Induced pluripotent stem cells (iPSCs) are a type of cell that reprogram the somatic cells into a pluripotent state. They have characteristics similar to embryonic stem cells and can differentiate into almost all cell types in the body. Therefore, scientists can use IPSC cells to generate various cell types in vitro for research and treatment, instead of using embryonic stem cells to achieve the experimental purposes.

iPSCs have broad clinical potential, including applications in cell therapy (e.g., for diabetes or heart disease treatment), tissue engineering (e.g., development of artificial skin or liver tissue), and personalized drug screening (e.g., selecting optimal treatments based on a patient’s specific cellular response). These applications may transform treatment methods, offering more effective and personalized medical services.

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.

The main difference lies in the duration and stability of gene expression: Transient cell line – The target gene is expressed temporarily in cells, typically lasting hours to days, and is suitable for short-term experiments. Stable cell line – The target gene is stably integrated into the cell genome, allowing long-term expression, suitable for extended research and production.

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

Monoclonal screening is the process of isolating a single clone from a mixed pool of cells and expanding that clone into a cell line. Monoclonal screening ensures that the cell lines used originate from a single cell, guaranteeing a high degree of genetic background consistency. After cells are gene-edited or genetically modified, the genetic background differences among the cells in the initial cell pool can be significant, making subsequent experimental results inaccurate. By using monoclonal screening, researchers can obtain cell populations with consistent genetic backgrounds and stable gene edits, allowing for stable and accurate monitoring of phenotypic changes.

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.

Cell selection can follow these principles:
1.It should align with the research objectives.
2.The genes targeted by the sgRNA library should correspond to the cell's lineage.
3.The cells should be capable of stable passaging.
4.The transfection efficiency should be high.
5.Avoid primary cells whenever possible. Primary cells cannot be stably passaged and may experience significant cell death during the library screening process, which can hinder experiment completion. If primary cells must be used for library screening, mitigating this risk can be achieved by lowering cell coverage and choosing a library with fewer gRNAs to minimize the cell pool size and shorten the experimental duration.

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.

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.

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.

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.

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.