Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia

Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse...

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Main Authors:	Seyedeh Zeinab Mirjalili Mohanna, Jack W. Hickmott, Siu Ling Lam, Nina Y. Chiu, Tess C. Lengyell, Beatrice M. Tam, Orson L. Moritz, Elizabeth M. Simpson
Format:	Article
Language:	English
Published:	Elsevier 2020-06-01
Series:	Molecular Therapy: Methods & Clinical Development
Online Access:	http://www.sciencedirect.com/science/article/pii/S2329050120300322

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spelling	doaj-72def171bf954da9a10a19c51103507a2020-11-25T02:39:55ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-06-0117478490Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of AniridiaSeyedeh Zeinab Mirjalili Mohanna0Jack W. Hickmott1Siu Ling Lam2Nina Y. Chiu3Tess C. Lengyell4Beatrice M. Tam5Orson L. Moritz6Elizabeth M. Simpson7Centre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, The University of British Columbia, Vancouver, BC, CanadaCentre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, The University of British Columbia, Vancouver, BC, CanadaCentre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, Vancouver, BC V5Z 4H4, CanadaCentre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, The University of British Columbia, Vancouver, BC, CanadaCentre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, Vancouver, BC V5Z 4H4, CanadaDepartment of Ophthalmology and Visual Sciences and Centre for Macular Research, The University of British Columbia, Vancouver, BC, CanadaDepartment of Ophthalmology and Visual Sciences and Centre for Macular Research, The University of British Columbia, Vancouver, BC, CanadaCentre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, Vancouver, BC V5Z 4H4, Canada; Department of Medical Genetics, The University of British Columbia, Vancouver, BC, Canada; Corresponding author: Elizabeth M. Simpson, Centre for Molecular Medicine and Therapeutics at British Columbia Children’s Hospital, The University of British Columbia, 3020-950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada.Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.http://www.sciencedirect.com/science/article/pii/S2329050120300322
collection	DOAJ
language	English
format	Article
sources	DOAJ
author	Seyedeh Zeinab Mirjalili Mohanna Jack W. Hickmott Siu Ling Lam Nina Y. Chiu Tess C. Lengyell Beatrice M. Tam Orson L. Moritz Elizabeth M. Simpson
spellingShingle	Seyedeh Zeinab Mirjalili Mohanna Jack W. Hickmott Siu Ling Lam Nina Y. Chiu Tess C. Lengyell Beatrice M. Tam Orson L. Moritz Elizabeth M. Simpson Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia Molecular Therapy: Methods & Clinical Development
author_facet	Seyedeh Zeinab Mirjalili Mohanna Jack W. Hickmott Siu Ling Lam Nina Y. Chiu Tess C. Lengyell Beatrice M. Tam Orson L. Moritz Elizabeth M. Simpson
author_sort	Seyedeh Zeinab Mirjalili Mohanna
title	Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia
title_short	Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia
title_full	Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia
title_fullStr	Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia
title_full_unstemmed	Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia
title_sort	germline crispr/cas9-mediated gene editing prevents vision loss in a novel mouse model of aniridia
publisher	Elsevier
series	Molecular Therapy: Methods & Clinical Development
issn	2329-0501
publishDate	2020-06-01
description	Aniridia is a rare eye disorder, which is caused by mutations in the paired box 6 (PAX6) gene and results in vision loss due to the lack of a long-term vision-saving therapy. One potential approach to treating aniridia is targeted CRISPR-based genome editing. To enable the Pax6 small eye (Sey) mouse model of aniridia, which carries the same mutation found in patients, for preclinical testing of CRISPR-based therapeutic approaches, we endogenously tagged the Sey allele, allowing for the differential detection of protein from each allele. We optimized a correction strategy in vitro then tested it in vivo in the germline of our new mouse to validate the causality of the Sey mutation. The genomic manipulations were analyzed by PCR, as well as by Sanger and next-generation sequencing. The mice were studied by slit lamp imaging, immunohistochemistry, and western blot analyses. We successfully achieved both in vitro and in vivo germline correction of the Sey mutation, with the former resulting in an average 34.8% ± 4.6% SD correction, and the latter in restoration of 3xFLAG-tagged PAX6 expression and normal eyes. Hence, in this study we have created a novel mouse model for aniridia, demonstrated that germline correction of the Sey mutation alone rescues the mutant phenotype, and developed an allele-distinguishing CRISPR-based strategy for aniridia.
url	http://www.sciencedirect.com/science/article/pii/S2329050120300322
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Germline CRISPR/Cas9-Mediated Gene Editing Prevents Vision Loss in a Novel Mouse Model of Aniridia

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