مقالات پذیرفته شده در پنجمین کنگره بین المللی زیست پزشکی
A novel tethering approach using monomeric streptavidin improves CRISPR-Cas9 genome editing efficiency in CHO cells
A novel tethering approach using monomeric streptavidin improves CRISPR-Cas9 genome editing efficiency in CHO cells
Mohammad Hassan Kheirandish,1Behnaz Rahmani,2Mohammad Ali Mazlomi,3Hossein Zarei Jaliani,4Fatemeh Davami,5,*
1. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 2. Medical Biotechnology Department, Semnan University of Medical Sciences, Semnan, Iran 3. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran 4. Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 5. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran
Introduction: CRISPR-Cas9 is becoming a leading tool to introduce defined changes into the pre-determined site of the genome by Cas9-mediated double strand break (DSB). Once the break is generated, knock-in can be occurred by homology-directed repair (HDR) mechanism when a repair template is provided at the DSB site. However, the low rate of HDR remains a key challenge in efficient knock-in at the most types of the cell. Recently has been found that tethering of donor template and Cas9 endonuclease and co-localizing them to the nucleus, improve the efficiency of knock-in via CRISPR-Cas9. In this regard, our study aimed to design a novel construct with the monomeric form of streptavidin that was genetically fused to the C-terminus of Cas9 protein and co-transfect it with the biotinylated donor to the Chinese hamster cell line.
Methods: Monomeric streptavidin (mSA) gene was amplified by polymerase chain reaction with primers complementary to the all-in-one (Cas9/sgRNA) vector backbone and mSA gene. First PCR using these primers was conducted to synthesize chimeric Cas9-NLS-mSA fragment. The product of the first PCR was gel purified and used as a mega primer in a quick-change PCR to synthesize the remaining parts of the all-in-one vector. The product was digested with DpnI restriction enzyme. Simultaneously we benefited from biotinylated primers to create biotinylated donor harboring GFP reporter and puromycin selection marker sequences flanked by long homology arms. Both constructs were transfected to CHO-K1 cell line using lipofectamine 3000 reagent. (Cells in the control group transfected by the circular donor and Cas9/sgRNA vector). Transfection efficiency has been evaluated by fluorescent microscopy and Flow cytometry for GFP expression. 72 hours after transfection, stable cell lines have been generated using 3.5 µg/mL of puromycin in the 6-well plate for 14 days. Cell pools of both groups were analyzed by 5'/3' junction PCR. Clonal selection has been done in 96-well. After 7 days each well including single-cell colonies that expressed GFP reporter were trypsinated and transferred to the 24-well. After genomic DNA extraction 5'/3' junction and out-out PCR analysis have been performed for each recovered clone to estimate overall knock-in efficiency.
Results: Monomeric streptavidin sequence with a suitable linker was fused to the 3' end of the Cas9 gene and cloned into the all-in-one vector by the restriction endonuclease free cloning method. The inserted sequences were confirmed by DNA sequencing. Cas9-mSA/sgRNA vector and biotinylated donor have been transfected to the CHO-K1 cell line and transfected cells were observed by fluorescent microscopy. Stable cell lines have been created after two weeks of the antibiotic selection procedure. 5'/3' junction PCR analysis on the cell pool showed desired bonds on 1% gel agarose electrophoresis. After limiting dilution, 39 single clones have been recovered for the tethering group and 49 clones for the control group. Each individual clones ubiquitously expressed GFP reporter, among which 20 clones of tethering and 10 clones of control groups were 5'/3' junction PCR positive. Further analysis showed that 15 of 39 and 6 of 49 clones were out-out PCR positive for tethering and control groups respectively.
Conclusion: In this study, we enhanced the targeted efficiency of knock-in via CRISPR-Cas9 in the CHO-K1 cell line as a leading host for biopharmaceutical production. With a tethering approach using a novel construct of Cas9-mSA/sgRNA and easy to create PCR amplified biotinylated donor, we achieved more than 3 fold enhancement of knock-in efficiency in CHO-K1 cells compared to the conventional CRISPR-Cas9 to insert the large transgene cassette (∼2 kb). This approach could propose a simple and efficient strategy for enhancing targeted knock-in efficiency in the Chinese hamster cell line.