Biology subjects

Santiago, J. G.

Publications and source records attributed to Santiago, J. G..

6 recordsLinked to original sources

bioRxiv2026

Experimental Methods for CRISPR Enzyme Assays with Fluorescence Readout

Fluorescence-based CRISPR diagnostic assays have become a popular platform for nucleic acid detection due to their programmability, configurability, specificity, and compatibility with standard laboratory equipment. However, reported enzymatic kinetic rates and limits of detection for CRISPR trans-cleavage assays vary by several orders of magnitude across the literature. This variation in performance parameters is coupled with and exacerbated by inconsistent calibration, incomplete correction of measurement biases, and nonstandardized or incomplete data-analysis procedures. We present an experimental protocol and quantitative analysis framework for fluorescence-based enzyme assays using routine laboratory instrumentation, including thermocyclers and fluorescence microplate readers. Building on previous studies of CRISPR enzyme kinetics and fluorescence calibration, we describe procedures for flat-field and background correction; comprehensive fluorescence calibration including correction for inner-filter-effect; quantification and implications of reporter degradation; extraction of Michaelis-Menten kinetic parameters; and determination of assay limits of detection. We provide step-by-step experimental guidelines and open-source Python implementations for each stage of the workflow. Using representative Cas12 trans-cleavage datasets, we demonstrate that explicit fluorescence calibration and correction procedures substantially reduce systematic bias in measured kinetic rates and improve consistency between experiments. Our framework aims to establish standardized practices for quantitative fluorescence-based CRISPR assays and provides researchers with practical tools for reproducible kinetic characterization and rational assay design.

biochemistry↗

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↗

bioRxiv2020

Electric-field-driven microfluidics for rapid CRISPR-based diagnostics and its application to detection of SARS-CoV-2

The rapid spread of COVID-19 across the world has revealed major gaps in our ability to respond to new virulent pathogens. Rapid, accurate, and easily configurable molecular diagnostic tests are imperative to prevent global spread of new diseases. CRISPR-based diagnostic approaches are proving to be useful as field-deployable solutions. In a basic form of this assay, the CRISPR-Cas12 enzyme complexes with a synthetic guide RNA (gRNA). This complex is activated when it highly specifically binds to target DNA, and the activated complex non-specifically cleaves single-stranded DNA reporter probes labeled with a fluorophore-quencher pair. We recently discovered that electric field gradients can be used to control and accelerate this CRISPR assay by co-focusing Cas12-gRNA, reporters, and target. We achieve an appropriate electric field gradient using a selective ionic focusing technique known as isotachophoresis (ITP) implemented on a microfluidic chip. Unlike previous CRISPR diagnostic assays, we also use ITP for automated purification of target RNA from raw nasopharyngeal swab sample. We here combine this ITP purification with loop-mediated isothermal amplification, and the ITP-enhanced CRISPR assay to achieve detection of SARS-CoV-2 RNA (from raw sample to result) in 30 min for both contrived and clinical nasopharyngeal swab samples. This electric field control enables a new modality for a suite of microfluidic CRISPR-based diagnostic assays. Significance statementRapid, early-stage screening is especially crucial during pandemics for early identification of infected patients and control of disease spread. CRISPR biology offers new methods for rapid and accurate pathogen detection. Despite their versatility and specificity, existing CRISPR-diagnostic methods suffer from the requirements of up-front nucleic acid extraction, large reagent volumes, and several manual steps--factors which prolong the process and impede use in low resource settings. We here combine on-chip electric-field control in combination with CRIPSR biology to directly address these limitations of current CRISPR-diagnostic methods. We apply our method to the rapid detection of SARS-CoV-2 RNA in clinical samples. Our method takes 30 min from raw sample to result, a significant improvement over existing diagnostic methods for COVID-19.

bioengineering↗

bioRxiv2017

Correlation of gene expressions between nucleus and cytoplasm reflects single-cell physiology

BackgroundEukaryotes transcribe RNAs in nuclei and transport them to the cytoplasm through multiple steps of post-transcriptional regulation. Existing single-cell sequencing technologies, however, are unable to analyse nuclear (nuc) and cytoplasmic (cyt) RNAs separately and simultaneously. Hence, there remain challenges to discern correlation, localisation, and translocation between them.\n\nResultsHere we report a microfluidic system that physically separates nucRNA and cytRNA from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA per cell, quantifies gene expression in the subcellular compartments and combines them to create a novel single-cell RNA-seq data enabled by our system, which we here term in-silico single cell.\n\nConclusionsLeveraging SINC-seq, we discovered three distinct natures of correlation among cytRNA and nucRNA that reflected the physiological state of single cells: The cell-cycle-related genes displayed highly correlated expression pattern with minor differences; RNA splicing genes showed lower nucRNA-to-cytRNA correlation, suggesting a retained intron may be implicated in inhibited mRNA transport; A chemical perturbation, sodium butyrate treatment, transiently distorted the correlation along differentiating human leukemic cells to erythroid cells. These data uniquely provide insights into the regulatory network of mRNA from nucleus toward cytoplasm at the single cell level.

genomics↗