Department of Medical Biotechnology, School of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran.
Fuller Laboratories, Fullerton, CA, USA.
Folia Histochem Cytobiol. 2022;60(1):13-23. doi: 10.5603/FHC.a2022.0007. Epub 2022 Feb 14.
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.
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.
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.
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.
乳腺癌是全球范围内的一个严峻挑战，因为它是导致女性死亡的主要原因之一。CD81 基因是四跨膜蛋白家族的成员，与人类癌症的发展有关。基因组编辑技术，特别是 CRISPR-Cas9 系统，在基因功能研究方面取得了快速进展。在这项研究中，我们旨在评估基于 CRISPR-Cas9 质粒系统修饰 MDA-MB-231 乳腺癌细胞系中 CD81 基因特定区域的能力。
使用生物信息学数据库搜索，设计了四个靶向 CD81 基因外显子 3 和外显子 5 的不同单链引导 RNA (sgRNA)。预期的 sgRNA 序列被克隆到表达质粒 pSpCas9(BB)-2A-GFP(PX458)中，该质粒带有 sgRNA 支架骨架、Cas9 和 EGFP 编码序列，通过菌落 PCR 和测序进行确认。通过荧光显微镜和流式细胞术分析确定转染效率。使用 T7E1 和 TIDE 软件分别定性和定量测量基因编辑效率。
我们的数据表明，表达构建体成功地引入 MDA-MB-231 细胞，转染效率可接受。TIDE 软件检测到两个 sgRNA 在其靶区域引入了显著的突变(p 值<0.05)。据我们所知，这项研究首次使用 CRISPR/Cas9 技术在这些细胞中研究了 CD81 基因编辑。
总之，我们的数据表明，CRISPR-Cas9 系统可以改变 MDA-MB-231 细胞靶区域的基因组序列。结合以前的研究，我们建议在使用基于 T7E1 的定量缺失估计时要深思熟虑，因为比较多个 gRNA 的活性与 T7E1 测定可能会导致不准确的结论。相反，建议通过 Sanger 测序和随后的 TIDE 分析估计非同源末端连接事件 (NHEJ)。
