Biology subjects

Savage, D.

Publications and source records attributed to Savage, D..

16 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↗

bioRxiv2026

Using Cryogenic Electron Tomography (cryoET) to Determine Rubisco Polymerization Constants in α-Carboxysomes

Bacteria microcompartments (BMCs) are pseudo-organelles comprised of a self-assembling, semi-permeable protein shell, most commonly enclosing components of enzymatic pathways. -Carboxysomes (-CBs) are anabolic BMCs known for their role in sequestering Rubisco, the enzyme responsible for carbon fixation in plants, algae and bacteria, along with an upstream enzyme and an assembly protein. Rubisco has low selectivity for its substrate, CO2, and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the -CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside -CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for in situ analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived -CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (n) and equilibrium polymerization constant (Kpol). The adopted modeling approach is consistent with in situ constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both in vitro and potentially in vivo biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography. Significance StatementCryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in -carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.

biophysics↗

bioRxiv2026

Architecting cis-regulation to quantitatively tune gene expression in cereals

Precise modulation of gene expression via cis-regulatory editing holds promise for non-transgenic crop improvement, but the sequence-to-function relationships that govern plant promoter activity remain poorly understood. Here, we develop a massively parallel reporter assay (MPRA) in Sorghum bicolor to systematically measure the effects of >30,000 CRISPR-like mutations-deletions, substitutions, and motif insertions-across entire native promoters and 5' untranslated regions (UTRs) of three photosynthesis genes: PsbS, Raf1, and SBPase. We find that gene expression is most tunable within a [~]500 base pair core promoter region, where mutational effects are reproducible across biological replicates and predictive of protein output. Within these regions, we identify compact deletions and motif insertions that strongly increase protein production (>30-fold relative to wild type), exceeding the performance of transgenic enhancer elements. Mutation-effect relationships are gene-specific, highlighting the need for tailored regulatory maps. Our results establish a high-throughput strategy for cis-regulatory fine-mapping that enables crop improvements via minimal, precise, and non-transgenic gene edits.

synthetic biology↗

bioRxiv2025

Random barcode transposon-site sequencing in Mycobacterium tuberculosis reveals the functions of uncharacterized genes

Mycobacterium tuberculosis (Mtb) is a human bacterial pathogen that establishes chronic infection in the lung. Although the genome of Mtb was sequenced nearly 25 years ago, the genetic basis of Mtbs success as a human pathogen remains to be fully elucidated. Large-scale genetic approaches to understanding gene function are hindered by the limited throughput of traditional transposon sequencing strategies used in mycobacteria. To create a resource for determining the function of genes, we generated a pooled random barcode transposon-site sequencing (RB-TnSeq) library in Mycobacterium tuberculosis (Mtb). A unique twenty-nucleotide barcode in the transposon allows for rapid, high-throughput genetic screening without the laborious protocol of standard bacterial TnSeq screens. We performed 95 RB-TnSeq screens on an array of carbon sources, nitrogen sources, stressors, and antibiotics. Using the resulting dataset, we examined phenotypes of PE/PPE genes, a mycobacterial gene family whose function has long been elusive, uncovering 187 novel phenotypes across 37 genes in this family. We propose a pathway for lactate utilization in which the ESX-5 type VII secretion system exports PPE3, facilitating the import of D- and L-lactate into the bacterial cell. Notably, we identify a candidate D-lactate dehydrogenase that may mediate this metabolic capability. Additionally, we find that the proton-pumping NADH dehydrogenase Nuo is required for utilization of propionate, highlighting the metabolic flexibility of Mtb. Lastly, we characterize a novel mutant that confers resistance to the new tuberculosis antibiotic pretomanid. Results from these genetic screens will facilitate the development of additional new hypotheses about the function of uncharacterized genes and will expand our knowledge of Mtb metabolism and resistance to stress.

