Biology subjects

Srikanth, S.

Publications and source records attributed to Srikanth, S..

9 recordsLinked to original sources

bioRxiv2026

Direct comparison of CRISPR knockout and interference with Perturb-seq

CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) are two workhorse technologies for loss-of-function studies, yet direct comparisons between the two are scant relative to their widespread adoption. Here, we establish benchmarking libraries for Cas9-based CRISPRko and CRISPRi screens using Perturb-seq as the read-out. For both modalities, we observe consistent transcriptional signatures among cells with the same genes perturbed, strong evidence of on-target signal. We also examine tradeoffs between modalities: while CRISPRi guides demonstrate heightened rates of off-target activity, we also observe artifacts stemming from the cellular response to double-stranded breaks with the use of CRISPRko. The libraries and analyses presented here will be a useful benchmarking and de-risking resource for any group preparing for a large-scale Perturb-seq screen.

genomics↗

bioRxiv2026

Optimized parameters for Cas9 CRISPR interference library design

CRISPR interference (CRISPRi) is a powerful technology for studying loss-of-function phenotypes, enabling transient and reversible control of gene expression without the introduction of double-stranded DNA breaks. The cost of conducting large-scale CRISPR screens necessitates the selection of effective and specific sgRNAs for the design of compact libraries. While several genome-wide Cas9 CRISPRi libraries have been created, updates to transcript annotations, the generation of higher-resolution chromatin accessibility datasets and the development of newer on-target prediction models motivate an updated CRISPRi library design approach. Here, we generate large CRISPRi datasets tiling essential and nonessential genes. We compare the performance of multiple KRAB domain systems, develop an updated CRISPRi-specific on-target scoring scheme, and quantitatively characterize off-target effects associated with seed sequence patterns. We leverage these findings to design an optimized Cas9 CRISPRi library, Katsano, and validate its performance with genome-wide viability screens.

bioengineering↗

bioRxiv2025

Serial Imaging of Tumor and microEnvironment (SITE) platform for live-cell ex vivo modeling of primary and metastatic cancer dynamics.

Understanding cancer initiation and progression is extremely challenging, in part due to experimental limitations in measuring and interpreting key signalling and tumour-microenvironment (TME) interactions that determine changes in cell and tissue behaviours over time. Here we developed SITE (Serial Imaging of Tumour and microEnvironment), a spatially-and temporally integrated, modular, ex vivo platform enabling quantitative analysis of TME interaction dynamics, signalling, and cell fate at single cell and tissue scales. Applied to modelling primary and lung metastatic breast cancer, SITE revealed tissue-specific TME interactions and ERK signalling patterns linked to distinct single-cell behaviours. We found that the earliest steps in tumour establishment and metastatic seeding involved active cell protrusion and the establishment of a multicellular niche interfacing tumour and host. Experimental and mathematical modelling showed that ERK signalling was co-influenced by these interactions, where cancer cluster formation increased signalling via the establishment of local signalling circuits. Disruption of these signalling circuits led to tissue-specific impacts on cancer intrinsic and TME interaction dynamics. Here, we modelled breast cancer as a test case, demonstrating the broad utility of SITE for quantitative exploration of TME interaction dynamics- closing a significant gap in experimental capabilities between in vivo models and in vitro systems.

cancer biology↗

bioRxiv2025

Balancing off-target and on-target considerations for optimized Cas9 CRISPR knockout library design

The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations yield library deprecation over time, further motivating an updated genome-wide design effort. Recently, we have developed an enhanced model, Rule Set 3, which leveraged an expansive training set and feature space to predict guide efficacy. However, the benefit of such advances to library design is limited by current approaches to balance predictions of on-target activity with off-target considerations. Here we present a guide selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides. We pair this model with strategic design choices to create Jacquere, an updated, optimized, and validated Cas9 CRISPR knockout (CRISPRko) genome-wide library for the human genome.

genomics↗

bioRxiv2025

Calcium Dependent Conformational Changes in Human Transglutaminase 2 and its Implications in Celiac Disease

Transglutaminase 2 (TG2) serves as a modifiable transamidating acyltransferase that precipitates calcium-induced protein alterations. The enzyme plays a crucial role in the cell and disease states, such as tissue repair, calcium signal transduction, celiac disease, and cancer. It is implicated in protein crosslinking and has been found in high concentrations in the small intestines of those with celiac disease. The function of TG2 hinges upon calcium ions binding to particular sites on the enzyme. In this study, we delve into the contribution of calcium-responsive transglutaminase 2 (TG2) in celiac disease, utilizing both molecular dynamics simulations and coarse-grained models, and investigate the impact of non-synonymous single nucleotide polymorphisms (nsSNPs) on TG2. Molecular dynamics reveal prominent conformational differences between the open and closed conformations. In the coarse-grained model, key residues are found adjacent to the active site in the open conformation, while in the closed conformation, key residues are distant from the active site. We further explore the functional impact of non-synonymous single nucleotide polymorphisms (nsSNPs) in TG2 using both sequence-based and structure-based computational tools. Through a consensus approach, we identify ten nsSNPs that are predicted to destabilize TG2 or alter its structural flexibility, with mutations such as R48H, E186Q, C277S, and E549G likely to influence active site accessibility and calcium coordination. The findings from this research enhances our understanding of the molecular processes underpinning celiac disease and helps facilitate innovative treatment approaches that target calcium-responsive TG2.

