Browsing by Author "Krohannon, Alexander"

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    CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion
    (BMC, 2022) Krohannon, Alexander; Srivastava, Mansi; Rauch, Simone; Srivastava, Rajneesh; Dickinson, Bryan C.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and Computing
    Background: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. Results: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Conclusions: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.
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    CRISPR Cas13 sg A Designer
    Krohannon, Alexander; Janga, Sarath
    Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (cas), has resulted in its widespread use for improved understanding of a variety of biological systems, by enabling large-scale perturbation of the genomes and transcriptomes. Cas13, a lesser studied cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The cas13 system utilizes base complementarity between a crRNA (crispr RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Additionally, transcripts exhibit complex three-dimensional structures and interact with multiple RBPs (RNA binding proteins), both of which further limit the scope of effective target sequences. As a result, there currently exists no method to predict whether a crRNA will be effective or not. This project aims to create a novel machine learning model to predict the efficacy of a crRNA; using publicly available RNA knockdown data from cas13 characterization experiments1 for 555 sgRNAs targeting the transcriptome in HEK293 cells. Numerous types of machine learning models were tested during development including ARD (Automatic Relevance Determination), Bayesian Ridge, Elastic Net, Huber, K-Nearest Neighbors, Linear, and SVM (Support Vector Machines). K-Nearest Neighbors showed the greatest accuracy, predicting knockdown value within 10% of the mean value in 39.1% of the instances. Despite their differences in accuracy, Elastic Net had the lowest precision error (0.0638) and SVM had the lowest recall error (0.0094). Implementation of this model will allow for rapid deployment of new types of screening the transcriptomes and enable potential treatments for diseases linked with aberrations in RNA regulatory processes.
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    Datawiz-IN: Summer Research Experience for Health Data Science Training
    (Research Square, 2024-03-29) Afreen, Sadia; Krohannon, Alexander; Purkayastha, Saptarshi; Janga, Sarath Chandra; Biomedical Engineering and Informatics, Luddy School of Informatics, Computing, and Engineering
    Background: Good science necessitates diverse perspectives to guide its progress. This study introduces Datawiz-IN, an educational initiative that fosters diversity and inclusion in AI skills training and research. Supported by a National Institutes of Health R25 grant from the National Library of Medicine, Datawiz-IN provided a comprehensive data science and machine learning research experience to students from underrepresented minority groups in medicine and computing. Methods: The program evaluation triangulated quantitative and qualitative data to measure representation, innovation, and experience. Diversity gains were quantified using demographic data analysis. Computational projects were systematically reviewed for research productivity. A mixed-methods survey gauged participant perspectives on skills gained, support quality, challenges faced, and overall sentiments. Results: The first cohort of 14 students in Summer 2023 demonstrated quantifiable increases in representation, with greater participation of women and minorities, evidencing the efficacy of proactive efforts to engage talent typically excluded from these fields. The student interns conducted innovative projects that elucidated disease mechanisms, enhanced clinical decision support systems, and analyzed health disparities. Conclusion: By illustrating how purposeful inclusion catalyzes innovation, Datawiz-IN offers a model for developing AI systems and research that reflect true diversity. Realizing the full societal benefits of AI requires sustaining pathways for historically excluded voices to help shape the field.
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    Epitranscriptomics in parasitic protists: Role of RNA chemical modifications in posttranscriptional gene regulation
    (Public Library of Science, 2022-12-22) Catacalos, Cassandra; Krohannon, Alexander; Somalraju, Sahiti; Meyer, Kate D.; Janga, Sarath Chandra; Chakrabarti, Kausik; BioHealth Informatics, School of Informatics and Computing
    "Epitranscriptomics" is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression and biological processes. There are several RNA base modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ), etc. that play pivotal roles in fine-tuning gene expression in almost all eukaryotes and emerging evidences suggest that parasitic protists are no exception. In this review, we primarily focus on m6A, which is the most abundant epitranscriptomic mark and regulates numerous cellular processes, ranging from nuclear export, mRNA splicing, polyadenylation, stability, and translation. We highlight the universal features of spatiotemporal m6A RNA modifications in eukaryotic phylogeny, their homologs, and unique processes in 3 unicellular parasites-Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. and some technological advances in this rapidly developing research area that can significantly improve our understandings of gene expression regulation in parasites.