Biology subjects

Ramachandran, A.

Publications and source records attributed to Ramachandran, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Differential Protection against Acetaminophen-induced Liver and Kidney Injury by N-Acetylcysteine and Fomepizole in Female Mice

Acetaminophen (APAP) overdose is the leading cause of acute liver failure and is frequently complicated by acute kidney injury (AKI), which worsens clinical outcomes. Although N-acetylcysteine (NAC) effectively attenuates APAP-induced hepatotoxicity by restoring hepatic glutathione (GSH), its efficacy against AKI remains limited, suggesting distinct mechanisms of renal injury. We investigated the role of GSH homeostasis and potential beneficial effects of NAC and Fomepizole (4-methylpyrazole) in APAP-induced liver and kidney injury using female C57BL/6J mice, which naturally lack renal CYP2E1 expression and therefore provide a unique model to examine nephrotoxicity independent of renal CYP2E1-mediated bioactivation. Female mice received APAP (300 or 600 mg/kg) with or without buthionine sulfoximine (BSO, 50 mg/kg) to inhibit GSH synthesis, NAC (500 mg/kg) to promote GSH recovery, or Fomepizole (50 mg/kg) to inhibit CYP2E1-mediated bioactivation. APAP overdose caused severe hepatotoxicity, whereas kidney injury remained minimal during the first 24 h. Although BSO markedly enhanced hepatic GSH depletion and liver injury, it did not significantly worsen renal dysfunction or tubular injury. In contrast, NAC substantially reduced liver injury and improved survival after severe APAP overdose; yet renal dysfunction progressed by 48 h, accompanied by tubular vacuolation, increased KIM-1 and osteopontin expression, and tubular cell death. Fomepizole substantially protected both organs from injury. These findings demonstrate that delayed APAP-induced AKI can develop independent of the severity of liver injury, even with effective NAC-mediated hepatoprotection. On the other hand, Fomepizole protects against AKI in female mice by a CYP2E1-independent mechanism.

pharmacology and toxicology↗

bioRxiv2026

Isolation of oxygen-dependent nicotine- and pseudooxynicotine-metabolizing enzymes

NicA2 is a flavin-bound amine dehydrogenase from Pseudomonas putida S16 that converts nicotine to the pharmacologically inactive N-methylmyosmine. In animal models of nicotine addiction, injection of NicA2 can decrease nicotine-seeking behavior 10-fold. Accordingly, NicA2-related enzymes have been investigated as smoking-cessation therapeutics. However, efficient catalysis by NicA2 in Pseudomonas putida relies on electron transfer to CycN, a cytochrome c, and not directly to O2. Impractically high amounts of NicA2 are thus necessary to achieve a pharmacological effect in the absence of CycN. Directed evolution has improved the ambient-O2 value of kcat from 0.007 s-1 to 1 s-1 for NicA2, but further improvements have been challenging. Here, we identify a strain of Peribacillus frigoritolerans NIC8 which encodes two flavin amine oxidoreductases, Ncox and Pnox. In the presence of oxygen, Ncox and Pnox act on nicotine and pseudooxynicotine respectively with apparent kcat values of 7.7 s-1 and 3.9 s-1. Transient kinetics establishes bimolecular rate constants of 51100 M-1s-1 and 81000 M-1s-1 for the half-reactions between Ncox and O2 and between Pnox and O2 respectively, consistent with Ncox and Pnox being bona-fide oxidases. Transcriptomics shows enhanced expression of Ncox and Pnox under nicotine-dependent growth as well as supporting the identification of downstream enzymes. Phylogenetic analysis suggests that Ncox and Pnox arose out of repurposing of homologous enzymes found in Bacillus species. The enzymes we describe may be useful for the development of nicotine addiction therapeutics and for bioconversion of nicotine in waste streams.

biochemistry↗

bioRxiv2026

Antibiotic tolerance due to filamentation shapes β-lactam pharmacodynamics in Escherichia coli

