We suggest that siRNA suppression plays an important role in regulating siRNA expression and preventing self-genes from aberrant epigenetic silencing. This article has an associated 'The people behind the papers' interview.

Modular safe-harbor transgene insertion for targeted single-copy and extrachromosomal array integration in Caenorhabditis elegans.

Efficient and reproducible transgenesis facilitates and accelerates research using genetic model organisms. Here, we describe a modular safe-harbor transgene insertion (MosTI) for use in Caenorhabditis elegans which improves targeted insertion of single-copy transgenes by homology directed repair and targeted integration of extrachromosomal arrays by nonhomologous end-joining. MosTI allows easy conversion between selection markers at insertion site and a collection of universal targeting vectors with commonly used promoters and fluorophores. Insertions are targeted at three permissive safe-harbor intergenic locations and transgenes are reproducibly expressed in somatic and germ cells. Chromosomal integration is mediated by CRISPR/Cas9, and positive selection is based on a set of split markers (unc-119, hygroR, and gfp) where only animals with chromosomal insertions are rescued, resistant to antibiotics, or fluorescent, respectively. Single-copy insertion is efficient using either constitutive or heat-shock inducible Cas9 expression (25-75%) and insertions can be generated from a multiplexed injection mix. Extrachromosomal array integration is also efficient (7-44%) at modular safe-harbor transgene insertion landing sites or at the endogenous unc-119 locus. We use short-read sequencing to estimate the plasmid copy numbers for 8 integrated arrays (6-37 copies) and long-read Nanopore sequencing to determine the structure and size (5.4 Mb) of 1 array. Using universal targeting vectors, standardized insertion strains, and optimized protocols, it is possible to construct complex transgenic strains which should facilitate the study of increasingly complex biological problems in C. elegans.

Targeted and Random Transposon-Assisted Single-Copy Transgene Insertion in C. elegans.

Transgenesis in model organisms is an essential tool for determining the function of protein-coding genes and non-coding regulatory regions. In Caenorhabditis elegans, injected DNA can be propagated as multicopy extra-chromosomal arrays, but transgenes in arrays are frequently mosaic, over-expressed in some tissues, and silenced in the germline. Here, we describe methods to insert single-copy transgenes into specific genomic locations (MosSCI) or random locations (miniMos) using Mos1 transposons. Single-copy insertions allow expression at endogenous levels, expression in the germline, and identification of active and repressed regions of the genome.

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.

A single-nucleotide change underlies the genetic assimilation of a plastic trait.

Genetic assimilation-the evolutionary process by which an environmentally induced phenotype is made constitutive-represents a fundamental concept in evolutionary biology. Thought to reflect adaptive phenotypic plasticity, matricidal hatching in nematodes is triggered by maternal nutrient deprivation to allow for protection or resource provisioning of offspring. Here, we report natural Caenorhabditis elegans populations harboring genetic variants expressing a derived state of near-constitutive matricidal hatching. These variants exhibit a single amino acid change (V530L) in KCNL-1, a small-conductance calcium-activated potassium channel subunit. This gain-of-function mutation causes matricidal hatching by strongly reducing the sensitivity to environmental stimuli triggering egg-laying. We show that reestablishing the canonical KCNL-1 protein in matricidal isolates is sufficient to restore canonical egg-laying. While highly deleterious in constant food environments, KCNL-1 V530L is maintained under fluctuating resource availability. A single point mutation can therefore underlie the genetic assimilation-by either genetic drift or selection-of an ancestrally plastic trait.

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.

SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors.

The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3'UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3'UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.

The Caenorhabditis elegans Transgenic Toolbox.

The power of any genetic model organism is derived, in part, from the ease with which gene expression can be manipulated. The short generation time and invariant developmental lineage have made Caenorhabditis elegans very useful for understanding, e.g., developmental programs, basic cell biology, neurobiology, and aging. Over the last decade, the C. elegans transgenic toolbox has expanded considerably, with the addition of a variety of methods to control expression and modify genes with unprecedented resolution. Here, we provide a comprehensive overview of transgenic methods in C. elegans, with an emphasis on recent advances in transposon-mediated transgenesis, CRISPR/Cas9 gene editing, conditional gene and protein inactivation, and bipartite systems for temporal and spatial control of expression.

Assessment and Maintenance of Unigametic Germline Inheritance for C. elegans.

The recent work of Besseling and Bringmann (2016) identified a molecular intervention for C. elegans in which premature segregation of maternal and paternal chromosomes in the fertilized oocyte can produce viable animals exhibiting a non-Mendelian inheritance pattern. Overexpression in embryos of a single protein regulating chromosome segregation (GPR-1) provides a germline derived clonally from a single parental gamete. We present a collection of strains and cytological assays to consistently generate and track non-Mendelian inheritance. These tools allow reproducible and high-frequency (>80%) production of non-Mendelian inheritance, the facile and simultaneous homozygosis for all nuclear chromosomes in a single generation, the precise exchange of nuclear and mitochondrial genomes between strains, and the assessments of non-canonical mitosis events. We show the utility of these strains by demonstrating a rapid assessment of cell lineage requirements (AB versus P1) for a set of genes (lin-2, lin-3, lin-12, and lin-31) with roles in C. elegans vulval development.

Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing.

The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

Chromosome-wide mechanisms to decouple gene expression from gene dose during sex-chromosome evolution.

Changes in chromosome number impair fitness by disrupting the balance of gene expression. Here we analyze mechanisms to compensate for changes in gene dose that accompanied the evolution of sex chromosomes from autosomes. Using single-copy transgenes integrated throughout the Caenorhabditis elegans genome, we show that expression of all X-linked transgenes is balanced between XX hermaphrodites and XO males. However, proximity of a dosage compensation complex (DCC) binding site (rex site) is neither necessary to repress X-linked transgenes nor sufficient to repress transgenes on autosomes. Thus, X is broadly permissive for dosage compensation, and the DCC acts via a chromosome-wide mechanism to balance transcription between sexes. In contrast, no analogous X-chromosome-wide mechanism balances transcription between X and autosomes: expression of compensated hermaphrodite X-linked transgenes is half that of autosomal transgenes. Furthermore, our results argue against an X-chromosome dosage compensation model contingent upon rex-directed positioning of X relative to the nuclear periphery.

An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline.

Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistant to position effect variegation and stochastic silencing in the germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.

Transposon-Assisted Genetic Engineering with Mos1-Mediated Single-Copy Insertion (MosSCI).

Transgenesis in model organisms is necessary to determine the function, expression, and subcellular localization of gene products. In Caenorhabditis elegans, injected DNA can be propagated as multicopy extrachromosomal arrays but transgenes in arrays are mosaic, over-expressed in some tissues and silenced in the germline. Here, a method to insert a transgene into a specific genomic location called Mos1-mediated single-copy insertion (MosSCI) is described. Single-copy insertion allows transgene expression at levels that approximate endogenous gene expression as well as expression in the germline.

Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon.

We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.