microbiology↗

bioRxiv2025

Exploring the deletion landscape of S. aureus Cas9 with SABER

Profiling tolerated amino acid deletions in proteins can elucidate structure-function relationships, reconstruct intermediate stages in protein evolution, and be used to engineer minimized versions of proteins with size-sensitive biotechnology applications. Despite advances in deletion library construction techniques over the past several decades, there are presently few methods available that are simultaneously efficient, precise, and easy to implement. Here we present SABER, a novel approach which utilizes SpRYCas9, a near-PAMless engineered SpCas9 variant, as a molecular biological tool for building deletion libraries with unprecedented speed and ease. We applied this technique to the small and structurally divergent Cas9 from Staphylococcus aureus (SaCas9) and mapped the set of deletions tolerated for DNA binding activity. We proceeded to use this information to design a set of minimal SaCas9-based effectors capable of CRISPRi transcriptional repression in bacterial cells. Our findings provide new insights into the function of certain structural elements in SaCas9, and we anticipate that our dSaCas9 deletion map may prove useful in further efforts to develop minimal Cas9-based effectors and gene editors.

molecular biology↗

bioRxiv2025

Mechanistic basis for protection against fatty liver disease by CIDEB loss-of-function mutations

Background & AimsSomatic and germline CIDEB mutations are associated with protection from chronic liver diseases. The mechanistic basis and whether CIDEB suppression would be an effective therapy against fatty liver disease remain unclear. Methods21 CIDEB somatic mutations were introduced into cells to assess functionality. In vivo screening was used to trace Cideb mutant clones in mice fed normal chow, western (WD), and choline-deficient, L-amino acid-defined, high-fat (CDA-HFD) diets. Constitutive and conditional Cideb knockout mice were generated to study Cideb in liver disease. Isotope tracing was used to evaluate fatty acid oxidation and de novo lipogenesis. Transcriptomics, lipidomics, and metabolic analyses were utilized to explore molecular mechanisms. Double knockout models (Cideb/Atgl and Cideb/Ppara) tested mechanisms underlying Cideb loss. ResultsMost CIDEB mutations showed that they impair function, and lineage-tracing showed that loss-of-function clones were positively selected with some, but not all fatty liver inducing diets. Cideb KO mice were protected from WD, CDA-HFD, and alcohol diets, but had the greatest impact on CDA-HFD induced liver disease. Hepatocyte-specific Cideb deletion could ameliorate disease after MASLD establishment, modeling the impact of therapeutic siRNAs. Cideb loss protected livers via increased {beta}-oxidation, specifically through ATGL and PPARa activation. ConclusionsCideb deletion is more protective in some types of fatty liver disease. {beta}-oxidation is an important component of the Cideb protective mechanism. CIDEB inhibition represents a promising approach, and somatic mutations in CIDEB might predict the patient populations that might benefit the most.

physiology↗

bioRxiv2025

Discovery of widespread activating mutations in a compact RNA-guided endonuclease

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the proteins mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

molecular biology↗

bioRxiv2023

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection

Many eukaryotic viruses require membrane-bound compartments for replication, but no such organelles are known to be formed by prokaryotic viruses1-3. Bacteriophages of the Chimalliviridae family sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of the viral protein ChmA4-10. Previously, we observed lipid membrane-bound vesicles in cells infected by Chimalliviridae, but due to the paucity of genetics tools for these viruses it was unknown if these vesicles represented unproductive, abortive infections or a bona fide stage in the phage life cycle. Using the recently-developed dRfxCas13d-based knockdown system CRISPRi-ART11 in combination with fluorescence microscopy and cryo-electron tomography, we show that inhibiting phage nucleus formation arrests infections at an early stage in which the injected phage genome is enclosed within a membrane-bound early phage infection (EPI) vesicle. We demonstrate that early phage genes are transcribed by the virion-associated RNA polymerase from the genome within the compartment, making the EPI vesicle the first known example of a lipid membrane-bound organelle that separates transcription from translation in prokaryotes. Further, we show that the phage nucleus is essential for the phage life cycle, with genome replication only beginning after the injected DNA is transferred from the EPI vesicle to the newly assembled phage nucleus. Our results show that Chimalliviridae require two sophisticated subcellular compartments of distinct compositions and functions that facilitate successive stages of the viral life cycle.

