Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome.

Numerous health benefits are attributed to the n-3 long-chain PUFA (n-3 LCPUFA); EPA and DHA. A systematic literature review was conducted to investigate factors, other than diet, that are associated with the n-3 LCPUFA levels. The inclusion criteria were papers written in English, carried out in adult non-pregnant humans, n-3 LCPUFA measured in blood or tissue, data from cross-sectional studies, or baseline data from intervention studies. The search revealed 5076 unique articles of which seventy were included in the qualitative synthesis. Three main groups of factors potentially associated with n-3 LCPUFA levels were identified: (1) unmodifiable factors (sex, genetics, age), (2) modifiable factors (body size, physical activity, alcohol, smoking) and (3) bioavailability factors (chemically bound form of supplements, krill oil v. fish oil, and conversion of plant-derived α-linolenic acid (ALA) to n-3 LCPUFA). Results showed that factors positively associated with n-3 LCPUFA levels were age, female sex (women younger than 50 years), wine consumption and the TAG form. Factors negatively associated with n-3 LCPUFA levels were genetics, BMI (if erythrocyte EPA and DHA levels are <5·6 %) and smoking. The evidence for girth, physical activity and krill oil v. fish oil associated with n-3 LCPUFA levels is inconclusive. There is also evidence that higher ALA consumption leads to increased levels of EPA but not DHA. In conclusion, sex, age, BMI, alcohol consumption, smoking and the form of n-3 LCPUFA are all factors that need to be taken into account in n-3 LCPUFA research.

This study aimed to assess the feasibility of conducting a nutrition trial in adult male prisoners. Adult male prisoners were recruited for a 16-week randomised control trial comparing the effect of ingestion of omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFA) and multivitamin supplements versus placebo on aggressive behaviour. The baseline and post-intervention assessments from the participant blood samples were the erythrocyte n-3 LCPUFA levels as well as measures of aggressive behaviour determined through institutional records of misconduct (IRM), the Inmate Behaviour Observation Scale (IBOS), and questionnaires. A total of 136 adult male prisoners consented to the study with a retention rate of 60%, and 93% of blood samples were successfully collected. The IRM and IBOS scores were collected for 100% of participants, whilst 82-97% of participants completed the questionnaires. From the baseline data, the Odds Ratio shows that prisoners are 4.3 times more likely to have an IBOS >2 if they are below the 6% cut off on the omega-3 index. Both groups improved across all outcome measures and, at the current sample size, no significant differences were seen between them. A power calculation suggests a total sample size of 600 participants is required to detect the effects of this dietary supplementation, and that this supplementation study is feasible in a Correctional Centre. Important criteria for the exclusion and consideration of logistics and compliance are presented.

Omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFA) supplementation in the cardiovascular field is effective if a certain Omega-3 index (O3I) is achieved or the daily n-3 LCPUFA dose is high enough. Whether this applies to studies on cognition in children and adolescents is unclear. The aims of the current review were to investigate whether: (1) a certain O3I level and (2) a minimum daily n-3 LCPUFA dose are required to improve cognition in 4-25 year olds. Web of Science and PubMed were searched. Inclusion criteria: placebo controlled randomized controlled trial; participants 4-25 years; supplementation with docosahexaenoic acid (DHA) and/or eicosapentaenoic acid (EPA); assessing cognition; in English and ≥10 participants per treatment arm. Thirty-three studies were included, 21 in typically developing participants, 12 in those with a disorder. A positive effect on cognitive measures was more likely in studies with an increase in O3I to >6%. Half of the studies in typically developing children with daily supplementation dose ≥450 mg DHA + EPA showed improved cognition. For children with a disorder no cut-off value was found. In conclusion, daily supplementation of ≥450 mg DHA + EPA per day and an increase in the O3I to >6% makes it more likely to show efficacy on cognition in children and adolescents.

