11 research outputs found
A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a 'self-cleaving' GFP-expression plasmid.
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
Industry-Scale Orchestrated Federated Learning for Drug Discovery
To apply federated learning to drug discovery we developed a novel platform
in the context of European Innovative Medicines Initiative (IMI) project
MELLODDY (grant n{\deg}831472), which was comprised of 10 pharmaceutical
companies, academic research labs, large industrial companies and startups. The
MELLODDY platform was the first industry-scale platform to enable the creation
of a global federated model for drug discovery without sharing the confidential
data sets of the individual partners. The federated model was trained on the
platform by aggregating the gradients of all contributing partners in a
cryptographic, secure way following each training iteration. The platform was
deployed on an Amazon Web Services (AWS) multi-account architecture running
Kubernetes clusters in private subnets. Organisationally, the roles of the
different partners were codified as different rights and permissions on the
platform and administrated in a decentralized way. The MELLODDY platform
generated new scientific discoveries which are described in a companion paper.Comment: 9 pages, 4 figures, to appear in AAAI-23 ([IAAI-23 track] Deployed
Highly Innovative Applications of AI
Downregulation of transposable elements extends lifespan in Caenorhabditis elegans
Mobility of transposable elements (TEs) frequently leads to insertional mutations in functional DNA regions. In the potentially immortal germline, TEs are effectively suppressed by the Piwi-piRNA pathway. However, in the genomes of ageing somatic cells lacking the effects of the pathway, TEs become increasingly mobile during the adult lifespan, and their activity is associated with genomic instability. Whether the progressively increasing mobilization of TEs is a cause or a consequence of ageing remains a fundamental problem in biology. Here we show that in the nematode Caenorhabditis elegans , the downregulation of active TE families extends lifespan. Ectopic activation of Piwi proteins in the soma also promotes longevity. Furthermore, DNA N 6 -adenine methylation at TE stretches gradually rises with age, and this epigenetic modification elevates their transcription as the animal ages. These results indicate that TEs represent a novel genetic determinant of ageing, and that N 6 -adenine methylation plays a pivotal role in ageing control