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Increased understanding of plant genetics and the development of powerful and easier-to-use gene editing tools over the past century have revolutionized humankind's ability to deliver precise genotypes in crops. Plant transformation techniques are well developed for making transgenic varieties in certain crops and model organisms, yet reagent delivery and plant regeneration remain key bottlenecks to applying the technology of gene editing to most crops. Typical plant transformation protocols to produce transgenic, genetically modified (GM) varieties rely on transgenes, chemical selection, and tissue culture. Typical protocols to make gene edited (GE) varieties also use transgenes, even though these may be undesirable in the final crop product. In some crops, the transgenes are routinely segregated away during meiosis by performing crosses, and thus only a minor concern. In other crops, particularly those propagated vegetatively, complex hybrids, or crops with long generation times, such crosses are impractical or impossible. This review highlights diverse strategies to deliver CRISPR/Cas gene editing reagents to regenerable plant cells and to recover edited plants without unwanted integration of transgenes. Some examples include delivering DNA-free gene editing reagents such as ribonucleoproteins or mRNA, relying on reagent expression from non-integrated DNA, using novel delivery mechanisms such as viruses or nanoparticles, using unconventional selection methods to avoid integration of transgenes, and/or avoiding tissue culture altogether. These methods are advancing rapidly and already enabling crop scientists to make use of the precision of CRISPR gene editing tools., Competing Interests: ZK and VC are employees of Centro de Tecnologia Canavieira., (Copyright © 2023 Kocsisova and Coneva.)

Understanding populations is important because they are a fundamental level of biological organization. Individual traits such as aging and lifespan interact in complex ways to determine birth and death and thereby influence population dynamics. However, we lack a deep understanding of the relationships between individual traits and population dynamics. To address this challenge, we established a laboratory population using the model organism C. elegans and an individual-based computational simulation informed by measurements of real worms. The simulation realistically models individual worms and the behavior of the laboratory population. To elucidate the role of aging in population dynamics, we analyzed old age as a cause of death and showed, using computer simulations, that it was influenced by maximum lifespan, rate of adult culling, and progeny number/food stability. Notably, populations displayed a tipping point for aging as the primary cause of adult death. Our work establishes a conceptual framework that could be used for better understanding why certain animals die of old age in the wild., Competing Interests: Declaration of Interest The authors declare no competing interests.

Aging animals display a broad range of progressive degenerative changes, and one of the most fascinating is the decline of female reproductive function. In the model organism Caenorhabditis elegans , hermaphrodites reach a peak of progeny production on day 2 of adulthood and then display a rapid decline; progeny production typically ends by day 8 of adulthood. Since animals typically survive until day 15 of adulthood, there is a substantial post reproductive lifespan. Here we review the molecular and cellular changes that occur during reproductive aging, including reductions in stem cell number and activity, slowing meiotic progression, diminished Notch signaling, and deterioration of germ line and oocyte morphology. Several interventions have been identified that delay reproductive aging, including mutations, drugs and environmental factors such as temperature. The detailed description of reproductive aging coupled with interventions that delay this process have made C. elegans a leading model system to understand the mechanisms that drive reproductive aging. While reproductive aging has dramatic consequences for individual fertility, it also has consequences for the ecology of the population. Population dynamics are driven by birth and death, and reproductive aging is one important factor that influences birth rate. A variety of theories have been advanced to explain why reproductive aging occurs and how it has been sculpted during evolution. Here we summarize these theories and discuss the utility of C. elegans for testing mechanistic and evolutionary models of reproductive aging., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Scharf, Pohl, Egan, Kocsisova and Kornfeld.)

C. elegans hermaphrodites display dramatic age-related decline of reproduction early in life, while somatic functions are still robust. To understand reproductive aging, we analyzed the assembly line of oocyte production that generates fertilized eggs. Aging germlines displayed both sporadic and population-wide changes. A small fraction of aging animals displayed endomitotic oocytes in the germline and other defects. By contrast, all animals displayed age-related decreases in germline size and function. As early as day 3 of adulthood, animals displayed fewer stem cells and a slower cell cycle, which combine to substantially decrease progenitor zone output. The C. elegans germline is the only adult tissue that contains stem cells, allowing the analysis of stem cells in aging. To investigate the mechanism of the decrease in stem cell number, we analyzed the Notch signaling pathway. The Notch effectors LST-1 and SYGL-1 displayed age-related decreases in expression domains, suggesting a role for Notch signaling in germline aging. The results indicate that although sporadic defects account for the sterility of some animals, population-wide changes account for the overall pattern of reproductive aging., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Mechanisms that control aging are important yet poorly defined. To discover longevity control genes, we performed a forward genetic screen for delayed reproductive aging in C. elegans. Here, we show that am117 is a nonsense mutation in the phm-2 gene, which encodes a protein homologous to human scaffold attachment factor B. phm-2(lf) mutant worms have an abnormal pharynx grinder, which allows live bacteria to accumulate in the intestine. This defect shortens lifespan on highly pathogenic bacteria but extends lifespan and health span on the standard E. coli diet by activating innate immunity pathways that lead to bacterial avoidance behavior and dietary restriction. eat-2(lf) mutants displayed a similar phenotype, indicating accumulation of live bacteria also triggers extended longevity in this mutant. The analysis of phm-2 elucidates connections between pathogen response and aging by defining a mechanism of longevity extension in C. elegans-bacterial colonization, innate immune activation, and bacterial avoidance behavior., (Copyright © 2019. Published by Elsevier Inc.)

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Background: The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E., Results: To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development., Conclusions: In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor cells is distinct from somatic cells, as it does not heavily rely on cyclic accumulation of classic cell cycle proteins.

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (∼5.2 Mb) is the smallest chromosome in Drosophila melanogaster , but it is substantially larger (>18.7 Mb) in D. ananassae To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes ( e.g. , larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae Compared to D. melanogaster , the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5' ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains., (Copyright © 2017 Leung et al.)

Nuclear receptors were originally defined as endocrine sensors in humans, leading to the identification of the nuclear receptor superfamily. Despite intensive efforts, most nuclear receptors have no known ligand, suggesting new ligand classes remain to be discovered. Furthermore, nuclear receptors are encoded in the genomes of primitive organisms that lack endocrine signaling, suggesting the primordial function may have been environmental sensing. Here we describe a novel Caenorhabditis elegans nuclear receptor, HIZR-1, that is a high zinc sensor in an animal and the master regulator of high zinc homeostasis. The essential micronutrient zinc acts as a HIZR-1 ligand, and activated HIZR-1 increases transcription of genes that promote zinc efflux and storage. The results identify zinc as the first inorganic molecule to function as a physiological ligand for a nuclear receptor and direct environmental sensing as a novel function of nuclear receptors., Competing Interests: The authors have declared that no competing interests exist.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25-50%) than euchromatic reference regions (3-11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11-27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4-3.6 vs. 8.4-8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu., (Copyright © 2015 Leung et al.)