Pharmacodynamic curves describe how changes in drug concentration affect pathogen growth. They are essential for designing treatments that promote pathogen eradication and minimize the evolution of antibiotic resistance. The classical function for modelling pharmacodynamics is a phenomenological, S-shaped curve with stable growth and death rates separated by a single drop. In this study, we characterized the pharmacodynamic curve of the {beta}-lactam antibiotic cefotaxime (CTX) acting against Escherichia coli. We found that the relationship between CTX concentration and net growth rate diverged from classical model predictions, instead yielding a two-step curve defined by distinct phases of growth, population maintenance, and killing. We hypothesized that the intermediate phase arose from antibiotic tolerance conferred by bacterial filaments. Microscopic assessment of treated cells indeed showed a difference in degree of filamentation with concentration. We further sought to explain this with a semi-mechanistic pharmacodynamic function, modelling the binding of CTX to its cellular targets, penicillin binding proteins (PBP) 1 and 3. By incorporating the preferential concentration-dependent binding of CTX to PBP3 and then PBP1, yielding filaments or lysed cells respectively, we replicated the two-step curve in silico. We also assessed the pharmacodynamics of CTX against mutants conferring resistance; these displayed further altered curves, in line with their fitness costs. Altogether, our results show that CTX has a two-step pharmacodynamic curve against E. coli arising from multiple targets separated in their affinity for the antibiotic. We present a model offering a mechanistically grounded framework for capturing such dynamics. These pharmacodynamic curves deserve careful consideration when defining optimal dosing.

microbiology↗

bioRxiv2026

Adenosine A2B Receptor Activation: A Novel Therapeutic Strategy for Accelerating Liver Recovery After Acetaminophen Overdose

An acetaminophen (APAP) overdose is the leading cause of drug-induced hepatotoxicity and acute liver failure (ALF) in the United States. While N-acetylcysteine (NAC), is highly effective when administered early after an overdose, its efficacy decreases with delayed administration. Since most patients present late to the clinic, there is an urgent need for novel late-acting therapeutic options to prevent progression to ALF. We previously demonstrated the benefit of delayed activation of the Adenosine A2B Receptor (A2BAR) in attenuating APAP-induced hepatotoxicity and this study focuses on its effects on liver recovery after injury. Fasted male C57BL/6J mice were treated with 300 mg/kg APAP, followed by activation of A2BAR 6 or 9 h later and sacrifice 24, 48 or 72 h post-APAP with evaluation of liver injury, the innate immune response and liver regeneration. Delayed activation of A2BAR significantly enhanced liver recovery, with accelerated repopulation of the liver by Kupffer cells, increased macrophage migration to the necrotic areas and their faster resolution. A2BAR activation also upregulated lipid metabolism-related genes in non-parenchymal cells and cell proliferation and metabolism genes in hepatocytes. Remarkably, genes such as Cidec and Plin2, crucial for lipid droplet formation, were upregulated, indicating that A2ABR activation enhances lipid metabolism which plays a key role in providing energy for liver regeneration. Overall, these findings highlight the potential of A2BAR activation not only in protecting against liver injury, but also in promoting and accelerating liver regeneration by modulating the innate immune responses and metabolic pathways.

pharmacology and toxicology↗

bioRxiv2026

Differentiating Hepatic and Renal Toxicity Reveals CYP-Independent Mechanisms of Acetaminophen-Induced Acute Kidney Injury

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF), with acute kidney injury (AKI) contributing substantially to morbidity and mortality in those patients. To determine whether APAP-induced AKI depends on hepatic CYP2E1-mediated bioactivation, we used CYP2E1^flox/flox^ mice treated with AAV8-TBG-Cre to selectively delete hepatic CYP2E1 while preserving renal metabolism. Male and female mice received APAP (600 mg/kg) and were evaluated up to 48 hours for liver and kidney injury. Liver-specific CYP2E1 deletion reduced APAP hepatotoxicity, confirming the absence of hepatic NAPQI formation. Despite this protection, both male and female mice treated with AAV8-TBG-Cre and APAP developed progressive renal injury, with marked increases in blood urea nitrogen (BUN) and creatinine, tubular vacuolation, and strong induction of KIM-1 and osteopontin, along with apoptotic cell death at 48 hours. Notably, female mice, lacking renal CYP2E1 and displaying no detectable renal protein adducts, still progressed to AKI, demonstrating that kidney injury can occur through CYP-independent mechanisms. Given that APAP-induced AKI is a delayed injury, we further considered p-aminophenol (PAP), a deacetylation product of APAP, as a potential CYP-independent contributor. These findings support the concept that non-CYP pathways, including PAP formation, may contribute to kidney injury during the later phase of toxicity, although this pathway likely represents only one component of a multifactorial injury process. Together, these results demonstrate that APAP-induced AKI is a kidney-intrinsic process that can develop independently of both hepatic and renal CYP2E1 activity, emphasizing the need for kidney-specific therapeutic strategies for preventing APAP-induced renal injury.

