Creating iPSC lines with Ribonucleoprotein (RNP): Nucleofection, Single-cell Sorting, Genotyping, and Line Maintenance Protocol v1

Gill, Kamaljot

doi:10.17504/protocols.io.4r3l2oeopv1y/v1

Cited by 2 publications

(1 citation statement)

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“…To engineer each iPSC line we used HiFi spCas9 protein (Macolabs, UC Berkeley) and two gRNAs ( Supplementary Table 1 ) to create an excision, using our published protocol 83 . gRNAs were designed to have no exact off-target matches and the lowest predicted off-targets using CRISPOR (Homo sapiens – USCS Dec. 2013 (GRCh38/hg38)) 46 .…”

Section: Methodsmentioning

confidence: 99%

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

Sckaff

Gill

Sachdev

et al. 2022

Preprint

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CRISPR gene editing holds promise to cure or arrest genetic disease, if we can find and implement curative edits reliably, safely and effectively. Expansion of a hexanucleotide repeat in C9orf72 is the leading known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We evaluated three approaches to editing the mutant C9orf72 gene for their ability to correct pathology in neurons derived from patient iPSCs: excision of the repeat region, excision of the mutant allele, and excision of regulatory region exon 1A. All three approaches normalized RNA abnormalities and TDP-43 pathology, but only repeat excision and mutant allele excision completely eliminated pathologic dipeptide repeats. Our work sheds light on the complex regulation of the C9orf72 gene and suggests that because of sense and anti-sense transcription, silencing a single regulatory region may not reverse all pathology. Our work also provides a roadmap for evaluating CRISPR gene correction using patient iPSCs.

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Section: Methodsmentioning

confidence: 99%

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

Sckaff

Gill

Sachdev

et al. 2022

Preprint

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Validated assays for the quantification of C9orf72 human pathology

Salomonsson,

Maltos,

Gill

et al. 2024

Sci Rep

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A repeat expansion mutation in the C9orf72 gene is the leading known genetic cause of FTD and ALS. The C9orf72-ALS/FTD field has been plagued by a lack of reliable tools to monitor this genomic locus and its RNA and protein products. We have validated assays that quantify C9orf72 pathobiology at the DNA, RNA and protein levels using knock-out human iPSC lines as controls. Here we show that single-molecule sequencing can accurately measure the repeat expansion and faithfully report on changes to the C9orf72 locus in what has been a traditionally hard to sequence genomic region. This is of particular value to sizing and phasing the repeat expansion and determining changes to the gene locus after gene editing. We developed ddPCR assays to quantify two major C9orf72 transcript variants, which we validated by selective excision of their distinct transcriptional start sites. Using validated knock-out human iPSC lines, we validated 4 commercially available antibodies (of 9 tested) that were specific for C9orf72 protein quantification by Western blot, but none were specific for immunocytochemistry. We tested 15 combinations of antibodies against dipeptide repeat proteins (DPRs) across 66 concentrations using MSD immunoassay, and found two (against poly-GA and poly-GP) that yielded a 1.5-fold or greater signal increase in patient iPSC-motor neurons compared to knock-out control, and validated them in human postmortem and transgenic mouse brain tissue. Our validated DNA, RNA and protein assays are applicable to discovery research as well as clinical trials.

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Creating iPSC lines with Ribonucleoprotein (RNP): Nucleofection, Single-cell Sorting, Genotyping, and Line Maintenance Protocol v1

Cited by 2 publications

References 0 publications

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

Validated assays for the quantification of C9orf72 human pathology

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