Ageing has a huge impact on human health and economy, but its molecular basis – regulation and mechanism – is still poorly understood. By today, more than three hundred genes (almost all of them function as protein-coding genes) have been related to human ageing. Although individual ageing-related genes or some small subsets of these genes have been intensively studied, their analysis as a whole has been highly limited. To fill this gap, for each human protein we extracted 21000 protein features from various databases, and using these data as an input to state-of-the-art machine learning methods, we classified human proteins as ageing-related or non-ageing-related. We found a simple classification model based on only 36 protein features, such as the “number of ageing-related interaction partners”, “response to oxidative stress”, “damaged DNA binding”, “rhythmic process” and “extracellular region”. Predicted values of the model quantify the relevance of a given protein in the regulation or mechanisms of the human ageing process. Furthermore, we identified new candidate proteins having strong computational evidence of their important role in ageing. Some of them, like Cytochrome b-245 light chain (CY24A) and Endoribonuclease ZC3H12A (ZC12A) have no previous ageing-associated annotations.

RNA interference (RNAi) technology used for the functional analysis of Caenorhabditis elegans genes frequently leads to phenotypes with low penetrance or even proves completely ineffective. The methods previously developed to solve this problem were built on mutant genetic backgrounds, such as those defective for rrf-3, in which endogenous RNAi pathways are overexpressed. These mutations, however, interferes with many other genetic pathways so that the detected phenotype cannot always be clearly linked to the RNAi-exposed gene. In addition, using RNAi-overexpressing mutant backgrounds requires time-consuming genetic crossing. Here, we present an improved RNAi vector that produces specific double-stranded RNA species only, and thereby significantly stronger phenotypes than the standard gene knockdown vector. The further advantage of the new RNAi vector is that the detected phenotype can be specifically linked to the gene silenced. We also created a new all-in-one C. elegans Cas9 vector whose spacer sequence is much easier to replace. Both new vectors include a novel CRISPR/Cas9-based auto-cloning vector system rendering needless the use of restriction and ligase enzymes in generating DNA constructs. This novel, efficient RNAi and auto-cloning Cas9 systems can be easily adapted to any other genetic model.

Understanding the molecular basis of ageing remains a fundamental problem in biology. In multicellular organisms, while the soma undergoes a progressive deterioration over the lifespan, the germ line is essentially immortal as it interconnects the subsequent generations. Genomic instability in somatic cells increases with age, and accumulating evidence indicates that the disintegration of somatic genomes is accompanied by the mobilisation of transposable elements (TEs) that, when mobilised, can be mutagenic by disrupting coding or regulatory sequences. In contrast, TEs are effectively silenced in the germ line by the Piwi-piRNA system. Here, we propose that TE repression transmits the persistent proliferation capacity and the non-ageing phenotype (e.g., preservation of genomic integrity) of the germ line. The Piwi-piRNA pathway also operates in tumorous cells and in somatic cells of certain organisms, including hydras, which likewise exhibit immortality. However, in somatic cells lacking the PiwipiRNA pathway, gradual chromatin decondensation increasingly allows the mobilisation of TEs as the organism ages. This can explain why the mortality rate rises exponentially throughout the adult life in most animal species, including humans.

SummaryDespite its medical, social, and economic significance, understanding what primarily causes aging, that is, the mechanisms of the aging process, remains a fundamental and fascinating problem in biology. Accumulating evidence indicates that a small RNA‐based gene regulatory machinery, the Piwi‐piRNA pathway, represents a shared feature of nonaging (potentially immortal) biological systems, including the germline, somatic cancer stem cells, and certain ‘lower’ eukaryotic organisms like the planarian flatworm and freshwater hydra. The pathway primarily functions to repress the activity of mobile genetic elements, also called transposable elements (TEs) or ‘jumping genes’, which are capable of moving from one genomic locus to another, thereby causing insertional mutations. TEs become increasingly active and multiply in the genomes of somatic cells as the organism ages. These characteristics of TEs highlight their decisive mutagenic role in the progressive disintegration of genetic information, a molecular hallmark associated with aging. Hence, TE‐mediated genomic instability may substantially contribute to the aging process.

The efficacies of guide RNAs (gRNAs), the short RNA molecules that bind to and determine the sequence specificity of the Streptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage vary dramatically. Thus, the selection of appropriate target sites, and hence spacer sequence, is critical for most applications. Here, we describe a simple, unparalleled method for experimentally pre-testing the efficiencies of various gRNAs targeting a gene. The method explores NHEJ-cloning, genomic integration of a GFP-expressing plasmid without homologous arms and linearized in-cell. The use of ‘self-cleaving’ GFP-plasmids containing universal gRNAs and corresponding targets alleviates cloning burdens when this method is applied. These universal gRNAs mediate efficient plasmid cleavage and are designed to avoid genomic targets in several model species. The method combines the advantages of the straightforward FACS detection provided by applying fluorescent reporter systems and of the PCR-based approaches being capable of testing targets in their genomic context, without necessitating any extra cloning steps. Additionally, we show that NHEJ-cloning can also be used in mammalian cells for targeted integration of donor plasmids up to 10 kb in size, with up to 30% efficiency, without any selection or enrichment.