pharmacology and toxicology↗

bioRxiv2026

FOXP3-engineered regulatory T cells restore intestinal barrier integrity in Crohn's disease enteroids via PDGF-AA

Epithelial regeneration and barrier integrity are impaired in inflammatory bowel diseases, including Crohns disease (CD), yet current therapies largely target immune inflammation without directly promoting mucosal repair. While regulatory T cells are classically immunomodulatory, their capacity to directly support human intestinal stem cells (ISCs) and barrier function remains unclear. In this study, we tested the hypothesis that FOXP3-expressing regulatory T cells--engineered CD4LVFOXP3 and thymic-derived Treg (tTreg)--directly support human ISC maintenance and restore epithelial barrier function independent of their immunomodulatory function. Using CD patient ISCs-derived enteroids that display disease-associated damage, we established co-culture with FOXP3-engineered Treg cell-CD4LVFOXP3 or thymic-derived Treg (tTreg). The presence of either CD4LVFOXP3 or tTreg cells enhanced enteroid growth, improved epithelial barrier function, and restored apical-basal polarity of ISCs, indicating reparative capacity. Conversely, activated conventional CD4+ T cells reduced barrier function and abrogated apical-basal polarity. Integrating secretome profiling with ligand add-back and receptor or ligand blockade, we identify the PDGF-AA-PDGFR axis as a key regulator of Treg-mediated intestinal epithelial barrier integrity, but dispensable for Treg suppressive capacity. Collectively, our data delineate a direct, human tissue-intrinsic role of FOXP3-driven Treg in the interaction with ISCs via PDGF-AA-PDGFR, enhancing epithelial barrier function and positioning CD4LVFOXP3 as a treatment approach coupling immunoregulation with epithelial repair. One Sentence SummaryRegulatory T cells improve the intestinal epithelial barrier function, primarily, through the PDGF-AA-PDGFR axis

immunology↗

bioRxiv2026

Epigenetic signature at FOXP3 distal enhancer affects regulatory T cell development in Kabuki syndrome

Kabuki syndrome (KS) is a congenital developmental disorder caused by germinal pathogenic variants in the lysine methyltransferase 2D (KMT2D, KS1) or lysine demethylase 6A (KDM6A, KS2) genes. Kabuki patients display mental retardation, multiorgan malformations and immune dysregulation - ranging from immunodeficiency to autoimmunity - which strongly compromises their life expectancy. We explored whether the complex immunological scenario of Kabuki syndrome 1 subjects (Ks) could be ascribed to an altered generation of CD4+FOXP3+ regulatory T cells (Tregs). We report that pediatric Ks carrying KMT2D pathogenic variants show a significant reduction of Tregs. DNA methylation analysis reveals a specific methylation pattern at the FOXP3 distal enhancer that correlates with decreased FOXP3 transcription early during Treg cell induction and promotes T helper (Th)-2 lineage differentiation. Finally, in vitro T cell demethylation rescues FOXP3 expression and Treg induction in Ks, offering a novel potential therapeutic perspective. Our findings connect KMT2D loss-of-function to the inhibition of human FOXP3 gene transcription and provide novel molecular insights to explain the immunological phenotype in Ks, thus pinpointing this syndrome as a novel Tregopathy.

