Biology subjects

Edgell, D.

Publications and source records attributed to Edgell, D..

7 recordsLinked to original sources

bioRxiv2025

PHYCUT: Scalable multiplex CRISPR/Cas9 editing forgenome engineering in the diatom Phaeodactylumtricornutum

Diatoms are globally significant microalgae that contribute [~] 20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom Phaeodactylum tricornutum has emerged as a promising synthetic biology platform for bioproduction of recombinant proteins, supported by a human-like N-linked glycosylation pathway. However, its (1,3)-linked core fucose is immunogenic in humans and thus limits biopharmaceutical applications. One hurdle to efficient genome engineering in P. tricornutum is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (Phaeodactylum tricornutum Csy4-Cas9 multiplex tool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multi-guide RNA arrays. To highlight PHYCUT applications, we demonstrate multiplex editing of all three FucT genes responsible for (1,3) fucosylation in P. tricornutum, yielding strains with markedly reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the P. tricornutum glycosylation pathway to support next-generation algal biotechnology.

synthetic biology↗

bioRxiv2025

Genetic entanglement enables ultra-stable biocontainment in the mammalian gut

Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability in vivo limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (Sequence enTAngLEd Multi lAyered geneTic buffEring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudo-essentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganu-clease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement simultaneously inactivated both biocontainment layers, leading to cell death by the ColE9 nuclease and the elimination of escape mutants. By lengthening the entangled region, refining ColE9 expression, and optimizing the TSM sequence against IS911 insertion, we achieved escape rates below 10-10 as compared to rates of 10-5 with the non-entangled TSM. The STALEMATE system contained plasmids in E. coli Nissle 1917 for over a week in the mouse gastrointestinal tract with nearly undetectable escape rates upon excretion. STALEMATE offers a modular and simple biocontainment approach to buffer against mutational inactivation in the mammalian gut without a requirement for engineered bacteria or exogenous signaling ligands.

synthetic biology↗

bioRxiv2025

PAM adenine methylation and flanking sequence regulate SaCas9 activity in bacteria

Cas9 nucleases are the effectors of the class 2 type II CRISPR system in bacteria and function to restrict invading DNA. They can also be used with single guide RNAs (sgRNAs) as antimicrobials and genome engineering tools in bacteria, yet applications are hindered by an incomplete understanding of Cas9-target interactions. Here, we generate large-scale SaCas9/sgRNA in vivo bacterial activity datasets and train a machine learning model (crisprHAL) to predict SaCas9 activity. The highest predictive performance was found when downstream sequence flanking the canonical NNGRRN PAM motif at positions [+1] and [+2] was included in model training, correlating with high in vivo activity on sites that included T-rich di-nucleotides in the [+1] and [+2] flanking positions. Strikingly, model predictions and experimentally determined activity in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium showed an [~]10-fold reduced SaCas9 activity at sites with 5'-NNGGAT[C]-3' PAM [+1] sequences. Cleavage assays using plasmid DNA isolated from E. coli inactivated for DNA adenine methyltransferase (DAM) and SaCas9/sgRNA combinations targeting sites with NNGGAT[C] PAM sequences confirmed that adenine methylation impacts SaCas9 cleavage. Moreover, ablation of a GATC DAM site in a PAM sequence enhanced SaCas9 in vitro activity, whereas creation of a DAM site reduced activity, providing a mechanistic link between adenine methylation and SaCas9 activity. Our results show that a general purpose machine learning architecture can provide biologically relevant insights into SaCas9-PAM interactions that can better inform activity predictions for bacterial applications. Avoidance of adenine methylated PAM sites by SaCas9 may be a mechanism of self versus non-self discrimination or reflect an evolutionary adaptation to counter methylation as an anti-restriction strategy by phage or plasmids.