The production of haploid gametes from diploid germ cells requires two rounds of meiotic chromosome segregation after one round of replication. Accurate meiotic chromosome segregation involves the remodeling of each pair of homologous chromosomes around the site of crossover into a highly condensed and ordered structure. We showed that condensin, the protein complex needed for mitotic chromosome compaction, restructures chromosomes during meiosis in Caenorhabditis elegans. In particular, condensin promotes both meiotic chromosome condensation after crossover recombination and the remodeling of sister chromatids. Condensin helps resolve cohesin-independent linkages between sister chromatids and alleviates recombination-independent linkages between homologues. The safeguarding of chromosome resolution by condensin permits chromosome segregation and is crucial for the formation of discrete, individualized bivalent chromosomes.

The targetable DNA endonuclease CRISPR-Cas9 has transformed analysis of biological processes by enabling robust genome editing in model and nonmodel organisms. Although rules directing Cas9 to its target DNA via a guide RNA are straightforward, wide variation occurs in editing efficiency and repair outcomes for both imprecise error-prone repair and precise templated repair. We found that imprecise and precise DNA repair from double-strand breaks (DSBs) is asymmetric, favoring repair in one direction. Using this knowledge, we designed RNA guides and repair templates that increased the frequency of imprecise insertions and deletions and greatly enhanced precise insertion of point mutations in Caenorhabditis elegans We also devised strategies to insert long (10 kb) exogenous sequences and incorporate multiple nucleotide substitutions at a considerable distance from DSBs. We expanded the repertoire of co-conversion markers appropriate for diverse nematode species. These selectable markers enable rapid identification of Cas9-edited animals also likely to carry edits in desired targets. Lastly, we explored the timing, location, frequency, sex dependence, and categories of DSB repair events by developing loci with allele-specific Cas9 targets that can be contributed during mating from either male or hermaphrodite germ cells. We found a striking difference in editing efficiency between maternally and paternally contributed genomes. Furthermore, imprecise repair and precise repair from exogenous repair templates occur with high frequency before and after fertilization. Our strategies enhance Cas9-targeting efficiency, lend insight into the timing and mechanisms of DSB repair, and establish guidelines for achieving predictable precise and imprecise repair outcomes with high frequency.

Mechanisms establishing higher-order chromosome structures and their roles in gene regulation are elusive. We analyzed chromosome architecture during nematode X chromosome dosage compensation, which represses transcription via a dosage-compensation condensin complex (DCC) that binds hermaphrodite Xs and establishes megabase-sized topologically associating domains (TADs). We show that DCC binding at high-occupancy sites (rex sites) defines eight TAD boundaries. Single rex deletions disrupted boundaries, and single insertions created new boundaries, demonstrating that a rex site is necessary and sufficient to define DCC-dependent boundary locations. Deleting eight rex sites (8rexΔ) recapitulated TAD structure of DCC mutants, permitting analysis when chromosome-wide domain architecture was disrupted but most DCC binding remained. 8rexΔ animals exhibited no changes in X expression and lacked dosage-compensation mutant phenotypes. Hence, TAD boundaries are neither the cause nor the consequence of DCC-mediated gene repression. Abrogating TAD structure did, however, reduce thermotolerance, accelerate aging, and shorten lifespan, implicating chromosome architecture in stress responses and aging.

The biological roles of nucleic acid methylation, other than at the C5-position of cytosines in CpG dinucleotides, are still not well understood. Here, we report genetic evidence for a critical role for the putative DNA demethylase NMAD-1 in regulating meiosis in C. elegans. nmad-1 mutants have reduced fertility. They show defects in prophase I of meiosis, which leads to reduced embryo production and an increased incidence of males due to defective chromosomal segregation. In nmad-1 mutant worms, nuclear staging beginning at the leptotene and zygotene stages is disorganized, the cohesin complex is mislocalized at the diplotene and diakinesis stages, and chromosomes are improperly condensed, fused, or lost by the end of diakinesis. RNA sequencing of the nmad-1 germline revealed reduced induction of DNA replication and DNA damage response genes during meiosis, which was coupled with delayed DNA replication, impaired DNA repair and increased apoptosis of maturing oocytes. To begin to understand how NMAD-1 regulates DNA replication and repair, we used immunoprecipitation and mass spectrometry to identify NMAD-1 binding proteins. NMAD-1 binds to multiple proteins that regulate DNA repair and replication, including topoisomerase TOP-2 and co-localizes with TOP-2 on chromatin. Moreover, the majority of TOP-2 binding to chromatin depends on NMAD-1. These results suggest that NMAD-1 functions at DNA replication sites to regulate DNA replication and repair during meiosis.