immunology↗

bioRxiv2025

Rutin derived microbiota metabolite 3,4 dihydroxybenzoic acid restores antibiotic susceptibility in XDR Gram negative pathogens with a system wide susceptibility signature

Antibiotic potentiators are a practical route to extend the utility of existing drugs against multidrug resistant Gram-negative pathogens, but most natural compound combinations remain mechanistically under defined and are rarely mapped at the level of whole cell susceptibility determinants. Here we apply a metabolite guided potentiator strategy based on gut microbiota derived breakdown products of the dietary polyphenol rutin, and we integrate potentiation phenotyping with system wide genetic susceptibility mapping to guide combination design. Three rutin metabolites, 3,4 dihydroxybenzoic acid (DHBA), 2,4,6 trihydroxybenzoic acid (THBA), and 3,4 dihydroxyphenylacetic acid (DOPAC), were screened against a CDC ARLG reference panel composed of multidrug resistant and extensively drug resistant (XDR) isolates, identifying DHBA as the most broadly active candidate, including against XDR Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. DHBA restored susceptibility to selected antibiotic classes in resistant Gram-negative pathogens, including an approximately sixteen-fold reduction in colistin MIC in mcr 1 positive E. coli. To better understand the potentiation mechanism, we performed a large-scale genetic susceptibility screen of 316 E. coli single gene knockouts and defined a DHBA response architecture enriched for envelope and transport determinants with additional contributions from central metabolism and information processing pathways. Comparative mapping under colistin exposure revealed a distinct susceptibility architecture with limited overlap, supporting the concept that both potentiators and antibiotics engage secondary cellular systems beyond canonical primary mechanisms. In a Mini Bioreactor Array gut community model, DHBA produced a more conserved community shift than colistin. Finally, DHBA-antibiotic combinations improved outcomes in infection relevant models, including improved survival in Galleria mellonella, reduced intestinal burden in Caenorhabditis elegans, and reduced bacterial burden in an ex vivo porcine burn wound infection model. Collectively, these findings support a systems-based framework for developing mechanistically informed potentiator antibiotic-combinations to extend the lifespan of existing antibiotics.

microbiology↗

bioRxiv2025

Co-adapting but not static predators facilitate prey adaptation to a fluctuating environment

Understanding how between-species interactions influence adaptation and population persistence in the face of environmental change is one of the greatest challenges for modern ecology and evolution, with important implications for conservation, and other applied fields. As predation is ubiquitous and may have strong demographic and selective impacts, it is likely to alter how prey respond to a changing environment. However, whether and how adaptation in prey depends on the way selection operates on predators remains little understood. We investigate this question by modeling the evolution of a preys trait whose optimum phenotype for fitness changes with the abiotic environment, and which also influences predation via its match with a trait of a predator species. We first show that when coevolutionary processes explicitly emerge from interactions among individuals, maladaptation in prey is not proportional to the difference between their optimum phenotype and that of predators, as assumed in most coevolutionary models. In a fluctuating environment, how well the prey track their moving optimum crucially depends on whether and how the predator evolves. Adaptive tracking of the optimum in prey is facilitated if predators track the same optimum, but hampered if predators have a fixed optimum, or cannot evolve. With eco-evolutionary dynamics, phenotypic mismatch reduces the population size of predators, thus decreasing the strength of predatory selection, but the qualitative influence of predation on prey maladaptation remains otherwise similar. Our findings highlight the importance of the evolutionary context of predators for their impacts on prey, and challenge conservation strategies for prey based on removing their predators. Significance statementSpecies in the wild face the dual challenge of adapting to their changing physical environment and coping with detrimental interactions with other species, including predators. While evolution by natural selection may jointly overcome both these challenges, the outcome of this process depends on whether the selective forces from predation and environmental change reinforce or oppose each other. Here, we show that the influence of coevolving predators on prey adaptation to a changing environment strongly depends on the selective scenario for the predators. Adaptation in prey is facilitated if predators also adapt to the changing environment, but hampered if selection favors a constant phenotype in predators. Our results challenge the relevance of predator removal as a conservation strategy to preserve prey.