cell biology↗

bioRxiv2022

Rubisco forms a lattice inside alpha-carboxysomes

Bacteria employ microcompartments to sequester enzymatic processes, either for purposes of protecting cellular contents from reactive intermediates or as a way of increasing reaction efficiency. In these structures, a cargo of enzymes and accessory proteins is encased within a semi-permeable protein shell that permits passage of substrates and products but restricts movement of intermediates. In addition to their importance as a component of many bacterial species metabolisms, microcompartments have recently become a target of protein engineering. The shells can be reassembled from purified proteins, and the full operons can be functionally expressed outside their native prokaryotes and can remain functional following purification. Despite the importance of microcompartments in prokaryotic biology and bioengineering, structural heterogeneity has prevented a complete understanding of their architecture, ultrastructure, and spatial organization. Here, we employ cryo electron tomography to image -carboxysomes, a pseudo-icosahedral microcompartment responsible for carbon fixation. We have solved a high-resolution subtomogram average of the Rubisco cargo in situ, and determined a novel arrangement of the enzyme. We find that the H. neapolitanus Rubisco polymerizes in vivo, mediated by the small Rubisco subunit. These fibrils can further pack to form a lattice with six-fold pseudo-symmetry. This arrangement preserves freedom of motion and accessibility around the Rubisco active site and the binding sites for two other carboxysome proteins, CsoSCA (a carbonic anhydrase) and the disordered CsoS2, even at Rubisco concentrations exceeding 800 M. This characterization of Rubisco cargo inside the -carboxysome provides new insight into the balance between order and disorder in microcompartment organization.

biophysics↗

bioRxiv2020

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

Proteins evolve through the modular rearrangement of elements known as domains. It is hypothesized that extant, multidomain proteins are the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) that comprehensively assays all possible deletions of a protein. Using MISER, we generated a deletion landscape for the CRISPR protein Cas9. We found that Cas9 can tolerate large single deletions to the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

molecular biology↗

bioRxiv2020

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUSs O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

molecular biology↗

bioRxiv2020

Detection of Prenatal Alcohol Exposure Using Machine Learning Classification of Resting-State Functional Network Connectivity Data

IntroductionPrevious work utilizing resting state fMRI to measure functional network connectivity in rodents with moderate prenatal alcohol exposure (PAE) revealed several sex- and region-dependent alterations in FNC implicating FNC as potential biomarker for PAE. Given that FNC is sensitive to neurological and psychiatric conditions in humans, here, we explore the use of previously acquired FNC data and machine learning methods to detect PAE among a sample of rodents exposed to moderate PAE and controls exposed to a saccharin solution. Materials & MethodsWe utilized previously acquired fMRI data from 48 adult rats 24 PAE (12 male 12 female) and 24 saccharin exposed (SAC) controls (12 male and 12 female) for classification. The entire data sample was utilized to perform binary classification (SAC or PAE) of FNC data with multiple support vector machine (SVM) kernels and out-of-sample cross-validation to evaluate classification performance. ResultsResults revealed accuracy rates of 62.5% for all samples, 58.3% for males, and 79.2% for females using a quadratic SVM kernel to classify moderate PAE from FNC data. In addition, brain networks localized to hippocampal and cortical regions contributed strongly to QSVM classifications. ConclusionOur results suggest overall modest classification performance of a QSVM to detect moderate PAE from FNC data gathered from adult rats, yet good performance among females. Further developments and refinement of the technique hold promise for the detection of PAE in earlier developmental time periods that potentially offer additional tools for the non-invasive detection of PAE from FNC data. IMPACT STATEMENTThe diagnosis of fetal alcohol spectrum disorders (FASDs) can be challenging in individuals who lack the hallmark facial dysmorphologies associated with heavy prenatal alcohol exposure (PAE). The absence of a diagnosis prevents individuals with PAE from receiving the treatment and services that improves quality of life outcomes. This research explores the use of preclinical functional network connectivity data and machine learning techniques as a novel and non-invasive means of detecting PAE. Our aim is to contribute basic science towards improving diagnostic strategies that can lead to securing timely and appropriate support for individuals with FASD and their caregivers.

