SUMMARY
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- Single-cell RNA-seq to measure editing efficiency across different CNS cell types
- Genome-wide off-target analysis in edited brain tissue
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## Background and Rationale
# CRISPR-based Gene Editing Efficiency and Safety Assessment in Central Nervous System Models for ALS Research
This falsification experiment aims to critically evaluate the efficacy and safety profile of CRISPR-Cas9 mediated gene editing across distinct central nervous system (CNS) cell types relevant to amyotrophic
METHODOLOGY NOTES
**Phase 1: Cell Line Preparation and Editing (Weeks 1-2)**
• Culture human iPSC-derived motor neurons (n=6 lines) and astrocytes (n=6 lines) representing ALS disease models
• Prepare CRISPR-Cas9 editing constructs targeting ALS-associated genes (SOD1, C9orf72, TARDBP)
• Perform electroporation-based gene editing with guide RNAs at 48-hour intervals
• Maintain edited and control cell lines in parallel cultures with standard media conditions
**Phase 2: Single-cell RNA-seq Analysis (Weeks 3-4)**
• Harvest cells at 7, 14, and 21 days post-editing (n=10,000 cells per timepoint per line)
• Process samples using 10x Genomics Chromium platform for scRNA-seq library preparation
• Sequence libraries to achieve >50,000 reads per cell with >80% saturation
• Perform bioinformatics analysis using Seurat pipeline for cell type identification and editing efficiency quantification
• Calculate on-target editing rates using CRISPResso2 analysis of aligned reads
**Phase 3: Genome-wide Off-target Detecti