evolutionary biology↗

bioRxiv2024

PATHOGEN GROWTH AND VIRULENCE DYNAMICS DRIVE THE HOST EVOLUTION AGAINST COINFECTIONS

Coinfections, or the simultaneous infection of hosts by multiple pathogens, are widespread in nature with significant negative impacts on global health. Can hosts evolve against such coinfections as effectively as they would against individual pathogens? Also, what roles do individual pathogens play during such evolution? Here, we combined theoretical models and experiments with Tribolium castaneum populations evolving against two coinfecting bacterial pathogens, with contrasting growth and virulence dynamics, to reveal that fast-growing pathogens inflicting rapid mortality surges (i.e., fast-acting) restrict adaptive success against coinfections. While hosts rapidly evolved better survival against slow-growing bacteria causing long-lasting infections, evolution against coinfection was significantly delayed and resembled slow adaptation against fast-acting pathogens. Moreover, limited scopes of immunomodulation against fast-acting pathogens during coinfections can drive the observed adaptive patterns. Overall, we provide new insights into how adaptive dynamics and mechanistic bases against coinfections are critically regulated by individual pathogens growth and virulence dynamics.

evolutionary biology↗

bioRxiv2023

Free-floating Bacteria Transcriptionally Respond to Shear Flow

Planktonic (free-floating) cells are typically assumed to be oblivious to any flow that carries them. Here we discover that planktonic bacteria can sense flow to induce gene expression changes that are beneficial in flow. Specifically, planktonic P. aeruginosa induce shear-rate-dependent genes that promote growth in low oxygen environments. Untangling this mechanism revealed that in flow, motile P. aeruginosa spatially redistribute, leading to cell density changes that activate quorum sensing, which in turn enhances oxygen uptake rate. In diffusion-limited environments, including those commonly encountered by bacteria, flow-induced cell density gradients also independently generate oxygen gradients that alter gene expression. Mutants deficient in this newly-discovered flow sensing mechanism exhibit decreased fitness in flow, suggesting that this dynamic coupling of biological and mechanical processes can be physiologically significant. One-Sentence SummaryFree-floating bacteria integrate biological and mechanical responses to adaptively alter gene expression in flow environments

microbiology↗

bioRxiv2023

Exogenous 4-1BB co-stimulation enhances memory B cell response during malaria.

T-dependent germinal center (GC) output, comprising plasma cells (PC) and memory B cells (MBC), is crucial for the clearance of Plasmodium infection and protection against reinfection. In this study, we examined the effect of an agonistic antibody targeting 4-1BB (CD137), a member of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF), during experimental malaria. Here we show that exogenous 4-1BB stimulation, despite delaying the effector GC response, surprisingly enhanced humoral memory recall and protection from reinfection. Single cell RNA and ATAC sequencing of MBCs from mice that received 4-1BB stimulation revealed distinct populations with transcriptional and epigenetic signatures indicative of superior recall and proliferative potential. Importantly, our results indicate that the effects of 4-1BB stimulation are dependent on IL-9R signaling in B cells but independent of parasite load during primary infection. Our study proposes an immunomodulatory approach to enhance the quality of the MBC pool, providing superior protection during infection and vaccination, particularly in the context of malaria.

immunology↗

bioRxiv2023

Klebsiella pneumoniae Co-infection Leads to Fatal Pneumonia in SARS-CoV-2-infected Mice

SARS-CoV-2 patients have been reported to have high rates of secondary Klebsiella pneumoniae infections. Klebsiella pneumoniae is a commensal that is typically found in the respiratory and gastrointestinal tracts. However, it can cause severe disease when a persons immune system is compromised. Despite a high number of K. pneumoniae cases reported in SARS-CoV-2 patients, a co-infection animal model evaluating the pathogenesis is not available. We describe a mouse model to study disease pathogenesis of SARS-CoV-2 and K. pneumoniae co-infection. BALB/cJ mice were inoculated with mouse-adapted SARS-CoV-2 followed by a challenge with K. pneumoniae. Mice were monitored for body weight change, clinical signs, and survival during infection. The bacterial load, viral titers, immune cell accumulation and phenotype, and histopathology were evaluated in the lungs. The co-infected mice showed severe clinical disease and a higher mortality rate within 48 h of K. pneumoniae infection. The co-infected mice had significantly elevated bacterial load in the lungs, however, viral loads were similar between co-infected and single-infected mice. Histopathology of co-infected mice showed severe bronchointerstitial pneumonia with copious intralesional bacteria. Flow cytometry analysis showed significantly higher numbers of neutrophils and macrophages in the lungs. Collectively, our results demonstrated that co-infection of SARS-CoV-2 with K. pneumoniae causes severe disease with increased mortality in mice.