bioinformatics↗

bioRxiv2024

A 96-Well Polyacrylamide Gel for Electrophoresis and Western Blotting

Western blotting is a stalwart technique for analyzing specific proteins and/or their post-translational modifications. However, it remains challenging to accommodate more than ~10 samples per experiment without substantial departure from trusted, established protocols involving accessible instrumentation. Here, we describe a 96-sample western blot that conforms to standard 96-well plate dimensional constraints and has little operational deviation from standard western blotting. The main differences are that (i) submerged polyacrylamide gel electrophoresis is operated horizontally (similar to agarose gels) as opposed to vertically, and (ii) a 6 mm thick gel is used, with 2 mm most relevant for membrane transfer (vs ~1 mm typical). Results demonstrate both wet and semi-dry transfer are compatible with this gel thickness. The major tradeoff is reduced molecular weight resolution, due primarily to less available migration distance per sample. We demonstrate proof-of-principle using gels loaded with molecular weight ladder, recombinant protein, and cell lysates. We expect the 96-well western blot will increase reproducibility, efficiency (cost and time ~8-fold), and capacity for biological characterization relative to established western blots.

biochemistry↗

bioRxiv2023

Comparative Analysis of Molecular Pathogenic Mechanisms and Antiviral Development Targeting Old and New World Hantaviruses

BackgroundHantaviruses - dichotomized into New World (i.e. Andes virus, ANDV; Sin Nombre virus, SNV) and Old-World viruses (i.e. Hantaan virus, HTNV) - are zoonotic viruses transmitted from rodents to humans. Currently, no FDA-approved vaccines against hantaviruses exist. Given the recent breakthrough to human-human transmission by the ANDV, an essential step is to establish an effective pandemic preparedness infrastructure to rapidly identify cell tropism, infective potential, and effective therapeutic agents through systematic investigation. MethodsWe established human cell model systems in lung (airway and distal lung epithelial cells), heart (pluripotent stem cell-derived (PSC-) cardiomyocytes), and brain (PSC-astrocytes) cell types and subsequently evaluated ANDV, HTNV and SNV tropisms. Transcriptomic, lipidomic and bioinformatic data analyses were performed to identify the molecular pathogenic mechanisms of viruses in different cell types. This cell-based infection system was utilized to establish a drug testing platform and pharmacogenomic comparisons. ResultsANDV showed broad tropism for all cell types assessed. HTNV replication was predominantly observed in heart and brain cells. ANDV efficiently replicated in human and mouse 3D distal lung organoids. Transcriptomic analysis showed that ANDV infection resulted in pronounced inflammatory response and downregulation of cholesterol biosynthesis pathway in lung cells. Lipidomic profiling revealed that ANDV-infected cells showed reduced level of cholesterol esters and triglycerides. Further analysis of pathway-based molecular signatures showed that, compared to SNV and HTNV, ANDV infection caused drastic lung cell injury responses. A selective drug screening identified STING agonists, nucleoside analogues and plant-derived compounds that inhibited ANDV viral infection and rescued cellular metabolism. In line with experimental results, transcriptome data shows that the least number of total and unique differentially expressed genes were identified in urolithin B- and favipiravir-treated cells, confirming the higher efficiency of these two drugs in inhibiting ANDV, resulting in host cell ability to balance gene expression to establish proper cell functioning. ConclusionsOverall, our study describes advanced human PSC-derived model systems and systems-level transcriptomics and lipidomic data to better understand Old and New World hantaviral tropism, as well as drug candidates that can be further assessed for potential rapid deployment in the event of a pandemic.

microbiology↗

bioRxiv2021

Theta oscillations in the prefrontal-hippocampal circuit do not couple to respiration-related oscillations

Oscillatory activity is thought to coordinate neural computations across brain regions, and theta oscillations are critical for learning and memory. Because the frequency of respiratory-related oscillations (RROs) in rodents can overlap with the frequency of theta in the prefrontal cortex (PFC) and the hippocampus, we asked whether odor-cued working memory may be supported by coupling between these two oscillations. We first confirmed that RROs are propagated to the hippocampus and PFC and that RRO frequency overlaps with canonical theta frequency. However, we found low coherence between RROs and local theta oscillations in the hippocampus-PFC network when the two types of oscillations overlapped in frequency. This effect was observed during all behavioral phases including during movement and while odors were actively sampled when stationary. Despite the similarity in frequency, RROs and theta oscillations therefore appear to be limited to supporting computation in distinct networks, which suggests that sustained long-range coordination between oscillation patterns that depend on separate pacemakers is not necessary to support at least one type of working memory.

neuroscience↗

bioRxiv2021

ORAI1 establishes resistance to SARS-CoV-2 infection by regulating tonic type I interferon signaling

ORAI1 and STIM1 are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of a cytosolic nucleic acid sensing pathway. Using ORAI1 and STIM1 knockout cells, we examined their contribution to the host response to SARS-CoV-2 infection. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection due to enhanced type I interferon response. On the contrary, ORAI1 knockout cells showed high susceptibility to SARS-CoV-2 infection as judged by increased expression of viral proteins and a high viral load. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic interferon signaling. Transcriptome analysis showed downregulation of multiple cellular defense mechanisms, including antiviral signaling pathways in ORAI1 knockout cells, which are likely due to reduced expression of the Ca2+-dependent transcription factors of the activator protein 1 (AP-1) family and MEF2C. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the baseline type I interferon level, which is a determinant of host resistance to SARS-CoV-2 infection.

microbiology↗