Biology subjects

Blount, J.

Publications and source records attributed to Blount, J..

4 recordsLinked to original sources

bioRxiv2026

Quantitative profiling of intrinsic dCas9-DNA recognition reveals key determinants of guide RNA performance

CRISPR technologies based on nuclease-deactivated Cas9 (dCas9) rely on programmable DNA binding rather than DNA cleavage, yet the intrinsic DNA-recognition properties that govern optimal guide RNA (gRNA) performance remain poorly understood. Existing approaches either measure genomic occupancy in cells or infer dCas9 behavior from cleavage-based Cas9 datasets, despite DNA binding being substantially more permissive than DNA cleavage. Here we introduce TANGO (Targeted Array-based Nucleic acid-Guided Occupancy), a high-density DNA-array platform that quantitatively profiles intrinsic dCas9:gRNA binding across tens of thousands of DNA targets in a cell-free system. TANGO captures established features of dCas9 target recognition, while providing substantially greater sensitivity than prior assays. Comparison with ChIP-seq data demonstrates that intrinsic DNA-binding specificity is a major driver of genomic occupancy and reveals that chromatin accessibility modulates the intrinsic binding affinity required for dCas9 recruitment. Across CRISPRi/a guides, TANGO identifies multiple independent biochemical determinants of guide performance--including on-target affinity, mismatch tolerance, and ribonucleoprotein assembly--and flags problematic and highly promiscuous guides overlooked by current specificity metrics. Unexpectedly, some guides retain substantial guide-directed DNA binding even in the absence of a protospacer-adjacent motif (PAM), revealing an additional dimension of dCas9 specificity. Together, these results establish intrinsic DNA recognition as a quantitative and experimentally accessible determinant of dCas9 function, providing a framework for improving guide selection and enhancing the precision of CRISPR technologies.

genomics↗

bioRxiv2026

Early electrical stimulation promotes functional recovery after volumetric muscle loss

Volumetric muscle loss (VML) injuries overwhelm the inherent regenerative capacity of skeletal muscle, causing persistent functional deficits with no routinely effective therapies. Electrical stimulation (ES) has been shown to preserve muscle structure in other injury models, but technical barriers have prevented daily delivery during the acute post-injury window when critical regenerative programs are established. Here, we developed a fully implantable bioelectronic system with nanoporous platinum-modified electrodes enabling daily therapeutic stimulation and electromyographic recording without repeated anesthesia in a rat tibialis anterior VML model. Animals receiving ES during the acute post-injury period (10 sessions over days 0-14) showed sustained functional improvement, reaching 90% of baseline torque at 8 weeks compared to 71% in unstimulated controls. This recovery reflected enhanced remodeling of injured muscle rather than synergistic muscle compensation. Histological analysis revealed coordinated early increases in vascularization, pro-regenerative macrophages, and satellite cells. These findings establish early ES as a promising intervention for promoting muscle regeneration after catastrophic injury.

bioengineering↗

bioRxiv2024

Strategic use of male alternative reproductive tactics in cooperatively breeding banded mongoose groups

Alternative reproductive tactics (ARTs) allow smaller or less competitive individuals to reproduce by avoiding direct fights through sneaky strategies. Within cooperatively breeding groups ARTs are rarely reported, potentially due to difficulties observing male reproductive behaviour in the wild, or reproductive suppression by fellow group members. In societies where all mating opportunities cannot be monopolised by one male, young males could use sneaky tactics as a stepping-stone to gain limited reproductive success while they grow in resource holding potential (RHP). Using 20 years of pedigree, weight, group demography, and behavioural data, we investigated the use of sneaky pesterer ARTs in wild banded mongooses. Instead of a stepping stone, pestering tactics were typically a rare back-up used by males displaced from mate guarding tactics by higher RHP rivals. Additionally, pesterers have lower siring success compared to mate guards, despite similar weight loss costs, which may explain why most males settle for reproductive inactivity rather than use pestering tactics. However, pestering allows younger males to gain access to older, higher fecundity females in the group and may even facilitate inbreeding avoidance. Overall, ARTs provide a viable option for reproductive fitness for some males that lose out on reproductive positions in highly male-skewed groups.

animal behavior and cognition↗

bioRxiv2024

Reproductive restraint to avoid the costs of reproductive conflict in a cooperatively breeding mammal

The costs of reproductive conflict can be an important factor shaping the evolution of life histories in animal societies. These costs may change as individuals age and grow, and with within-group competition. Social costs of reproductive conflict have been invoked to explain why females might gain from delaying maturity or ceasing reproduction midway through life, but not in males. Here we analyze more than 20 years of data to understand how individual male banded mongooses adjust their reproductive activity in response to the costs of reproductive conflict. In banded mongooses groups multiple female breeders enter oestrus synchronously and males compete to mate guard the females. The oldest and heaviest males in the group gained the greatest share of paternity, while younger and lighter males obtained little or no paternity. Those lighter males that are reproductively active paid disproportionate survival costs. Our results suggest that by delaying or suppressing reproduction early in life, males may reduce their mortality. Males may also avoid inflicting costs on their male kin by queuing. These results from an egalitarian cooperative breeding mammal may provide a rare example of voluntary reproductive restraint, although we cannot discount reproductive suppression by rival males.

animal behavior and cognition↗