Abstract: TH-PO0399
Elucidation of Proteinase-3 Regulation Through Clustered Regularly Interspaced Short Palindromic Repeats Interference (CRISPRi)-Screening Using a Novel Genetic Reporter
Session Information
- Glomerular Diseases: Autoimmune Diseases
October 22, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Taus, Patrick J., The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States
- Humphreys, Kaitlyn M., The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
- Snipes, Stephen Andrew, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States
- Liu, Hongwei, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina, United States
- Falk, Ronald, The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
- Ciavatta, Dominic J., The University of North Carolina at Chapel Hill Kidney Center, Chapel Hill, North Carolina, United States
Background
Anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis is a highly morbid and potentially fatal autoimmune disease that is initiated by autoantibody engagement of the neutrophil proteins, proteinase-3 (PR3) or myeloperoxidase. We sought to develop a transcriptional reporter to comprehensively identify the factors required for PR3 expression using CRISPRi screening.
Methods
A reporter of PRTN3 transcription was constructed and validated as shown in Figure 1. Reporter lines were screened using a Gene Expression library at 1000X coverage. 6 days later cells were sorted based on mCherry intensity and sgRNAs enriched in the lowest decile identified using Robust Rank Aggregation (RRA) analysis.
Results
7 genes with an FDR <0.05, log-2-fold enrichment >0.3, and known DNA-binding motifs and no obvious associations with the generic cellular transcription machinery were further investigated. From this cohort, SPI1 and GATA2 depletion had the greatest impact on PRTN3 expression (Fig 2A). This phenotype held true in a second cell line, HL60, and in both lines depletion of SPI1 or GATA2 did not repress the other, suggesting they act in parallel pathways that are significant for PRTN3 expression.
Conclusion
CRISPRi screening utilizing a novel PRTN3 reporter confirmed the essential role of SPI1 in PRTN3 expression and identified a second parallel pathway involving GATA2 necessary for PRTN3 expression.
Figure 1: Validation of a PRTN3 reporter. A) Diagram of the final reporter expression cassette. B) Fluorescent images of THP1 reporter cells 7 days after transfection with indicated sgRNAs. C) qPCR results from mRNA extracted from cells treated as in (B). C) Flow cytometry histograms of mCherry intensity from cells treated as in (B).
Figure 2: Validation of screen hits: (A) PRTN3 mRNA levels following transduction with the indicated sgRNAs and blasticidin selection (n=3 +/- SEM). B) mRNA expression levels of indicated genes following transduction with indicated sgRNAs and blasticidin selection in THP-1 cells (Left) or HL60 cells (right) (n=3 +/- SEM). C) Cell lysates from HL60 cells treated as in (B) was subjected to immunoblotting with indicated antibodies.
Funding
- NIDDK Support