Time and Cost-Effective Genome Editing Protocol for Simultaneous Caspase 8 Associated Protein 2 Gene Knock in/out in Chinese Hamster Ovary Cells Using CRISPR-Cas9 System.

IF 1.6 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Soofia Sorourian, Abbas Behzad Behbahani, Mohsen Forouzanfar, Mojtaba Jafarinia, Fatemeh Safari
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引用次数: 0

Abstract

Background: CHO cells are preferred for producing biopharmaceuticals, and genome editing technologies offer opportunities to enhance recombinant protein production. Targeting apoptosis-related genes, such as Caspases 8-Associated Protein 2 (CASP8AP2), improves CHO cell viability and productivity. Integrating robust strategies with the CRISPR-Cas9 system enables its application in CHO cell engineering.

Objectives: This study was performed to develop a cost-effective protocol using the CRISPR-Cas9 system combined with the HITI strategy for simultaneous CASP8AP2 gene deletion/insertion in CHO cells and to assess its impact on cell viability and protein expression.

Materials and methods: We developed an efficient protocol for CHO cell engineering by combining CRISPR/Cas9 with the HITI strategy. Two distinct sgRNA sequences were designed to target the 3' UTR region of the CASP8AP2 gene using CHOPCHOP software. The gRNAs were cloned into PX459 and PX460-1 vectors and transfected into CHO cells using the cost-effective PEI reagent. A manual selection system was employed to streamline the process of single-cell cloning. MTT assays assessed gene silencing and cell viability at 24, 48, and 72 hours. Flow cytometry evaluated protein expression in CASP8AP2-silenced CHO cells.

Results: The study confirmed the robustness of combining CRISPR-Cas9 with the HITI strategy, achieving a high 60% efficiency in generating knockout clones. PEI transfection successfully delivered the constructs to nearly 65% of the clones, with the majority being homozygous. The protocol proved feasible for resource-limited labs, requiring only an inverted fluorescent microscope. CASP8AP2 knockout (CHO-KO) cells exhibited significantly extended cell viability compared to CHO-K1 cells when treated with NaBu, with IC50 values of 7.28 mM and 14.25 mM at 48 hours, respectively (P-value 24 hours ≤ 0.0001, 48 hours ≤ 0.0001, P-value 72 hours = 0.0007). CHO CASP8AP2-silenced cells showed a 1.3-fold increase in JRed expression compared to native cells.

Conclusions: CRISPR-Cas9 and HITI strategy was used to efficiently engineer CHO cells for simultaneous CASP8AP2 gene deletion/insertion, which improved cell viability and protein expression.

利用 CRISPR-Cas9 系统在中国仓鼠卵巢细胞中同时敲入/敲出 Caspase 8 相关蛋白 2 基因的省时、省钱基因组编辑方案。
背景:CHO 细胞是生产生物制药的首选细胞,基因组编辑技术为提高重组蛋白的生产提供了机会。靶向凋亡相关基因,如 Caspases 8-Associated Protein 2 (CASP8AP2),可提高 CHO 细胞的活力和生产率。将稳健的策略与 CRISPR-Cas9 系统相结合,可将其应用于 CHO 细胞工程:本研究旨在利用 CRISPR-Cas9 系统结合 HITI 策略开发一种经济有效的方案,用于在 CHO 细胞中同时进行 CASP8AP2 基因缺失/插入,并评估其对细胞活力和蛋白质表达的影响:我们开发了一种将CRISPR/Cas9与HITI策略相结合的高效CHO细胞工程方案。我们使用 CHOPCHOP 软件设计了两种不同的 sgRNA 序列来靶向 CASP8AP2 基因的 3' UTR 区域。gRNA 被克隆到 PX459 和 PX460-1 载体中,并使用经济有效的 PEI 试剂转染到 CHO 细胞中。为了简化单细胞克隆过程,采用了人工选择系统。MTT 检测法评估了基因沉默和细胞在 24、48 和 72 小时内的存活率。流式细胞术评估了 CASP8AP2 沉默的 CHO 细胞的蛋白质表达:研究证实了将 CRISPR-Cas9 与 HITI 策略相结合的稳健性,在产生基因敲除克隆方面达到了 60% 的高效率。PEI 转染成功地将构建体传递给了近 65% 的克隆,其中大部分为同源基因。事实证明,该方案对资源有限的实验室来说是可行的,只需要一台倒置荧光显微镜。与 CHO-K1 细胞相比,CASP8AP2 基因敲除(CHO-KO)细胞经 NaBu 处理后,细胞存活率明显提高,48 小时的 IC50 值分别为 7.28 mM 和 14.25 mM(24 小时 P 值≤0.0001,48 小时 P 值≤0.0001,72 小时 P 值 = 0.0007)。与原生细胞相比,CHO CASP8AP2沉默细胞的JRed表达量增加了1.3倍:结论:CRISPR-Cas9和HITI策略被用于高效设计CHO细胞,使其同时进行CASP8AP2基因缺失/插入,从而提高了细胞活力和蛋白质表达。
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来源期刊
Iranian Journal of Biotechnology
Iranian Journal of Biotechnology BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
2.60
自引率
7.70%
发文量
20
期刊介绍: Iranian Journal of Biotechnology (IJB) is published quarterly by the National Institute of Genetic Engineering and Biotechnology. IJB publishes original scientific research papers in the broad area of Biotechnology such as, Agriculture, Animal and Marine Sciences, Basic Sciences, Bioinformatics, Biosafety and Bioethics, Environment, Industry and Mining and Medical Sciences.
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