Gene Editing

Gene editing is a molecular technology developed to modify specific sites within the genome through gene deletions, insertions or conversions for the purpose of studying functionally unknown genes or conducting gene therapy. As modified genetic information is inherited to the offspring, gene editing is frequently employed to modify biological traits of organisms to establish new varieties or improve the economic return of agriculture and livestock production.

Several gene editing techniques have been developed chronologically, which include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas 9) (CRISPR/Cas9). All these techniques use a specific nuclease or nuclease complex to recognize and cleave DNA at specific target sites to achieve DNA knock out or replacement through DNA repair machinery. In this review, the principles of gene editing were introduced, the advantages and disadvantages of different editing techniques were discussed, and their applications in the field of animal husbandry was reviewed and prospected.

MCE has not independently verified the accuracy of these methods. They are for reference only.

1. Design and synthesis of sgRNA and genotype primers

1.1 sgRNA design: Analyze the target gene sequence, screen suitable target sites, and design one sgRNA for each target site. In general, targeting Cas9 to sgRNAs that encode exons of functional protein domains is more likely to eliminate gene function than targeting the 5' exon alone.

1.2 PCR primer design: Design PCR primers based on the target site for subsequent target site DNA mutation detection.

2. Vector construction

2.1 Synthesize Oligo DNA according to the designed sgRNA sequence. Digest the empty vector with Cas9 gene (with fluorescent tag (GFP) and selection gene (puromycin)) to obtain linearized plasmid, mix Oligo DNA and linearized empty vector, ligate with T4 ligase, add buffer and ddH₂O , connect overnight at 16°C.

3. Plasmid verification

3.1 Transfer the ligation product into DH5α E. coli and culture it at 37°C overnight. Single colonies were selected for amplification, plasmids were extracted, and enzyme digestion was performed for identification.

3.2 Perform agarose gel electrophoresis on the digested products, and select the digested products with the correct size for sequencing verification.

4. Plasmid extraction and quality detection

4.1 Transfer the verified plasmid into new DH5α competent E. coli and culture it at 37°C overnight. Single colonies were picked for amplification and culture, and plasmids were extracted. Detect endotoxin; perform its absorbance detection (the ratio of 260 nm/280 nm is 1.8~2.0); and detect the integrity of the plasmid through agarose electrophoresis.

5. Before transfection

5.1 One day before transfection, place well-growing 293T cells in a 6-well plate. The cells in each well are about 1.0 x 10⁵ -3.0 x 10⁵ indivual. Culture in a cell culture incubator overnight to 90%~95% cell density.

6. Cell transfection

6.1 Preheat the reagents required for transfection (including Opti-MEM, plasmid) in a 37°C water bath.

6.2 Take two centrifuge tubes, add 4 μg plasmid and 100 μL Opti-MEM to one tube, and add 4 μL Lipo-2000 and 100 μL Opti-MEM to the other tube. Let stand for 5 minutes, mix well, and let stand for 20-25 minutes. Add 200 μL to each well of a 6-well plate, mix gently, and continue culturing.

7. Single clone screening and identification

7.1 After 24 hours of transfection, add puromycin at a final concentration of 2~3 μg/mL to the culture dish for screening. Generally, the screening lasts for 2~3 days.

7.2 After washing away floating dead cells and serum with preheated PBS, add 200 μL 1×trypsin for digestion.

7.3 Terminate digestion by adding 2 mL of culture medium with 10% serum, then transfer to a sterile 15 mL centrifuge tube.

7.4 Centrifuge cells at 800g for 3 minutes.

7.5 Resuspend the cell pellet in 5 mL of 10% serum medium.

7.6 Count the number of viable cells in the hemocytometer to determine cell density. Use trypan blue to exclude dead cells, which absorb the dye and turn blue.

7.7 In a 50 mL sterile centrifuge tube, dilute cells to a density of 1-2 cells/200 μL in 40 mL of complete growth medium. Perform 10-fold serial dilutions from the starting suspension to achieve this cell density.

7.8 Plate the diluted cells into a 96-well plate. To ensure that knockout cells can be obtained, one well of the 6-well plate corresponds to at least two 96-well plates.

7.9 After 3 to 5 days, observe the 96-well plate under a light microscope and mark the wells containing single cells.

7.10 Wait until the cell density in the well reaches 70%~90%, and digest with 20 μL of 1× trypsin.

7.11 Terminate digestion by adding 200 μL of 10% serum medium. Pipette 100 μL of cell suspension and transfer to a 1.5 mL EP tube. The remaining cell suspension was left in the 96-well plate for continued culture.

7.12 Use the CRISPR/Cas9 genotyping kit to process the collected cells, amplify them with the previously designed PCR primers, and sequence the amplified products.

7.13 Based on the sequencing results, screen single clones with effective mutations.

8. Expansion of monoclonal cells

8.1 When the confluence of positive clone cells reaches more than 70%, transfer to 48-well plate, 24-well plate, 12-well plate, and 6-well plate for expansion culture.

8.2 Record the growth of monoclonal cells and evaluate the stability of the cell line.

8.3 Use the monoclonal antibody corresponding to the protein of the target gene to detect the expression product of the cells, lyse the cells, extract the total protein of the cells, transfer to PVDF membrane after SDS-PAGE electrophoresis, block with blocking solution for 1 hour, and incubate with primary and secondary antibodies. , take photos and analyze under chemiluminescence imager.

1. sgRNA verification: Before starting the experiment, you can use sgRNA online verification to screen high-efficiency sgRNA for the construction of knockout cell lines.

2. Gene copy number: It is recommended to determine the copy number of the target gene before starting the experiment. Many immortalized cell lines, especially cancer cell lines, are not diploid and therefore can have 3 or more copies of chromosomes.

3. Quality of the plasmid: It is critical to use high quality endotoxin-free plasmid DNA. Determine DNA concentration by reading absorbance at 260 nm. Measure DNA purity by using the 260 nm/280 nm ratio (the ratio should be in the range of 1.8 to 2.0). Check the integrity of the plasmid by agarose gel electrophoresis.

4. Cell condition: Use high-quality cells that are well maintained and routinely certified, including testing for bacterial, fungal, or mycoplasmal contamination. If cells are from freshly recovered cells, complete at least 2 passages before use.