Biology subjects

Thornton, B.

Publications and source records attributed to Thornton, B..

3 recordsLinked to original sources

bioRxiv2026

Reprogramming Cas9 PAM Recognition for Allele-Specific Editing

The therapeutic potential of CRISPR-Cas9 genome editing is fundamentally constrained by the requirement for specific short DNA sequences (PAMs) flanking the target site, limiting access to many clinically relevant genomic loci. This stringent PAM requirement is particularly problematic in applications which require precise positioning, such as base editing and allele-specific editing. Although PAM-relaxed variants have expanded the targetable genome, they incur trade-offs in on-target activity, off-target editing, and cleavage kinetics. This highlights an unmet need for variants that are re-targeted to alternative PAMs in order to maintain the specificity and enzymatic performance inherent to stringent dinucleotide PAM recognition. To overcome these limitations, we developed a yeast selection platform to engineering SpCas9 variants with re-specified PAM recognition. Using a clinically relevant Huntingtons disease gene (HTT) SNP as a proof-of-concept target, we engineered variants with reciprocal NGC and NGT PAM selectivity, as a step toward allele-specific editing in a large percentage of Huntingtons disease patients. These yeast-selected SpCas9 variants retained their modified activity across multiple endogenous HEK293T loci, demonstrating that this specificity is robust across diverse genomic contexts. The variants surpassed PAM-broadened variants on their respective on-target PAM while displaying broad loss of activity across alternative PAMs, effectively re-specifying PAM recognition toward a single dinucleotide sequence. Retargeted variants recovered on-target cleavage kinetics approaching that of wild-type SpCas9, even under competing substrate conditions, demonstrating that PAM re-specification can simultaneously restore catalytic efficiency and improve specificity. Beyond NGC and NGT, we leveraged our high-throughput platform to engineer Cas9 with re-specified activity across multiple additional non-canonical PAMs in yeast, further demonstrating its utility as a general and programmable framework for expanding the therapeutic reach of precision genome editing.

molecular biology↗

bioRxiv2024

CLONALITY AND POLYPLOIDY CONTRIBUTE TO THE SPREAD OF AVRAINVILLEA LACERATA (BRYOPSIDALES, CHLOROPHYTA) IN HAWAI'I

The relative rates of sexual versus asexual reproduction influence the partitioning of genetic diversity within and among populations. During range expansions, uniparental reproduction often facilitates colonization and establishment. The arrival of the green alga Avrainvillea lacerata has caused shifts in habitat structure and community assemblages since its discovery in 1981 offshore of west Oahu, Hawai i. Field observations suggest this species is spreading via vegetative reproduction. To characterize the reproductive system of A. lacerata in Hawai i, we developed seven microsatellite loci and genotyped 321 blades collected between 2018 and 2023 from two intertidal sites at Maunalua Bay and Ewa Beach. We found one to four alleles at multiple loci, suggesting A. lacerata is tetraploid. Each site was characterized by high genotypic richness (R > 0.8). However, clonal rates were also high at both sites, suggesting vegetative spread of A. lacerata plays a significant role. The importance of clonal reproduction for the persistence of A. lacerata in Hawai i is consistent with the ecological data collected for this species, and observations of other abundant macroalgal invaders in Hawaii and other regions of the world. These data demonstrate the necessity for implementing appropriate population genetic methods and provide insights into the biology of this alga that will contribute to future studies on effective management strategies incorporating its reproductive system. This study represents one of the few investigating green algal population genetic patterns and contributes to our understanding of algal reproductive system evolution.

evolutionary biology↗

bioRxiv2018

Reciprocal carbon subsidies between autotrophs and bacteria in stream food webs under stoichiometric constraints

O_LISoils are currently leaching out their organic matter at an increasing pace and darkening aquatic ecosystems due to climate and land use change, or recovery from acidification. The implications for stream biogeochemistry and food webs remain largely unknown, notably the metabolic balance (biotic CO2 emissions), reciprocal subsidies between autotrophs and bacteria, and trophic transfer efficiencies.\nC_LIO_LIWe use a flow food web approach to test how a small addition of labile dissolved organic matter affects the strength and dynamics of the autotrophs-bacteria interaction in streams. Our paired streams whole-ecosystem experimental approach combined with continuous whole-stream metabolism and stable isotope probing allowed to unravel carbon fluxes in the control and treatment streams.\nC_LIO_LIWe increased the natural supply of dissolved organic matter for three weeks by only 12% by continuously adding 0.5 mg L-1 of sucrose with a {delta}13C signature different from the natural organic matter. Both photosynthesis and heterotrophic respiration increased rapidly following C addition, but this was short lived due to N and P stoichiometric constraints. The resulting peak in heterotrophic respiration was of similar magnitude to natural peaks in the control observed when soils were hydrologically connected to the streams and received soil derived carbon.\nC_LIO_LICarbon reciprocal subsidies between autotrophs and bacteria in the control stream accounted for about 50% of net primary production and 75% of bacterial production, under low flow conditions when stream water was hydrologically disconnected from soil water. The reciprocal subsidies were weaker by 33% (autotrophs to bacteria) and 55% (bacteria to autotrophs) in the treatment relative to the control. Net primary production relied partly (11% in the control) on natural allochthonous dissolved organic carbon via the CO2 produced by bacterial respiration.\nC_LIO_LIMany large changes in ecosystem processes were observed in response to the sucrose addition. The light use efficiency of the autotrophs increased by 37%. Ecosystem respiration intensified by 70%, and the metabolic balance became relatively more negative, i.e. biotic CO2 emissions increased by 125%. Heterotrophic respiration and production increased by 89%, and this was reflected by a shorter (-40%) uptake length (SwOC) and faster (+92%) mineralisation velocity of organic carbon. The proportion of DOC flux respired and organic carbon use efficiency by bacteria increased by 112%.\nC_LIO_LIMacroinvertebrate consumer density increased by 72% due to sucrose addition and consumer production was 1.8 times higher in the treatment than in the control at the end of the experiment. The trophic transfer efficiencies from resources to consumers were similar between the control and the treatment (2-5%).\nC_LIO_LISynthesis. Part of the carbon derived from natural allochthonous organic matter can feed the autotrophs via the CO2 produced by stream bacterial respiration, intermingling the green and brown webs. The interaction between autotrophs and bacteria shifted from mutualism to competition with carbon addition under nutrient limitation (N, P) increasing biotic CO2 emissions. Without nutrient limitation, mutualism could be reinforced by a positive feedback loop, maintaining the same biotic CO2 emissions. A small increase in dissolved organic carbon supply from climate and land use change could have large effects on stream food web and biogeochemistry with implications for the global C cycle under stoichiometric constraints.\nC_LI

ecology↗