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BRAIN Publication-derived

CRISPR antiviral targeting JC polyomavirus in 2D and 3D culture models using dual-gRNA SaCas9

Source Rocchi et al., 2025 · Temple University Lewis Katz School of Medicine, Department of Microbiology, Immunology and Inflammation, Center for Neurovirology and Gene Editing · 10.1016/j.omtn.2025.102556

👤 Angela Rocchi, Shuren Liao, Hong Liu, Chen Chen, Senem Çakır, Anna Bellizzi, Hassen S. Wollebo, Ilker K. Sariyer, Kamel Khalili ⏱ 150 days 📋 8 phases 🧫 Human iPSC (cerebral organoid-derived) and SVG astrocytes

Abstract

This protocol demonstrates the development and validation of a dual-gRNA SaCas9 CRISPR antiviral targeting JC virus large tumor antigen (LT-Ag) and capsid protein VP1. The treatment achieves complete excision of a 2,336 bp viral genome segment in both 2D SVGA cell cultures and 3D cerebral organoids, reducing viral load, protein expression, and infectivity of viral progeny. This approach provides a preclinical foundation for treating progressive multifocal leukoencephalopathy (PML).

Cell source
Human iPSC (cerebral organoid-derived) and SVG astrocytes
Application
Disease modeling and therapeutic validation for JCV/PML

Protocol overview

64 steps across 8 phases

CRISPR construct design and packaging 1-14
  1. 1 gRNA design using Benchling bioinformatic software
  2. 2 Generate seven gRNA construct combinations
  3. 3 Design split-construct arrangement
  4. 4 Plasmid DNA extraction and purification
  5. 5 Lentiviral packaging using 293T/HEK cells
  6. 6 Lentiviral concentration by ultracentrifugation
  7. 7 Lentiviral titer determination
SVGA cell line creation, selection, and validation 15-35
  1. 1 Maintain SVGA cells in culture
  2. 2 Lentiviral transduction of SVGA cells
  3. 3 Puromycin selection of transduced cells
  4. 4 Expansion and maintenance of selected cell lines
  5. 5 RT-qPCR confirmation of construct expression
  6. 6 Western blot confirmation of SaCas9 protein expression
2D culture infection and excision validation 36-60
  1. 1 Produce JCV Mad-1 viral stock
  2. 2 Infect SVGA cell lines with Mad-1
  3. 3 Sample collection for viral load analysis
  4. 4 DNA extraction from cell lysate and media
  5. 5 Excision PCR (ePCR) to detect viral genome excision
  6. 6 Sanger sequencing of ePCR amplicons
  7. 7 qPCR quantification of viral genomic copy number
  8. 8 Western blot analysis of viral protein expression
  9. 9 Time-course analysis and linear regression
Adoptive transfer to assess viral infectivity 61-75
  1. 1 Harvest media from infected cell lines
  2. 2 Quantify viral genomic copy number in media samples
  3. 3 Expose naive SVGA cells to harvested media at three MOI levels
  4. 4 Culture exposed cells for 10 days
  5. 5 Harvest cells and extract nucleic acids
  6. 6 qPCR quantification of viral genomic copy number in exposed cells
  7. 7 Western blot analysis of viral proteins in exposed cells
Cerebral organoid differentiation and characterization 1-120
  1. 1 Obtain human induced pluripotent stem cells
  2. 2 Initiate cerebral organoid differentiation (days 1-40)
  3. 3 Maturation phase (days 41-120)
  4. 4 Cell counting for MOI calculation
  5. 5 Characterization by immunofluorescence
  6. 6 Characterization by electron microscopy
3D organoid infection model development 121-150
  1. 1 Infect 120-day-old organoids with Mad-1
  2. 2 Maintain infected organoids in culture
  3. 3 Harvest organoids and media at 10 and 20 dpi
  4. 4 DNA extraction from organoid lysate and media
  5. 5 qPCR quantification of viral genomic copy number in organoids
  6. 6 Western blot analysis of viral protein expression in organoids
  7. 7 Immunohistochemistry of viral proteins in organoids
  8. 8 Co-immunostaining to identify infected cell types
3D organoid infection-treatment model 1-30
  1. 1 Prepare organoids for infection-treatment study
  2. 2 Infect 50-day-old organoids with Mad-1 (day 0 of treatment timeline)
  3. 3 Lentiviral transduction with CRISPR constructs (day 1 of treatment timeline)
  4. 4 Maintain organoids and collect media (days 1-15)
  5. 5 Endpoint harvest of organoids (day 15 post-transduction)
  6. 6 RT-qPCR confirmation of construct expression (day 15)
  7. 7 Excision PCR (ePCR) on organoid DNA (day 15)
  8. 8 Sanger sequencing and ICE analysis of ePCR products
  9. 9 qPCR quantification of viral genomic copy number in organoid lysate
  10. 10 Time-course qPCR of extracellular viral load in media
  11. 11 Immunofluorescence quantification of viral proteins in organoids
  12. 12 Adoptive transfer of organoid-derived media to naive SVGA cells
  13. 13 qPCR and Western blot of exposed SVGA cells
Data analysis and statistical interpretation Throughout study
  1. 1 Compile qPCR data across timepoints
  2. 2 Perform Student's t-test comparisons
  3. 3 Linear regression analysis of time-course data
  4. 4 Quantify fold reduction in viral load
  5. 5 Quantify immunofluorescence signal intensity
  6. 6 Quantify Western blot band intensity
  7. 7 Generate publication-quality graphs
  8. 8 Interpret results in context of treatment efficacy

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Rocchi et al., 2025. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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