Biology subjects

Casini, A.

Publications and source records attributed to Casini, A..

3 recordsLinked to original sources

bioRxiv2023

Eukaryotic-driven directed evolution of Cas9 nucleases

Full exploitation of the natural reservoir of CRISPR-Cas nucleases from prokaryotes for genome editing is limited by the suboptimal activity of these enzymes in mammalian cells. Here we developed a Eukaryotic Platform to Improve Cas Activity (EPICA) to steer weakly active Cas9 nucleases into highly active enzymes by directed evolution. The EPICA platform is obtained by coupling Cas nuclease activity with yeast auxotrophic selection followed by mammalian cell selection through a sensitive reporter system. EPICA was validated with a poorly efficient Cas9 nuclease from Campylobacter jejuni, CjCas9, generating an enhanced variant, UltraCjCas9, following directed evolution rounds. UltraCjCas9 was up to 12-fold more active in mammalian endogenous genomic loci, while preserving high genome-wide specificity. Here we report a eukaryotic pipeline allowing enhancement of Cas9 systems, setting the ground to unlock the multitude of RNA-guided nucleases existing in nature.

bioengineering↗

bioRxiv2023

Organometallic Pillarplexes that bind DNA 4-way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination and repair) and are dynamic structures which adopt either open or closed conformations, with the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core giving them an ideal structure to interact with the central cavities of open DNA junctions. Combining experimental studies and MD simulations we show that an Au pillarplex can bind DNA 4-way junctions (Holliday junctions) in their open form, a binding mode not accessed by synthetic agents before. The Au pillarplexes can bind designed 3-way junctions too but their large size leads them to open up and expand that junction, disrupting the base pairing which manifests in an increase in hydrodynamic size and a lower junction thermal stability. At high loading they re-arrange both 4-way and 3-way junctions into Y-shaped DNA forks to increase the available junction-like binding sites. The structurally related Ag pillarplexes show similar DNA junction binding behaviour, but a lower solution stability. This pillarplex binding contrasts with (but complements) that of the metallo-supramolecular cylinders, which prefer 3-way junctions and we show can rearrange 4-way junctions into 3-way junction structures. The pillarplexes ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures where they are key interconnecting components. Studies in human cells, confirm that the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction-binders into organometallic chemistry. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/522759v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@10fac36org.highwire.dtl.DTLVardef@1f32f9forg.highwire.dtl.DTLVardef@bb8d72org.highwire.dtl.DTLVardef@1433de5_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Identifying and validating the presence of Guanine-Quadruplexes (G4) within the blood fluke parasite Schistosoma mansoni.

BackgroundSchistosomiasis is a neglected tropical disease that currently affects over 250 million individuals worldwide. In the absence of an immunoprophylactic vaccine and the recognition that mono-chemotherapeutic control of schistosomiasis by praziquantel has limitations, new strategies for managing disease burden are urgently needed. A better understanding of schistosome biology could identify previously undocumented areas suitable for the development of novel interventions. Methodology/Principal findingsHere, for the first time, we detail the presence of G-quadruplexes (G4) and putative quadruplex forming sequences (PQS) within the Schistosoma mansoni genome. We find that G4 are present in both intragenic and intergenic regions of the seven autosomes as well as the sex- defining allosome pair. Amongst intragenic regions, G4 are particularly enriched in 3 UTR regions. Gene Ontology (GO) term analysis evidenced significant G4 enrichment in the wnt signalling pathway (p<0.05) and PQS oligonucleotides synthetically derived from wnt-related genes resolve into parallel and hybrid G4 motifs as elucidated by circular dichroism (CD) spectroscopy. Finally, utilising a single chain anti-G4 antibody called BG4, we confirm the in situ presence of G4 within both adult female and male worm nuclei. Conclusion/SignificanceThese results collectively suggest that G4-targeted compounds could be tested as novel anthelmintic agents and highlights the possibility that G4-stabilizing molecules could be progressed as candidates for the treatment of schistosomiasis. Author SummarySchistosoma mansoni causes schistosomiasis, a parasitic disease that affects millions of people living in resource-deprived areas of developing countries. No vaccine exists and the current drug treatment has limitations, notably inefficacy against the larval stages of the parasite. New drugs are, therefore, needed to sustainably control schistosomiasis. A further understanding of parasite biology will uncover new targets and lead to the development of novel therapies. Here, we identify the presence of G-Quadruplexes (G4s) in S. mansoni. G4s are four-stranded DNA structures that can affect gene function and, to date, have not been previously found in any parasitic helminth. Computational analysis predicted potential G4 folding sequences within the S. mansoni genome, several of which were confirmed to fold by circular dichroism spectroscopy. Analysis of G4-containing protein coding genes found an enrichment within the wnt signalling pathway, a developmental pathway crucial for axial development in the parasite. Additionally, G4s could be detected within adult worms using a fluorescent antibody that selectively recognises quadruplex structures in nucleic acids. This research describes the presence of a previously unknown structure within the parasite, which could present a new target for developing novel treatments.

microbiology↗