neuroscience↗

bioRxiv2020

Functional reconstitution of a bacterial CO2 concentrating mechanism in E. coli

Many photosynthetic organisms employ a CO2 concentrating mechanism (CCM) to increase the rate of CO2 fixation via the Calvin cycle. CCMs catalyze ≈50% of global photosynthesis, yet it remains unclear which genes and proteins are required to produce this complex adaptation. We describe the construction of a functional CCM in a non-native host, achieved by expressing genes from an autotrophic bacterium in an engineered E. coli strain. Expression of 20 CCM genes enabled E. coli to grow by fixing CO2 from ambient air into biomass, with growth depending on CCM components. Bacterial CCMs are therefore genetically compact and readily transplanted, rationalizing their presence in diverse bacteria. Reconstitution enabled genetic experiments refining our understanding of the CCM, thereby laying the groundwork for deeper study and engineering of the cell biology supporting CO2 assimilation in diverse organisms.One Sentence Summary A bacterial CO2 concentrating mechanism enables E. coli to fix CO2 from ambient air.Competing Interest StatementD.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. A.B.-E. is co-founder of b.fab. These companies were not involved in this research in any way. All other authors declare no competing interests.View Full Text

synthetic biology↗

bioRxiv2020

Discovery and characterization of a novel family of prokaryotic nanocompartments involved in sulfur metabolism

Prokaryotic nanocompartments, also known as encapsulins, are a recently discovered proteinaceous organelle in prokaryotes that compartmentalize cargo enzymes. While initial studies have begun to elucidate the structure and physiological roles of encapsulins, bioinformatic evidence suggests that a great diversity of encapsulin nanocompartments remains unexplored. Here, we describe a novel encapsulin in the freshwater cyanobacterium Synechococcus elongatus PCC 7942. This nanocompartment is upregulated upon sulfate starvation and encapsulates a cysteine desulfurase enzyme via an N-terminal targeting sequence. Using cryoelectron microscopy, we have determined the structure of the nanocompartment complex to 2.2 [A] resolution. Lastly, biochemical characterization of the complex demonstrated that the activity of the cysteine desulfurase is enhanced upon encapsulation. Taken together, our discovery, structural analysis, and enzymatic characterization of this prokaryotic nanocompartment provide a foundation for future studies seeking to understand the physiological role of this encapsulin in various bacteria.

biochemistry↗

bioRxiv2018

Revisiting tradeoffs in Rubisco kinetic parameters

Rubisco is the most abundant enzyme in the biosphere and one of the best-characterized enzymes. Based on correlations between Rubisco kinetic parameters, it is widely posited that tradeoffs embedded in the catalytic mechanism constrain its specificity and maximum catalytic rate. However, the reasoning that established this view was based on data from {approx}20 organisms. We re-examine these tradeoff models using a dataset from {approx}300 organisms. Most correlations are substantially attenuated, with the inverse relationship between carboxylation kcat and specificity SC/O being a key example. Only one tradeoff model survives in our dataset. In this model, increasing catalytic efficiency (kcat/KM) for carboxylation requires increased catalytic efficiency for the competing oxygenation reaction, evidenced by strong power-law correlation between catalytic efficiencies. Our results imply that Rubisco evolution is constrained primarily by the physicochemical limits of O2/CO2 discrimination, which should reframe efforts to engineer this very central enzyme.

systems biology↗

bioRxiv2017

Rapid and Programmable Protein Mutagenesis Using Plasmid Recombineering

Comprehensive and programmable protein mutagenesis is critical for understanding structure-function relationships and improving protein function. However, current techniques enabling comprehensive protein mutagenesis are based on PCR and require in vitro reactions involving specialized protocols and reagents. This has complicated efforts to rapidly and reliably produce desired comprehensive protein libraries. Here we demonstrate that plasmid recombineering is a simple and robust in vivo method for the generation of protein mutants for both comprehensive library generation as well as programmable targeting of sequence space. Using the fluorescent protein iLOV as a model target, we build a complete mutagenesis library and find it to be specific and unbiased, detecting 99.8% of our intended mutations. We then develop a thermostability screen and utilize our comprehensive mutation data to rapidly construct a targeted and multiplexed library that identifies significantly improved variants, thus demonstrating rapid protein engineering in a simple one-pot protocol.

synthetic biology↗