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Updated: May 19, 2026

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries
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Arrayed dual-gRNA CRISPR screening platform for C9orf72 repeat expansion excision in patient iPSCs.

Olubankole Aladesuyi Arogundade1, Katie Jing Kay Lam1, Katherine A Brown1

  • 1Weill Institute for Neurosciences, University of California, San Francisco, San Francisco, CA 94158, USA.

Molecular Therapy. Advances
|May 18, 2026
PubMed
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Researchers identified effective CRISPR-Cas9 guide RNAs (gRNAs) for excising the C9orf72 repeat expansion, a key cause of frontotemporal dementia and amyotrophic lateral sclerosis (C9-FTD/ALS). This advances targeted gene therapy development for these devastating neurodegenerative diseases.

Area of Science:

  • Genetics
  • Neuroscience
  • Molecular Biology

Background:

  • The C9orf72 intronic hexanucleotide repeat expansion is the primary genetic driver of frontotemporal dementia and amyotrophic lateral sclerosis (C9-FTD/ALS).
  • Previous work demonstrated CRISPR-Cas9's potential to reverse C9-FTD/ALS pathological hallmarks in patient-derived induced pluripotent stem cells (iPSCs).

Purpose of the Study:

  • To discover highly efficient and safe guide RNA (gRNA) pairs for CRISPR-spCas9 dual-gRNA mediated excision of the C9orf72 repeat expansion.
  • To establish a screening framework for dual-gRNA strategies in patient iPSCs, applicable to other repeat expansion disorders.

Main Methods:

  • Screened 120 gRNA pairs using ddPCR and single-molecule sequencing in patient iPSCs.
  • Compared bi-allelic intronic repeat region excisions with allele-specific mutant allele excisions.
Keywords:
C9orf72CRISPRallele-specificamyotrophic lateral sclerosis, ALSarrayed CRISPR gRNA screendementiadual-gRNAfrontotemporal dementia, FTDgene therapyiPSCsmotor-neuron diseaserepeat expansion

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  • Utilized whole genome sequencing and INDUCE-seq to assess off-target effects of top-performing gRNAs.

    • Main Results:

    • Bi-allelic intronic excision of the repeat region proved more efficient than allele-specific excision.
    • Excision efficiency was dictated by individual gRNA activity, not repeat expansion length.
    • Only one off-target mutation was detected among the most efficient gRNAs, as predicted by computational tools.

    Conclusions:

    • Identified optimal gRNA pairs for dual-gRNA CRISPR-Cas9 excision of the C9orf72 repeat expansion.
    • Demonstrated the feasibility and safety of this approach for potential C9-FTD/ALS therapies.
    • Established a scalable screening platform for developing gene-editing therapies for repeat expansion disorders.