immunology↗

bioRxiv2023

PandoGen: Generating complete instances of future SARS-CoV2 sequences using Deep Learning

One of the challenges in a viral pandemic is the emergence of novel variants with different phenotypical characteristics. An ability to forecast future viral individuals at the sequence level enables advance preparation by characterizing the sequences and closing vulnerabilities in current preventative and therapeutic methods. In this article, we explore, in the context of a viral pandemic, the problem of generating complete instances of undiscovered viral protein sequences, which have a high likelihood of being discovered in the future using protein language models. Current approaches to training these models fit model parameters to a known sequence set, which does not suit pandemic forecasting as future sequences differ from known sequences in some respects. To address this, we develop a novel method, called PandoGen, to train protein language models towards the pandemic protein forecasting task. PandoGen combines techniques such as synthetic data generation, conditional sequence generation, and reward-based learning, enabling the model to forecast future sequences, with a high propensity to spread. Applying our method to modeling the SARS-CoV-2 Spike protein sequence, we find empirically that our model forecasts twice as many novel sequences with five times the case counts compared to a model that is thirty times larger. Our method forecasts unseen lineages months in advance, whereas models 4x and 30x larger forecast almost no new lineages. When trained on data available up to a month before the onset of important Variants of Concern, our method consistently forecasts sequences belonging to those variants within tight sequence budgets. PandoGen is available at: https://github.com/UIUC-ChenLab/PandoGen

bioinformatics↗

bioRxiv2022

Loss of FOXP3 function causes expansion of two pools of autoreactive T cells in patients with IPEX syndrome.

The monogenic autoimmune disease Immunedysregulation polyendocrynopathy entheropathy X-linked syndrome (IPEX) has elucidated the essential function of the transcription factor FOXP3 and of thymic-derived regulatory T (Treg) cells in controlling autoimmunity. However, the presence of autoreactive T cells in IPEX remains undetermined, thus representing a crucial gap in understanding the origin of autoimmunity in a FOXP3 deficient immune system. Combining epigenetic analysis as a lineage marker of Treg identity and TCR sequencing to assess the self-reactive clones, we showed that IPEX patients have two pools of expanded autoreactive T cells. The first originates from the expansion of autoreactive effector T cells (Teff), likely due to loss of Treg suppressive function since it is absent in carrier mothers, in whom Treg cells are functional. The second pool originates, unexpectedly, from Treg cells which lose their phenotypic markers, including CD25 and FOXP3. We call these loss of identity Treg cells and show that they are i) suppressed by healthy donor Treg in a patient post hematopoietic transplantation despite low donor chimerism, and ii) not detectable in patients with Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome (APECED), a monogenic autoimmune disease of thymic origin. Moreover, we demonstrate that FOXP3 knock-out in Treg cells leads to increased Treg expansion and production of Th17 and Th2 cytokines, known to be increased in IPEX patients. These results suggest that the loss of identity Treg cells could directly contribute to immune dysregulation in IPEX. Collectively, we provide a better understanding of autoimmunity and novel ways to monitor the effects of Treg cell therapies in IPEX disease or other autoimmune diseases. One Sentence SummaryMutations of FOXP3 gene in humans cause expansion of autoreactive T cells originating from both effector T cells and regulatory T cells which gain effector function.

immunology↗

bioRxiv2022

Detection and discrimination of single nucleotide polymorphisms by quantification of CRISPR-Cas catalytic efficiency

