Investigation on Gene Editing of Tumor Cells Based on Advanced Engineering Transfection Method


Student thesis: Doctoral Thesis

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Award date13 Jun 2022


Gene therapy with engineered nucleases offers promising solutions to genetic disorder by editing DNA sequence or modulation gene expression. The CRISPR-Cas9 nuclease system represents an efficient tool for genome editing with unprecedented potential. The system uses a single guide RNA (sgRNA) to direct the Cas9 nuclease to complementary regions, where Cas9 cleaves the recognized DNA and generates double-stranded breaks (DSBs), leading to insertions or deletions at specific target genomic loci. The critical technology for successful gene editing is cell transfection. Traditional transfection methods can be broadly divided into three types: physical, chemical and biological methods. Transfection efficiency also depends on the cell type, and different types of cells often have different transfection efficiency. In this thesis study, the high-throughput single cell transfection systems based on engineering technology is introduced, and the specific gene function is investigated by applying the CRISPR-Cas9 system. This research is conducted in the following aspects.

First, the high-throughput robotic microinjection technology is used to generate gene knock-in HepG2 cells through CRISPR-Cas9 system. A case study was conducted to microinject Cas9 and sgRNA expression cassettes encoded plasmids and donor template plasmid encoding a fused enhanced green fluorescent protein (eGFP) sequences to HepG2 cells. Homology-directed repair mediated eGFP knocked-in was observed with an efficiency of 41%. Assessed by T7E1 assay, eGFP knock-in cells showed no detectable changes at potential off-target sites. The eGFP knock-in cells were able to form in vivo tumor model after injecting to zebrafish embryos. The results demonstrated a high level of target gene knock-in achieved by using the combination of robotic microinjection and CRISPR-Cas9 mediated knock-in system. The method has a great application potential in biomedical engineering and gene targeting therapy for mutation correction of disease genes.

Second, microinjection technology is used to generate functional gene expression macrophage cell lines. Microinjection of the expression plasmid carrying a mouse-derived toll-like-receptor 4 (tlr4) gene into a mouse macrophage cell line (Raw264.7) can construct a new stable cell line overexpressing the target gene. The expression efficiency of the target gene in the injected Raw264.7 cells reached 90%, which was measured by injecting a particular plasmid carrying eGFP gene fragment with the Tlr4 gene and counting the proportion of cells that emitted green fluorescence. Assessment of the messenger RNA (mRNA) and protein of produced by the Tlr4 gene indicated that its expression was up-regulated remarkably in successfully injected cells. The expression of downstream genes of Tlr4 in injected cells was higher-compared with that in untouched cells. Microinjection can avoid polarization effects, which are common when traditional transfection methods are used. The success of this study verify that microinjection can be an efficient and sage method in macrophage cell transfection applications.

Third, TRIM72 gene function is investigated through microdroplet-based single cell transfection. The improved high-throughput microdroplet-based single cell transfection method provided as an alternative method for delivering genome-editing reagents into single living cells. By accurately controlling the number of exogenous plasmids in microdroplets, this method can achieve high-efficiency delivery of nucleic acids to different types of single cells. The transfection efficiency of cells in different concentrations of DNA in microdroplets was measured. Under the optimized transfection conditions, the method was used to construct gene-knockout cancer cell lines to determine specific gene functions through the CRISPR-Cas9 system. In a case study, the migration ability of TRIM72 knockout cancer cells was inhibited, and the tumorigenicity of cells in a zebrafish tumor model was reduced. A single-cell microfluidic chip was designed to achieve CRISPR-Cas9 DNA transfection, which dramatically improved the transfection efficiency of difficult-to-transfect cells. Therefore, the microdroplet method developed had a unique advantage in CRISPR-Cas9 gene-editing applications.

In summary, due to the safety issue, the non-viral delivery of the nucleases into mammalian cells is more welcome, but the common delivery approaches, such as liposome-related transfection and electroporation, face major challenges to overcome drawbacks of low efficiency, high toxicity, and cell-type dependency. The alternative methods, such as microinjection and microfluidic chip, are capable of directly delivering genome-editing reagents into single living cells with high efficiency. In particular, microinjection can precisely introduce extracellular nucleic acid substances into the nucleus or cytoplasm under well controlled. The engineered methods could achieve high-efficiency delivery of different macromolecular substance to single cells while maintaining high cell viability. In this study, microinjection and microdroplet transfection methods were used to construct eGFP knock-in HepG2 cells, Tlr4 overexpression macrophage cell line Raw264.7, and TRIM72 knockout tumor cell lines. The biological functions of wild-type cells and gene-edited cells were further analyzed to prove the important role of specific genes in different cell types. Knockout of TRIM72 can effectively inhibit cell growth, indicating that these genes are necessary for tumor cell growth and migration, and the efficiency of gene editing through CRISPR-Cas9 is greatly improved. The engineering transfection technologies have opened up new horizons for basic biomedical research, and provides a useful platform for studying specific gene functions through CRISPR-Cas9 system.

    Research areas

  • CRISPR-Cas9, Gene editing, Cell microinjection, Microdroplet-based transfection