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ORIGINAL RESEARCH article
Front. Genome Ed.
Sec. Genome Editing in Human Health and Disease
Volume 7 - 2025 | doi: 10.3389/fgeed.2025.1581743
This article is part of the Research TopicInsights in Genome Editing in Human Health and Disease 2023/2024View all articles
Efficient GBA1 editing via HDR with ssODNs by outcompeting pseudogene-mediated gene conversion upon CRISPR/Cas9 cleavage
Provisionally accepted- Genome engineering & Stem cell Center, McDonnell Genome Institute, Department of Genetics, Washington University in St. Louis School of Medicine, St. Louis, United States
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CRISPR/Cas9-edited induced pluripotent stem cells (iPSCs) are valuable research models for mechanistic studies. However, gene conversion between a gene-pseudogene pair that share high sequence identity and form direct repeats in proximity on the same chromosome can interfere with the precision of gene editing. Mutations in the human beta-glucocerebrosidase gene (GBA1) are associated with Gaucher disease, Parkinson's disease, and Lewy body dementia. During the creation of a GBA1 KO iPSC line, we detected about 70% gene conversion from its pseudogene GBAP1. These events maintained the reading frame and resulted from GBA1-specific cleavage by CRISPR/Cas9, and NAHR-mediated deletion between the GBA1 and GBAP1. To increase the percentage of alleles with out-of-frame indels for triggering nonsense-mediated decay of the GBA1 mRNA, we supplied the cells with two single-stranded oligodeoxynucleotide (ssODN) donors as homology-directed repair (HDR) templates. We demonstrate that HDR using the ssODN templates effectively competes with gene conversion and enabled biallelic KO clone isolation, whereas the NAHR-based deletion rate remained the same. Here, we report a generalizable method to direct cellular DNA repair of double-strand breaks at a target gene towards the HDR pathway using exogenous ssODN templates, allowing specific editing of one gene in a gene-pseudogene pair without disturbing the other.
Keywords: gene conversion (GC), Nonallelic homologous recombination (NAHR), knock out and knock in, gene editing (CRISPR/Cas9), Stem cell engineering, Pseudogenes
Received: 23 Feb 2025; Accepted: 18 Apr 2025.
Copyright: © 2025 Sentmanat, Lagas and Cui. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Xiaoxia Cui, Genome engineering & Stem cell Center, McDonnell Genome Institute, Department of Genetics, Washington University in St. Louis School of Medicine, St. Louis, United States
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