The specificity of CRISPR-Cas12 assays is attractive for the detection of single nucleotide polymorphisms (SNPs) implicated in, e.g., SARS-CoV-2 variants. Such assays often employ endpoint measurements of SNP or wild type (WT) activated Cas12 trans-cleavage activity; however, the fundamental kinetic effects of SNP versus WT activation remain unknown. We here show that endpoint-based assays are limited by arbitrary experimental choices (like used reporter concentration and assay duration) and work best for known target concentrations. More importantly, we show that SNP (versus WT) activation results in measurable shifts in the Cas12 trans-cleavage substrate affinity (KM) and apparent catalytic efficiency [Formula]. To address endpoint-based assay limitations, we then develop an assay based on the quantification of Michalis-Menten parameters and apply this assay to a 20-base pair WT target of the SARS-CoV-2 E gene. We find that the [Formula] measured for WT is 130-fold greater than the lowest [Formula] among all 60 measured SNPs (compared to a 4.8-fold for endpoint fluorescence of the same SNP). KM also offers strong ability to distinguish SNPs, varies 27-fold over all the cases, and is insensitive to target concentration. Lastly, we point out trends among kinetic rates and SNP base and location within the CRISPR-Cas12 targeted region.

bioengineering↗

bioRxiv2022

Fundamental limits of amplification-free CRISPR-Cas12 and Cas13 diagnostics

Interest in CRISPR diagnostics continues to increase. CRISPR-Cas12 and -Cas13 based detection are particularly interesting as they enable highly specific detection of nucleic acids. The fundamental sensitivity limits of Cas12 and Cas13 enzymes are governed by their kinetic rates and are critical to develop amplification-free assays. However, these kinetic rates remain poorly understood and their reporting has been inconsistent. We here measure kinetic parameters for several enzymes (LbCas12a, AsCas12a, AapCas12b, LwaCas13a and LbuCas13a) and evaluate their limits of detection (LoD) for amplification-free target detection. Collectively, we here present quantitation of enzyme kinetics for 14 gRNAs and nucleic acid targets for a total of 50 sets of kinetic rate parameters and 25 LoDs. Importantly, we also validate the self-consistency our measurements by comparing trends and limiting behaviors with a Michaelis-Menten, trans-cleavage reaction kinetics model. Our measurements reveal that activated Cas12 and Cas13 enzymes exhibit typical trans-cleavage catalytic efficiencies between order 105 and 106 M-1 s-1. Moreover, for assays that use fluorescent reporter molecules (ssDNA and ssRNA) for target detection, we find most CRISPR enzymes have an amplification-free LoD in the picomolar range. We find also that successful detection of target requires cleavage (by activated CRISPR enzyme) of at least ~0.1% of the fluorescent reporter molecules. This fraction of cleaved reporters is required to differentiate signal from background, and we hypothesize that this fraction is largely independent of the detection method (i.e., endpoint vs reaction velocity). Our results provide a map of the feasible application range and highlight areas of improvement for the emerging field of CRISPR diagnostics.

bioengineering↗

bioRxiv2021

Enzyme kinetics of CRISPR molecular diagnostics

CRISPR diagnostic assays have gained significant interest in the last few years. This interest has grown rapidly during the current COVID-19 pandemic where CRISPR diagnostics have been frontline contenders for rapid testing solutions. This surge in CRISPR diagnostics research prompts the following question: What exactly are the achievable limits of detection and associated assay times enabled by the kinetics of Cas12 and Cas13 enzymes? To address this question, we here present a model based on Michaelis-Menten enzyme kinetics theory applied to Cas enzymes. We use the model to develop analytical solutions for reaction kinetics and develop back-of-the{-} envelope criteria to validate and check for consistency in reported enzyme kinetics parameters. We applied our analyses to all studies known to us which report Michaelis-Menten-type kinetics data for CRISPR associated enzymes. These studies include all subtypes of Cas12 and Cas13 and orthologs. We found all studies but one clearly violate at least two of our three rules of consistency. We further use our model to explore ranges of reaction time scales and degree of reaction completion for practically relevant target concentrations applicable to CRISPR-diagnostic assays.

bioengineering↗