Both iPSCs and embryonic stem cells (ESCs) have pluripotency, but iPSCs are obtained by reprogramming the somatic cells, while ESCs come from early embryos. IPSC does not involve the use of embryos and does not violate ethical requirements, so it can also avoid immune rejection issues in scientific research. Therefore, iPSC is considered to be a preferred choice.

EDITGENE’s advantages in gene knock-in technology include: Guaranteed results: With 10 years of CRISPR gene editing experience and a team of PhDs from world-renowned institutions offering one-on-one support. High precision: EDITGENE’s optimized tools reduce off-target effects, enhancing editing accuracy. High efficiency: EDITGENE’s technology platform improves knock-in success rates, accelerating experimental progress. Customized service: Tailored knock-in solutions to meet specific research or therapeutic goals.

Gene knock-in plays a crucial role in drug development. It is used in target validation by introducing specific genes into cell lines or animal models to confirm drug target efficacy. It also aids in establishing disease models, testing drug efficacy and safety in these models, and supporting drug screening through high-throughput screening in knock-in cell lines to identify potential drug candidates. Additionally, gene knock-in helps uncover drug mechanisms, optimize drug structure, and improve dosing strategies, expediting drug development while enhancing efficacy and safety.

EDITGENE brings 10 years of CRISPR-based cell editing experience and offers one-on-one support from a team of PhDs from globally recognized institutions.

Gene overexpression refers to using various techniques to significantly increase the expression level of a specific gene in cells or organisms. This is often achieved by introducing additional gene copies or using strong promoters to drive gene expression.

Gene overexpression aids in studying the function of specific genes, revealing their role within the organism. It is also commonly used in drug screening, vaccine development, and protein production. For example, by overexpressing a therapeutic protein, researchers can evaluate its efficacy in disease models.

EDITGENE’s 3D single-cell printing technology employs non-contact operation, avoiding mechanical damage and background contamination, which helps maintain cell integrity and biological activity. This technology also minimizes human error in the traditional limited dilution method of monoclonal selection, ensuring the reliability of screening results.

EDITGENE utilizes industry-leading 3D single-cell printing technology, which enables precise isolation and positioning of individual cells, significantly increasing the success rate and efficiency of monoclonal screening. This technology is widely applied in biomedicine research, antibody development, drug screening, and therapeutic selection, showcasing broad application prospects in cell research.

CRISPR libraries can be divided into whole-genome libraries and subgenomic libraries. If the goal is to perform screenings across the entire genome, a whole-genome library is the best choice. Such libraries typically contain sgRNAs targeting the entire genome. If the research focus is specific, such as targeting only particular gene families or specific signaling pathways, a subgenomic library can be chosen to reduce unnecessary screening workload and costs.

Maintaining the activity of cell cultures is crucial. Cells should not be allowed to reach confluence for more than 24 hours. Frozen new cells can restore transfection activity. The optimal cell plating density varies for different cell types or applications; however, for adherent cells, a confluence of 70% to 90% or a density of 5×10^5 to 2×10^6 suspended cells/ml typically yields good transfection results. It is important to ensure that cells are not fully confluent or in a fixed phase during transfection.

The main differences among Cas9, Cas12, and Cas13 lie in their action mechanisms:
· Cas12 is activated to cleave ssDNA trans-cleaving after binding with guide RNA and target DNA.
· Cas13 is activated to cleave ssRNA trans-cleaving after binding with guide RNA and target RNA.
· Cas9 has not been reported to exhibit trans-cleaving activity.

CRISPR detection reagents:
1.The RPA isothermal amplification kit can be stored at -20°C for long-term storage.
2.Target plasmids can be stored at -20°C for long-term use.
3.Cas proteins are sensitive to repeated freeze-thaw cycles; it is recommended to aliquot into multiple tubes and store at -80°C, retrieving them as needed for experiments. For short-term use, they can be stored at -20°C.
4.crRNA is prone to degradation and should be stored at -80°C if not used in the short term.
5. Probes, being double-stranded DNA, are relatively stable and can be stored at -20°C.

1.Design an efficient crRNA sequence. Proper design and structure prediction using online resources can help select suitable crRNA to achieve good trans-cleavage activity of the Cas enzyme.
2.Choose an appropriate signal reporter substrate. Research indicates that using a 15 nt single-stranded DNA (ssDNA) as a reporter substrate maximizes the cleavage reaction rate of Cas12a, significantly enhancing the reaction rate compared to the commonly used 5-nt ssDNA.
3.Optimize reaction conditions and buffers. Adjusting the CRISPR reaction parameters, such as the ratio of Cas enzyme to crRNA, the concentration of the Cas enzyme, and the reaction temperature, can improve detection performance to some extent.

1.The design process can follow these steps:
1.Identify the target gene sequence.
2.Specify the Cas protein being used. Different Cas proteins require corresponding PAM (Protospacer Adjacent Motif) sequences; for instance, Cas12a needs the "TTTV" PAM sequence for target recognition.
3.Select the crRNA targeting region. Choose a 20 nt nucleotide sequence on the target gene that is adjacent to the PAM site and pairs with the complementary strand of the crRNA.
4.Combine the selected 20 nt target sequence (variable part) with the scaffold sequence (fixed part) to design the crRNA sequence.
5.Use online tools such as CRISPR design tools (e.g., CRISPOR, Benchling, etc.) to assist in designing crRNA. These tools can predict the efficiency and specificity of the sgRNA, helping to avoid potential off-target effects.
6.After completing the design, the synthetic crRNA sequence can be ordered from a synthetic biology company.

Selecting a suitable gene delivery system requires a comprehensive assessment based on specific experimental conditions, research objectives, and cell types. Quantitatively comparing various systems in terms of delivery efficiency, cytotoxicity, and stability is an important step in determining the choice.
Viral delivery systems are suitable for experiments that require high delivery efficiency and sustained gene expression, especially when cells can tolerate higher levels of cytotoxicity and immune responses. If lower cytotoxicity and immune response, along with ease of use and cost-effectiveness, are priorities, then a liposome-based gene delivery system should be chosen. For high delivery efficiency that involves delivering large DNA fragments, and if the user can accept a higher operational complexity, a gene gun delivery system is an optional method. If high delivery efficiency is needed while maintaining relative simplicity and no special equipment is required, then the electroporation delivery system may be a suitable choice.