open access

Vol 60, No 1 (2022)
Original paper
Submitted: 2021-10-13
Accepted: 2022-02-05
Published online: 2022-02-14
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Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line

Kasra Arbabi Zaboli1, Hossein Rahimi1, Jose Thekkiniath2, Amir Hossein Taromchi1, Saeed Kaboli31
·
Pubmed: 35157300
·
Folia Histochem Cytobiol 2022;60(1):13-23.
Affiliations
  1. Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
  2. Fuller Laboratories, Fullerton, CA, USA
  3. Cancer Gene Therapy Research Center, Zanjan University of Medical Sciences, Zanjan 4513956111, Iran

open access

Vol 60, No 1 (2022)
ORIGINAL PAPERS
Submitted: 2021-10-13
Accepted: 2022-02-05
Published online: 2022-02-14

Abstract

Introduction. Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line. Materials and methods. Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively. Results. Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique. Conclusions. Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

Abstract

Introduction. Breast cancer has been represented a challenging issue worldwide as it is one of the major leading causes of death among women. CD81 gene, a member of the tetraspanin protein family, has been associated with the development of human cancers. Genome editing technologies, particularly the CRISPR-Cas9 system, have shown rapid progress in gene function studies. In this study, we aimed to evaluate the ability of the CRISPR-Cas9 plasmid-based system to modify specific regions of the CD81 gene in the MDA-MB-231 breast cancer cell line. Materials and methods. Using bioinformatics database search, four different single guide RNAs (sgRNAs) to target exon 3 and exon 5 of the CD81 gene were designed. The intended sgRNAs sequences were cloned into the expression plasmid pSpCas9(BB)-2A-GFP (PX458) bearing sgRNA scaffold backbone, Cas9, and EGFP coding sequences, which was confirmed by colony PCR and sequencing. Transfection efficiency was determined by fluorescence microscopy and flow cytometry analysis. Gene editing efficiency was measured qualitatively and quantitatively using the T7E1 and TIDE software, respectively. Results. Our data show that expression constructs were successfully introduced into MDA-MB-231 cells with an acceptable transfection efficiency. Two sgRNAs that were afforded to introduce significant mutations in their target regions were detected by TIDE software (p-value < 0.05). To the best of our knowledge, CD81 gene editing in these cells has been investigated for the first time in this study using the CRISPR/Cas9 technique. Conclusions. Taken together, our data show that the CRISPR-Cas9 system can change the genomic sequence in the target area of MDA-MB-231 cells. Along with previous studies, we propose forethought when using T7E1-based quantitative indel estimates, as comparing activities of multiple gRNAs with the T7E1 assay may lead to inaccurate conclusions. Instead, estimating non-homologous end-joining events (NHEJ) by Sanger sequencing and subsequent TIDE analysis is recommended.

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Keywords

CRISPR-Cas9; gene editing; CD81 gene; MDA-MB-231 cells; transfection efficiency

About this article
Title

Plasmid-based CRISPR-Cas9 system efficacy for introducing targeted mutations in CD81 gene of MDA-MB-231 cell line

Journal

Folia Histochemica et Cytobiologica

Issue

Vol 60, No 1 (2022)

Article type

Original paper

Pages

13-23

Published online

2022-02-14

Page views

6128

Article views/downloads

1134

DOI

10.5603/FHC.a2022.0007

Pubmed

35157300

Bibliographic record

Folia Histochem Cytobiol 2022;60(1):13-23.

Keywords

CRISPR-Cas9
gene editing
CD81 gene
MDA-MB-231 cells
transfection efficiency

Authors

Kasra Arbabi Zaboli
Hossein Rahimi
Jose Thekkiniath
Amir Hossein Taromchi
Saeed Kaboli

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