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BRAIN Publication-derived

CRISPR/Cas9-mediated excision of ALS/FTD-causing hexanucleotide repeat expansion in C9ORF72

Source Meijboom et al., 2022 · University of Massachusetts Medical School and University of Miami Miller School of Medicine · 10.1038/s41467-022-33332-7

👤 Katharina E. Meijboom, Abbas Abdallah, Nicholas P. Fordham, Hiroko Nagase, Tomás Rodriguez, Carolyn Kraus, Tania F. Gendron, Gopinath Krishnan, Rustam Esanov, Nadja S. Andrade, Matthew J. Rybin, Melina Ramic, Zachary D. Stephens, Alireza Edraki, Meghan T. Blackwood, Aydan Kahriman, Nils Henninger, Jean-Pierre A. Kocher, Michael Benatar, Michael H. Brodsky, Leonard Petrucelli, Fen-Biao Gao, Erik J. Sontheimer, Robert H. Brown, Zane Zeier, Christian Mueller ⏱ 60 days 📋 5 phases 🧫 Patient-Derived iPSC, Primary cortical neurons from C9ORF72 transgenic mice

Abstract

This protocol describes AAV9-mediated CRISPR/Cas9 gene-editing for excision of the pathogenic GGGGCC hexanucleotide repeat expansion from the C9ORF72 locus in patient-derived iPSC-derived motor neurons, primary cortical neurons, and transgenic mouse brain tissue. Successfully edited cells and tissues show dramatic reduction of RNA foci, toxic poly-dipeptides, and restoration of C9ORF72 transcript levels.

Cell source
Patient-Derived iPSC, Primary cortical neurons from C9ORF72 transgenic mice
Application
Disease modeling and therapeutic development for C9ORF72 ALS/FTD

Protocol overview

42 steps across 5 phases

gRNA Design, Cloning, and Validation in HEK293T Cells Day 1-10
  1. 1 Design gRNAs flanking the C9ORF72 HRE
  2. 2 Clone guide sequences into expression plasmids
  3. 3 Test gRNA activity in HEK293T cells using GFP reporter
  4. 4 Screen gRNA combinations in HEK293T cells
  5. 5 Estimate editing efficiency by Sanger sequencing
  6. 6 Analyze off-target effects by amplicon deep sequencing
  7. 7 Perform UDiTaS analysis for comprehensive editing assessment
  8. 8 Package validated gRNA pairs into AAV9 vectors
In Vitro Editing in Primary Cortical Neurons Day 1-15
  1. 1 Prepare primary cortical neurons from transgenic mouse embryos
  2. 2 Dissociate cortical tissue
  3. 3 Pellet and resuspend cells
  4. 4 Prepare Neurobasal medium with supplements
  5. 5 Seed cells onto poly-d-Lysine coated plates and slides
  6. 6 Culture neurons to DIV 4 for AAV transduction
  7. 7 Transduce primary neurons with AAV9 vectors (DIV 4)
  8. 8 Harvest cells for analysis at DIV 10
In Vivo Editing in Transgenic Mouse Brain Day 1-60
  1. 1 Prepare young adult transgenic mice
  2. 2 Anesthetize mice and position on stereotaxic frame
  3. 3 Perform bilateral stereotaxic striatal injections
  4. 4 Administer post-operative pain management
  5. 5 Allow vector transduction and editing (8 weeks post-injection)
  6. 6 Harvest striatal tissue (8 weeks post-injection)
  7. 7 Prepare tissue for FISH analysis
Editing of Patient-Derived iPSC Motor Neurons and Brain Organoids Day 1-50
  1. 1 Culture and maintain patient-derived iPSCs
  2. 2 Transduce undifferentiated iPSCs with AAV9-Cas9 and AAV9-gRNA2,4
  3. 3 Isolate single edited iPSC clones
  4. 4 Subclone edited iPSC lines for clonality confirmation
  5. 5 Differentiate iPSCs into induced motor neurons (iMNs)
  6. 6 Transduce differentiated iMNs at day 42 of differentiation
  7. 7 Generate brain organoids from C9ORF72 iPSCs
  8. 8 Transduce brain organoids with AAV9 vectors
  9. 9 Harvest organoids for analysis at DIV 32
Molecular and Cellular Analyses Day 1-30 (concurrent with phases 2-4)
  1. 1 Extract genomic DNA and perform PCR validation
  2. 2 Perform repeat-primed PCR to confirm HRE excision
  3. 3 Measure poly-dipeptide levels by sandwich immunoassay
  4. 4 Perform RNA fluorescence in situ hybridization (FISH)
  5. 5 Quantify RNA foci by image acquisition and manual counting
  6. 6 Extract RNA and perform droplet digital PCR (ddPCR)
  7. 7 Perform Western blot for C9ORF72 protein levels
  8. 8 Assess Cas9 and gRNA transduction by RNAscope
  9. 9 Perform UDiTaS library preparation for comprehensive editing assessment
  10. 10 Perform PacBio No-Amp targeted long-read sequencing

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Meijboom et al., 2022. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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