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CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics
The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogeni...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10106585/ https://www.ncbi.nlm.nih.gov/pubmed/37077890 http://dx.doi.org/10.3389/fgeed.2023.1034720 |
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author | Polikarpova, Anna V. Egorova, Tatiana V. Lunev, Evgenii A. Tsitrina, Alexandra A. Vassilieva, Svetlana G. Savchenko, Irina M. Silaeva, Yuliya Y. Deykin, Alexey V. Bardina, Maryana V. |
author_facet | Polikarpova, Anna V. Egorova, Tatiana V. Lunev, Evgenii A. Tsitrina, Alexandra A. Vassilieva, Svetlana G. Savchenko, Irina M. Silaeva, Yuliya Y. Deykin, Alexey V. Bardina, Maryana V. |
author_sort | Polikarpova, Anna V. |
collection | PubMed |
description | The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts. |
format | Online Article Text |
id | pubmed-10106585 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-101065852023-04-18 CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics Polikarpova, Anna V. Egorova, Tatiana V. Lunev, Evgenii A. Tsitrina, Alexandra A. Vassilieva, Svetlana G. Savchenko, Irina M. Silaeva, Yuliya Y. Deykin, Alexey V. Bardina, Maryana V. Front Genome Ed Genome Editing The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts. Frontiers Media S.A. 2023-04-03 /pmc/articles/PMC10106585/ /pubmed/37077890 http://dx.doi.org/10.3389/fgeed.2023.1034720 Text en Copyright © 2023 Polikarpova, Egorova, Lunev, Tsitrina, Vassilieva, Savchenko, Silaeva, Deykin and Bardina. https://creativecommons.org/licenses/by/4.0/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) and the copyright owner(s) 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. |
spellingShingle | Genome Editing Polikarpova, Anna V. Egorova, Tatiana V. Lunev, Evgenii A. Tsitrina, Alexandra A. Vassilieva, Svetlana G. Savchenko, Irina M. Silaeva, Yuliya Y. Deykin, Alexey V. Bardina, Maryana V. CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics |
title | CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics |
title_full | CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics |
title_fullStr | CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics |
title_full_unstemmed | CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics |
title_short | CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics |
title_sort | crispr/cas9-generated mouse model with humanizing single-base substitution in the gnao1 for safety studies of rna therapeutics |
topic | Genome Editing |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10106585/ https://www.ncbi.nlm.nih.gov/pubmed/37077890 http://dx.doi.org/10.3389/fgeed.2023.1034720 |
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