Biology subjects

Castillo, S. R.

Publications and source records attributed to Castillo, S. R..

5 recordsLinked to original sources

bioRxiv2026

Systematic Engineering of Loss-of-Function Alleles in the Zebrafish Mitochondrial Proteome

Pathogenic variants in the 13 protein-coding genes of the mitochondrial genome underlie clinically and biochemically heterogeneous disorders. Most mtDNA-encoded genes lack defined loss-of-function (LOF) models in vivo. To address this gap, we have generated Z-Terminator, a systematic in vivo atlas of loss-of-function alleles covering all the mtDNA-encoded OXPHOS subunits in zebrafish (Danio rerio). We used mitochondrial TALE base editors to introduce premature termination codon (PTC) alleles via C-to-T transitions across Complexes I, III, IV, and V. Larvae harboring PTC alleles displayed bioenergetic defects and elevated lactate. While mtDNA mutations are associated with sensorineural hearing loss, the cellular basis has remained unclear. We show that engineered mtDNA LOF alleles directly impair hair cell function in proportion to heteroplasmy in a living vertebrate. We investigated the germline transmission and tissue-specific heteroplasmy of these LOF alleles and observed that a subset of variants was transmitted to the F1 generation and displayed distinct mutant loads across organs. These findings establish Z-Terminator as a vertebrate platform for interrogating the role of mtDNA protein-coding genes in cellular dysfunction and for elucidating the pathophysiology of mitochondrial disorders.

genetics↗

bioRxiv2025

Antitachycardia Pacing is More Effective When Delivered to the Left Bundle Branch Area Compared to the Right Ventricle in a Pre-clinical Model of Ischemic Ventricular Tachycardia

BackgroundAnti-tachycardia pacing (ATP) delivered from implantable cardioverter defibrillators (ICDs) provides critically timed pacing pulses to terminate ventricular tachycardia (VT). Physiological pacing through left bundle branch area (LBBA) pacing has emerged as a clinically relevant alternative to induce synchronous activation of the ventricles. The main objective of this study was to compare the efficacy and safety of ATP delivered to an LBBA lead and a conventional RV lead. MethodsUsing a preclinical animal model (n=7), pacing leads were implanted in the RV apex and LBBA and connected to ICDs. The left anterior descending artery was occluded for two hours to cause an ischemia-reperfusion injury (IRI). Four days following IRI, VT episodes were induced using programmed electrical stimulation (PES), and burst ATP therapy was delivered to the RV or LBBA leads for each VT episode. ResultsVT was induced 80 times, with a mean VT cycle length (VT CL) of 180.0{+/-}30.0 ms. ATP delivered to the LBBA terminated VT more often than RV ATP (70.2% vs 47.3%, P = 0.04), and there was no significant difference in negative outcomes of VT acceleration or VF induction. Activation sequences from the basket catheter were determined for the pre-therapy VT and captured ATP beats, and correlation coefficients were computed to the activation sequences during ATP delivery. The number of ATP pulses required for the activation sequences to correlate with the captured ATP pattern rather than the pre-therapy VT pattern was lower for LBBA ATP compared to RV ATP (4.1 vs. 5.1 beats, P = 0.04). Successful LBBA ATP demonstrated earlier and more frequent Purkinje activations preceding myocardial activation than successful RV ATP. ConclusionImproved performance of LBBA ATP compared to RV ATP provides further incentive for the wide use of LBBA leads in patients who need cardiac electrotherapy.

bioengineering↗

bioRxiv2024

Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs

DddA-derived cytosine base editors (DdCBEs) enable the targeted introduction of C*G-to-T*A conversions in mitochondrial DNA (mtDNA). DdCBEs are often deployed as pairs, with each arm comprised of a transcription activator-like effector (TALE), a split double-stranded DNA deaminase half, and a uracil glycosylase inhibitor. This pioneering technology has helped improve our understanding of cellular processes involving mtDNA and has paved the way for the development of models and therapies for genetic disorders caused by pathogenic mtDNA variants. Nonetheless, given the intrinsic properties of TALE proteins, several target sites in human mtDNA remain out of reach to DdCBEs and other TALE-based technologies. Specifically, due to the conventional requirement for a thymine immediately upstream of the TALE target sequences (i.e., the 5-T constraint), over 150 loci in the human mitochondrial genome are presumed to be inaccessible to DdCBEs. Previous attempts at circumventing this constraint, either by developing monomeric DdCBEs or utilizing DNA-binding domains alternative to TALEs, have resulted in suboptimal specificity profiles with reduced therapeutic potential. Here, aiming to challenge and elucidate the relevance of the 5-T constraint in the context of DdCBE-mediated mtDNA editing, and to expand the range of motifs that are editable by this technology, we generated DdCBEs that contain modified TALE proteins engineered to recognize all 5 bases. Notably, 5-T-noncompliant, canonical DdCBEs efficiently edited mtDNA at diverse loci. However, DdCBEs were frequently outperformed by DdCBEs, which consistently displayed significant improvements in activity and specificity, regardless of the 5-most bases of their TALE binding sites. Furthermore, we showed that DdCBEs are compatible with DddAtox and its derivatives DddA6, and DddA11, and we validated TALE shifting with DdCBEs as an effective approach to optimize base editing outcomes at a single target site. Overall, DdCBEs enable efficient, specific, and unconstrained mitochondrial base editing.

bioengineering↗

bioRxiv2024

Engineering sequestration-based biomolecular classifiers with shared resources

Constructing molecular classifiers that enable cells to recognize linear and non-linear input patterns would expand the biocomputational capabilities of engineered cells, thereby unlocking their potential in diagnostics and therapeutic applications. While several biomolecular classifier schemes have been designed, the effect of biological constraints such as resource limitation and competitive binding on the function of those classifiers has been left unexplored. Here, we first demonstrate the design of a sigma factor-based perceptron as a molecular classifier working on the principles of molecular sequestration between the sigma factor and its anti-sigma molecule. We then investigate how the output of the biomolecular perceptron, i.e., its response pattern or decision boundary, is affected by the competitive binding of sigma factors to a pool of shared and limited resources of core RNA polymerase. Finally, we reveal the influence of sharing limited resources on multi-layer perceptron neural networks and outline design principles that enable the construction of non-linear classifiers using sigma-based biomolecular neural networks in the presence of competitive resource-sharing effects.

synthetic biology↗

bioRxiv2021

The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mtDNA and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci, the first example of majority mtDNA heteroplasmy induction in any system. Gene editing specificity as precise as a single nucleotide was observed in vivo for a protein-coding gene. Non-destructive genotyping enables single animal mtDNA analyses for downstream biological functional genomics applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

genetics↗