Haplotype editing with CRISPR/Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL DOI Creative Commons

Prerna Dua,

Babette Simon,

Chiara B.E. Marley

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 22, 2024

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene haplo-sufficient. We previously edited dominant NEFL missense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, multitude different mutations cause disease. Here, we addressed this challenge targeting common single-nucleotide polymorphisms cis for excision. validated haplotype two demonstrated its therapeutic potential iPSC-motor Surprisingly, our analysis revealed that inversion, frequent byproduct excision editing, failed reliably disrupt mutant allele expression. deployed alternative strategies novel molecular assays increase outcomes while maintaining specificity allele. Finally, population genetics power enable development greatest number patients. Our data serve as an important case study many disorders amenable approach.

Language: Английский

DNA damage and its links to neuronal aging and degeneration DOI
Ilse Delint‐Ramírez, Ram Madabhushi

Neuron, Journal Year: 2025, Volume and Issue: 113(1), P. 7 - 28

Published: Jan. 1, 2025

Language: Английский

Citations

3
Packaged delivery of CRISPR–Cas9 ribonucleoproteins accelerates genome editing DOI Creative Commons
Hannah Karp, Madeline Zoltek, Kevin Wasko

et al.

Nucleic Acids Research, Journal Year: 2025, Volume and Issue: 53(5)

Published: Feb. 27, 2025

Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target editing, and cytotoxicity. Clinical use Cas9 RNPs currently entails electroporation into cells ex vivo, but no systematic comparison this method packaged RNP delivery has been made. Here we compared two strategies, enveloped vehicles (EDVs), investigate dosage requirements for editing. Using fluorescence correlation spectroscopy, determined that >1300 per nucleus are typically required productive EDV-mediated was >30-fold more efficient than electroporation, occurs at least 2-fold faster EDV comparable total doses. We hypothesize differences in efficacy between these methods result part from increased duration nuclear residence resulting delivery. Our results directly compare showing could dramatically reduce amount experimental or clinical

Language: Английский

Citations

0
Packaged delivery of CRISPR-Cas9 ribonucleoproteins accelerates genome editing DOI Creative Commons
Hannah Karp, Madeline Zoltek, Kevin Wasko

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 19, 2024

Effective genome editing requires a sufficient dose of CRISPR-Cas9 ribonucleoproteins (RNPs) to enter the target cell while minimizing immune responses, off-target and cytotoxicity. Clinical use Cas9 RNPs currently entails electroporation into cells

Language: Английский

Citations

0
Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins DOI Creative Commons
Da Xu,

Swen Besselink,

Gokul N. Ramadoss

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 27, 2024

Abstract Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, large size of CRISPR-based poses a challenge to their broad use biomedical research and as future therapies. Here, we present Robust ENveloped Delivery Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) activator (TET1-dCas9) ribonucleoprotein complexes into human modulate expression. After rational engineering, show that RENDER induces durable silencing endogenous genes across various cell types, including primary T cells. Additionally, apply epigenetically repress stem cell-derived neurons, reduction neurodegenerative disease associated V337M-mutated Tau protein. Together, our platform advances delivery cells, broadening editing fundamental therapeutic applications.

Language: Английский

Citations

0
Haplotype editing with CRISPR/Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL DOI Creative Commons

Prerna Dua,

Babette Simon,

Chiara B.E. Marley

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 22, 2024

Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene haplo-sufficient. We previously edited dominant NEFL missense mutation with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, multitude different mutations cause disease. Here, we addressed this challenge targeting common single-nucleotide polymorphisms cis for excision. validated haplotype two demonstrated its therapeutic potential iPSC-motor Surprisingly, our analysis revealed that inversion, frequent byproduct excision editing, failed reliably disrupt mutant allele expression. deployed alternative strategies novel molecular assays increase outcomes while maintaining specificity allele. Finally, population genetics power enable development greatest number patients. Our data serve as an important case study many disorders amenable approach.

Language: Английский

Citations

0