DNA topology and alternative DNA structures are implicated in regulating diverse biological processes. Although biomechanical properties of these structures have been studied extensively in vitro, characterization in vivo, particularly in multicellular organisms, is limited. We devised new methods to map DNA supercoiling and single-stranded DNA in Caenorhabditis elegans embryos and diapause larvae. To map supercoiling, we quantified the incorporation of biotinylated psoralen into DNA using high-throughput sequencing. To map single-stranded DNA, we combined permanganate treatment with genome-wide sequencing of induced double-stranded breaks. We found high levels of negative supercoiling at transcription start sites (TSSs) in embryos. GC-rich regions flanked by a sharp GC-to-AT transition delineate boundaries of supercoil propagation. In contrast to TSSs in embryos, TSSs in diapause larvae showed dramatic reductions in negative supercoiling without concomitant attenuation of transcription, suggesting developmental-stage-specific regulation. To assess whether alternative DNA structures control chromosome architecture and gene expression, we examined DNA supercoiling in the context of X-Chromosome dosage compensation. We showed that the condensin dosage compensation complex creates negative supercoils locally at its highest-occupancy binding sites but found no evidence for large-scale supercoiling domains along X Chromosomes. In contrast to transcription-coupled negative supercoiling, single-strandedness, which is most pronounced at transcript end sites, is dependent on high AT content and symmetrically positioned nucleosomes. We propose that sharp transitions in sequence composition at functional genomic elements constitute a common regulatory code and that DNA structure and propagation of torsional stress at regulatory elements are critical parameters in shaping important developmental events.

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.

Interventions to better manage aggressive behaviour and reduce recidivism are a primary concern for corrective services. Nutritional interventions to correct prisoner behaviour have been largely overlooked in the literature. Emerging evidence suggests that dietary intake influences aggressive behaviours and that nutritional supplementation with omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFA) could attenuate both the severity and frequency of aggressive behaviour.

In preeclampsia, maternal insulin resistance leads to defective expansion of adipocytes, enhanced adipocyte lipolysis, up-regulation of very low density lipoprotein synthesis, maternal hypertriglyceridaemia and the potential for ectopic fat storage. Our aim was to quantitate and compare the total amount and type of lipid in placenta from pregnancies complicated with preeclampsia and healthy pregnancies. Quantitative lipid analysis of lipid extracts from full thickness placental biopsies was carried out by shotgun lipidomics. Placental lipid profiles from pregnancies complicated by preeclampsia (n = 23) were compared to healthy pregnancies (n = 68), and placenta from intrauterine growth restriction pregnancies (n = 10) were used to control for gross differences in placental pathology. Placentae from pregnancies complicated with preeclampsia had higher neutral lipid content than healthy placentae (40% higher triacyglycerol (P = 0.001) and 33% higher cholesteryl ester (P = 0.004)) that was specific to preeclampsia and independent of maternal gestation.

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.

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.

Self-regulation, the regulation of behaviour in early childhood, impacts children's success at school and is a predictor of health, wealth, and criminal outcomes in adulthood. Self-regulation may be optimised by dietary supplementation of omega-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs). The aim of the "Omega Kid" study is to investigate the feasibility of a protocol to investigate whether n-3 LCPUFA supplementation enhances self-regulation in preschool-aged children. The protocol assessed involved a double-blind, randomised, placebo-controlled trial of 12 weeks duration, with an intervention of 1.6 g of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) per day (0.3 g EPA and 1.3 g DHA) in a microencapsulated powder compared to placebo. Children (n = 78; 40 boys and 38 girls) aged 3-5 years old were recruited and randomly allocated to the treatment (n = 39) or placebo group (n = 39). The HS-Omega-3 Index® served as a manipulation check on the delivery of either active (n-3 LCPUFAs) or placebo powders. Fifty-eight children (76%) completed the intervention (28-30 per group). Compliance to the study protocol was high, with 92% of children providing a finger-prick blood sample at baseline and high reported-adherence to the study intervention (88%). Results indicate that the protocol is feasible and may be employed in an adequately powered clinical trial to test the hypothesis that n-3 LCPUFA supplementation will improve the self-regulation of preschool-aged children.

