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  Abstract View: 81

Research Article

Multiplex Genome Editing in Chinese Hamster Ovary Cell Line Using All-in-One and HITI CRISPR Technology

Fatemeh Safari 1,2 ORCID logo, Safar Farajnia 3* ORCID logo, Younes Ghasemi 4, Nosratollah Zarghami 1, Mazyar Barekati‑Mowahed 5

1 Medical Biotechnology Department, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran.
2 Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
3 Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.
4 Department of Pharmaceutical Biotechnology, School of Pharmacy, and Pharmaceutical Sciences Research Centre, Shiraz University of Medical Sciences, Shiraz, Iran.
5 Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, Ohio, USA.

Abstract

Purpose: CRISPR/Cas9 gene editing technology has revolutionized gene manipulation by providing the opportunity of gene knock out/in, transcriptional modification and base editing. The application of this system extended into different eras of biology, from cell development to animal modeling. Various generations of CRISPR technology have been developed to make genome editing easy which resulted in rapid protocols for amelioration of a large genome.
Methods: we established a simple protocol for gene manipulation in Chinese hamster ovary (CHO) cells to achieve a Caspase 7 deficient cell line by using combination of all-in-one CRISPR technology and CRISPR/ Cas9 homology-independent targeted integration (CRISPR HITI). Results: the findings of this study indicated that using CRISPR knocking in/out technology facilitates genomic manipulation in CHO cells. Integration of EGFP in target locus of caspase 7 gene made the selection of knockout CHO cell line easy which achieved by cell sorting and single-cell cloning.
Conclusion: this system introduces an effective targeting strategy for multiplex genome engineering, coinciding gene integration which simplified the selection of desired genomic characteristics.
Keywords: Apoptosis, CHO cell, Caspase 7, CRISPR-Associated Protein 9
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Submitted: 11 Jan 2020
Revision: 08 Mar 2020
Accepted: 15 Apr 2020
ePublished: 15 Apr 2020
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