Browsing by Subject "Protein expression"

Now showing 1 - 3 of 3
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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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    In Vivo siRNA Delivery and Rebound of Renal LRP2 in Mice
    (Hindawi Publishing Corporation, 2017) Eadon, Michael T.; Cheng, Ying-Hua; Hato, Takashi; Benson, Eric A.; Ipe, Joseph; Collins, Kimberly S.; De Luca, Thomas; El-Achkar, Tarek M.; Bacallao, Robert L.; Skaar, Todd C.; Dagher, Pierre C.; Medicine, School of Medicine
    siRNA stabilized for in vivo applications is filtered and reabsorbed in the renal proximal tubule (PT), reducing mRNA expression transiently. Prior siRNA efforts have successfully prevented upregulation of mRNA in response to injury. We proposed reducing constitutive gene and protein expression of LRP2 (megalin) in order to understand its molecular regulation in mice. Using siRNA targeting mouse LRP2 (siLRP2), reduction of LRP2 mRNA expression was compared to scrambled siRNA (siSCR) in mouse PT cells. Mice received siLRP2 administration optimized for dose, administration site, carrier solution, administration frequency, and administration duration. Kidney cortex was collected upon sacrifice. Renal gene and protein expression were compared by qRT-PCR, immunoblot, and immunohistochemistry (IHC). Compared to siSCR, siLRP2 reduced mRNA expression in PT cells to 16.6% ± 0.6%. In mouse kidney cortex, siLRP2 reduced mRNA expression to 74.8 ± 6.3% 3 h and 70.1 ± 6.3% 6 h after administration. mRNA expression rebounded at 12 h (160.6 ± 11.2%). No megalin renal protein expression reduction was observed by immunoblot or IHC, even after serial twice daily dosing for 3.5 days. Megalin is a constitutively expressed protein. Although LRP2 renal mRNA expression reduction was achieved, siRNA remains a costly and inefficient intervention to reduce in vivo megalin protein expression.
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    Per2-Mediated Vascular Dysfunction Is Caused by the Upregulation of the Connective Tissue Growth Factor (CTGF).
    (PLOS, 2016) Jadhav, Vaishnavi; Luo, Qianyi; M. Dominguez, James; Al-Sabah, Jude; Chaqour, Brahim; Grant, Maria B.; Bhatwadekar, Ashay D.; Department of Ophthalmology, IU School of Medicine
    Period 2-mutant mice (Per2m/m), which possess a circadian dysfunction, recapitulate the retinal vascular phenotype similar to diabetic retinopathy (DR). The vascular dysfunction in Per2m/m is associated with an increase in connective tissue growth factor (CTGF/CCN2). At the molecular level, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. The nuclear binding of β-catenin to a transcription factor, lymphoid enhancer binding protein (Lef)/