We demonstrate how RNA binding protein FOX-1 functions as a dose-dependent X-signal element to communicate X-chromosome number and thereby determine nematode sex. FOX-1, an RNA recognition motif protein, triggers hermaphrodite development in XX embryos by causing non-productive alternative pre-mRNA splicing of xol-1, the master sex-determination switch gene that triggers male development in XO embryos. RNA binding experiments together with genome editing demonstrate that FOX-1 binds to multiple GCAUG and GCACG motifs in a xol-1 intron, causing intron retention or partial exon deletion, thereby eliminating male-determining XOL-1 protein. Transforming all motifs to GCAUG or GCACG permits accurate alternative splicing, demonstrating efficacy of both motifs. Mutating subsets of both motifs partially alleviates non-productive splicing. Mutating all motifs blocks it, as does transforming them to low-affinity GCUUG motifs. Combining multiple high-affinity binding sites with the twofold change in FOX-1 concentration between XX and XO embryos achieves dose-sensitivity in splicing regulation to determine sex.

The prostate is surrounded by periprostatic adipose tissue (PPAT), the thickness of which has been associated with more aggressive prostate cancer (PCa). There are limited data regarding the functional characteristics of PPAT, how it compares to subcutaneous adipose tissue (SAT), and whether in a setting of localized PCa, these traits are altered by obesity or disease aggressiveness. PPAT and SAT were collected from 60 men (age: 42-78 years, BMI: 21.3-35.6 kg/m2) undergoing total prostatectomy for PCa. Compared to SAT, adipocytes in PPAT were smaller, had the same basal rates of fatty acid release (lipolysis) yet released less polyunsaturated fatty acid species, and were more sensitive to isoproterenol-stimulated lipolysis. Basal lipolysis of PPAT was increased in men diagnosed with less aggressive PCa (Gleason score (GS) ≤ 3 + 4) compared to men with more aggressive PCa (GS ≥ 4 + 3) but no other measured adipocyte parameters related to PCa aggressiveness. Likewise, there was no difference in PPAT lipid biology between lean and obese men. In conclusion, lipid biological features of PPAT do differ from SAT; however, we did not observe any meaningful difference in ex vivo PPAT biology that is associated with PCa aggressiveness or obesity. As such, our findings do not support a relationship between altered PCa behavior in obese men and the metabolic reprogramming of PPAT.

Gestational diabetes mellitus (GDM) affects approximately one in six pregnancies, causing a significant burden on maternal and infant health. Lifestyle interventions are first-line therapies to manage blood glucose levels (BGLs) and prevent future cardiometabolic complications. However, women with GDM experience considerable barriers to lifestyle interventions; thus, the aim of this study was to determine how women with GDM manage their condition and to identify the primary supports and barriers to lifestyle intervention participation.

The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (∼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.

We show that multiple, functionally specialized cohesin complexes mediate the establishment and two-step release of sister chromatid cohesion that underlies the production of haploid gametes. In C. elegans, the kleisin subunits REC-8 and COH-3/4 differ between meiotic cohesins and endow them with distinctive properties that specify how cohesins load onto chromosomes and then trigger and release cohesion. Unlike REC-8 cohesin, COH-3/4 cohesin becomes cohesive through a replication-independent mechanism initiated by the DNA double-stranded breaks that induce crossover recombination. Thus, break-induced cohesion also tethers replicated meiotic chromosomes. Later, recombination stimulates separase-independent removal of REC-8 and COH-3/4 cohesins from reciprocal chromosomal territories flanking the crossover site. This region-specific removal likely underlies the two-step separation of homologs and sisters. Unexpectedly, COH-3/4 performs cohesion-independent functions in synaptonemal complex assembly. This new model for cohesin function diverges from that established in yeast but likely applies directly to plants and mammals, which utilize similar meiotic kleisins.