microbiology↗

bioRxiv2024

Harnessing RNA-based DNA repair pathways for targeted gene editing

Recent studies have revealed a role for RNA in the repair of DNA double-strand breaks. Here, we show that the asymmetric DNA overhangs generated by the small TevSaCas9 dual nuclease informs a simple and robust editing strategy in human cells whereby Pol{theta} and Rad52 are recruited to repair the double-strand break. The 2-nt, 3 DNA overhang generated by the I-TevI nuclease domain of TevSaCas9 hybridizes with the 3 end of a co-localized repair template guide RNA to specifically license repair. Substitutions that destabilize the repair duplex reduce editing efficiency. Targeted RNA-templated repair (rep-editing) harnesses cellular RNA-based DNA repair pathways to introduce precise nucleotide edits, deletions and insertions in human cells with high efficiency and fidelity independent of co-delivered repair functions. The small size of TevSaCas9 and RNA repair template offers delivery advantages over size-constrained or multi-component editing systems.

molecular biology↗

bioRxiv2023

Intern-based thermoregulated meganucleases for biocontainment of genetic material

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18{degrees}C and not at higher temperatures. We constructed an orthogonal set of temperature sensitive-meganucleases, or TSMs, by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18{degrees}C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. We demonstrated that TSMs eliminated plasmids from the E. coli Nissle 1917 strain after passage through the mouse gut when fecal resuspensions were incubated at 18{degrees}C but not at 37{degrees}C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.

synthetic biology↗

bioRxiv2020

Cas9-generated auxotrophs of Phaeodactylum tricornutum are characterized by small and large deletions that can be complemented by plasmid-based genes

The model diatom Phaeodactylum tricornutum is an attractive candidate for synthetic biology applications. Development of auxotrophic strains of P. tricornutum would provide alternative selective markers to commonly used antibiotic resistance genes. Here, using CRISPR/Cas9, we show successful editing of genes in the uracil, histidine, and tryptophan biosynthetic pathways. Editing events are characterized by loss of heterozygosity and by the occurrence of large deletions of up to ~2.7-kb centred on the editing site. The uracil and histidine-requiring phenotypes can be complemented by plasmid-based copies of the intact genes after curing of the Cas9-editing plasmid. Growth of uracil auxotrophs on media supplemented with 5-FOA and uracil results in loss of the complementing plasmid, providing a facile method for plasmid curing with potential applications in strain engineering and CRISPR editing. Metabolomic characterization of uracil auxotrophs revealed changes in cellular orotate concentrations consistent with partial or complete loss of orotate phosphoribosyl transferase activity in knockout strains. Our results expand the range of P. tricornutum auxotrophic strains and demonstrate that auxotrophic complementation markers provide a viable alternative to traditionally used antibiotic selection markers. Plasmid-based auxotrophic markers should expand the range of genome engineering applications and provide a means for biocontainment of engineered P. tricornutum strains.

synthetic biology↗

bioRxiv2018

Designer Sinorhizobium meliloti strains and multi-functional vectors for direct inter-kingdom transfer of high G+C content DNA

Storage and manipulation of large DNA fragments is crucial for synthetic biology applications, yet DNA with high G+C content can be unstable in many host organisms. Here, we report the development of Sinorhizobium meliloti as a new universal host that can store DNA, including high G+C content, and mobilize DNA to Escherichia coli, Saccharomyces cerevisiae, and the eukaryotic microalgae Phaeodactylum tricornutum. We deleted the S. meliloti hsdR restriction-system to enable DNA transformation with up to 1.4 x 105 efficiency. Multi-host and multi-functional shuttle vectors (MHS) were constructed and shown to stably replicate in S. meliloti, E. coli, S. cerevisiae, and P. tricornutum, with a copy-number inducible E. coli origin for isolating plasmid DNA. Crucially, we demonstrated that S. meliloti can act as a universal conjugative donor for MHS plasmids with a cargo of at least 62 kb of G+C rich DNA derived from Deinococcus radiodurans.

synthetic biology↗