Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation

doi:10.13523/j.cb.2102023

China Biotechnology

2021, Vol. 41

Issue (5): 17-26 DOI: 10.13523/j.cb.2102023

Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation

HU Xuan^1,²,WANG Song¹,YU Xue-ling²,ZHANG Xiao-peng^2,^**(

)

1 Science and Technology on Advanced Ceramic Fibers and Composites Laboratory, National University of Defense Technology, Changsha 410073, China
2 Beijing Institute of Biotechnology, Academy of Military Medical Science, Beijing 100071, China

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Abstract

Objective: To meet the requirements of rapid and efficient gene editing detection in high-throughput screening applications, it is of great significance to establish an in situ evaluation method on cells. EGFP can be used to evaluate the gene editing performance mediated by the CRISPR system, but the efficiency is limited by the long half-life of EGFP. Methods: A version of destabilized EGFP (EGFP-PEST) was constructed by fusing degradation domain of ornithine decarboxylase (containing PEST motif) to EGFP. The EGFP-PEST gene was introduced into the chromosome of HEK-293T cells by lentivirus, and the copy number of EGFP-PEST gene was measured by RT-qPCR. Finally, cell strains with single copy of EGFP-PEST transgene were established. Results: Compared to unmodified EGFP, the cellular fluorescence of EGFP-PEST significantly decreased within 4 h, suggesting efficient PEST-mediated protein degradation. The cell model was used to evaluate the potential of three commercial lipids to deliver CRISPR/Cas9 complex. Data showed that gene editing was detected in 2-4 days by quantitative or qualitative measurements. Conclusion: This cell model can be used in high-throughput screening for new CRISPR tools or novel delivery systems by indicating the gene editing rapidly and sensitively.

Key words： Fluorescent protein Single copy CRISPR system Cell model Gene editing

Received: 22 February 2021 Published: 01 June 2021

ZTFLH:

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Corresponding Authors: Xiao-peng ZHANG E-mail: zxp8565@aliyun.com

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	Xuan HU
	Song WANG
	Xue-ling YU
	Xiao-peng ZHANG

Cite this article:

HU Xuan,WANG Song,YU Xue-ling,ZHANG Xiao-peng. Construction of a Destabilized EGFP Cell Model for Gene Editing Evaluation. China Biotechnology, 2021, 41(5): 17-26.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2102023 OR https://manu60.magtech.com.cn/biotech/Y2021/V41/I5/17

Fig.1 The diagram of the pLVX-EGFP-PEST construction principle

Fig.2 Puromycin sensitivity test (0-3 μg/mL) in HEK-293T cells for 7 days in a 24-well plate

Fig.3 Construction of pLVX-EGFP-PEST (a) Electrophoresis after EcoRI/BamHI digestion (b) Colony PCR identification for positive screening

Fig.4 Relationship between the proportion of fluorescent HEK-293T cells and the volume of virus stock solution (a) Fluorescence microscopy images and flow cytometry histograms (b) Linear fitting calculation of the experimental data to plot diagram for the fitting function

Fig.5 Poisson distribution mathematical model of virus-infected cells

Fig.6 Amplification plot, standard curve and melt curve of RT-qPCR Rn: Normalized reporter fluorescence value; Sample 1-9: 10 times the concentration gradient dilution of standard sample (5.244×10⁹-5.244×10 copies/μL)

Table 1 qRT-PCR analysis of the copy number of the EGFP-PEST transgenes

Fig.7 EGFP expression level assessed by flow cytometry

Fig.8 Flow cytometric analysis of the fluorescence stabilities for (a) 293T.EGFP-PEST cells and (b) 293T-EGFP cells

Fig.9 Flow cytometry histograms at different times of 293T.EGFP-PEST cells after transfection with jetCRISPR/Cas9/sgRNA

Fig.10 Flow cytometry histograms of 293T.EGFP-PEST cells 4 days after transfection with RNAiMAX/Cas9/sgRNA and Lipo2000/Cas9/sgRNA


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