An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing

(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerat...

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Autores principales:	Nannan Duan, Shuqing Tang, Baitao Zeng, Zhiqing Hu, Qian Hu, Lingqian Wu, Miaojin Zhou, Desheng Liang
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	Orip/EBNA1 CRISPR/Cas12a gene editing DMD iPSCs Science Q
Acceso en línea:	https://doaj.org/article/4484a438e67a4695b4475dc04efca9c6
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spelling	oai:doaj.org-article:4484a438e67a4695b4475dc04efca9c62021-11-25T18:11:39ZAn Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing10.3390/life111112622075-1729https://doaj.org/article/4484a438e67a4695b4475dc04efca9c62021-11-01T00:00:00Zhttps://www.mdpi.com/2075-1729/11/11/1262https://doaj.org/toc/2075-1729(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (<i>DMD</i>) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.Nannan DuanShuqing TangBaitao ZengZhiqing HuQian HuLingqian WuMiaojin ZhouDesheng LiangMDPI AGarticleOrip/EBNA1CRISPR/Cas12agene editingDMDiPSCsScienceQENLife, Vol 11, Iss 1262, p 1262 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Orip/EBNA1 CRISPR/Cas12a gene editing DMD iPSCs Science Q
spellingShingle	Orip/EBNA1 CRISPR/Cas12a gene editing DMD iPSCs Science Q Nannan Duan Shuqing Tang Baitao Zeng Zhiqing Hu Qian Hu Lingqian Wu Miaojin Zhou Desheng Liang An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing
description	(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (<i>DMD</i>) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.
format	article
author	Nannan Duan Shuqing Tang Baitao Zeng Zhiqing Hu Qian Hu Lingqian Wu Miaojin Zhou Desheng Liang
author_facet	Nannan Duan Shuqing Tang Baitao Zeng Zhiqing Hu Qian Hu Lingqian Wu Miaojin Zhou Desheng Liang
author_sort	Nannan Duan
title	An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing
title_short	An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing
title_full	An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing
title_fullStr	An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing
title_full_unstemmed	An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing
title_sort	episomal crispr/cas12a system for mediating efficient gene editing
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/4484a438e67a4695b4475dc04efca9c6
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An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing

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