CRISPR Cas9 Gene Editing - CRISPR Library Screening - Gene Knockout Cell Line - Knock in Cell Line - Cas12a – EDITGENE

Gene interference (gene knockdown) is a technique that partially suppresses the expression of target genes using small RNA molecules (such as siRNA or shRNA) or CRISPRi systems:
siRNA/shRNA: The siRNA associates with the cellular RISC complex to degrade target mRNA, thereby blocking protein translation.
CRISPRi: A modified dCas9 protein binds to a guide RNA (gRNA) and blocks the gene promoter, repressing transcription without cleaving DNA.
Applications: Gene interference is widely used for functional genomics studies, disease mechanism investigation, and drug target validation.

Technology	Applications	Delivery Format
siRNA transfection	Rapid validation (effects observed within 72-96 hours)	Cells post-interference/experimental report
shRNA viral vectors	Long-term suppression (stable cell line screening)	Viral particles/stable knockdown cell lines
CRISPRi	High-specificity gene inhibition	dCas9-expressing cell line and gRNA vector

Parameter	siRNA	shRNA (viral vector)	CRISPRi
Duration of Effect	Transient (5-7 days)	Long-term (stably maintained through passages)	Long-term and stable
Cell Type Compatibility	Easily transfected cells	Broad spectrum, including hard-to-transfect cells	Requires pre-established dCas9 cell line
Off-target Risk	Medium to high	Medium	Very low
Typical Use	Rapid validation	Animal or cell models	High-precision gene regulation

Recommendations:
For Rapid Validation: Use siRNA. Suitable for quickly testing the function of 1–2 genes in easily transfected cells. This approach is cost-effective and fast.
For Stable Gene Knockdown in Cells or In Vivo: Use shRNA (viral delivery). A well-established technique that enables long-term and stable gene silencing, widely applied in the construction of cellular and animal models.
For High Specificity, Low Off-target, Reversible Regulation, Multi-gene Manipulation, Non-coding RNA Targeting: Use CRISPRi (viral delivery to establish stable cell lines).

Lentivirus is a gene delivery tool that introduces exogenous genes into cells. By using tool cells such as 293T, lentiviral vectors carrying target DNA fragments are packaged into lentiviral particles with cell-infectious activity. This packaging process includes constructing lentiviral vectors, preparing packaging plasmids, culturing tool cells, transfecting plasmids, collecting viral particles, purifying and concentrating viral particles, and titration.

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.

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.

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.

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 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.

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.

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 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.

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.