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CRISPR Library Screening
CRISPR library screening is a high-throughput gene screening approach based on CRISPR/Cas9 technology. It involves inducing single-gene perturbations within a pooled cell population, followed by functional screening methods such as drug treatment or viral infection. High-throughput sequencing is then used to assess the genes affected by the perturbation, allowing for the identification of genes associated with specific phenotypes of interest.
The types of perturbations are diverse, including gene knockout, gene activation, gene silencing, and point mutations. Additionally, the screening scope can cover the entire genome or be focused on specific pathways. CRISPR library screening has broad applications in molecular and cell biology, genetic disorders, cancer, immune regulation, and microbiology. It can be used to identify cancer-related genes, investigate tumor-specific biological processes, and discover novel immune-regulatory genes, among other functions.
Service Details
| Cell Types |
Various cell types including tumor cells and stem cells, etc. Click to view the Comprehensive Cell List |
|---|---|
| Services | Custom sgRNA library design, amplification, and quality control / Cas9 stable cell line construction / Library viral packaging / Library cell pool construction / Functional screening assays / NGS analysis |
| Turnaround/Price |
 Consult online for details |
EDITGENE has over a decade of expertise in gene editing, specializing in cellular gene editing techniques. We offer a comprehensive suite of CRISPR library services, including custom CRISPR library design, amplification, and quality control, lentiviral packaging, library cell pool construction, positive or negative screening, high-throughput sequencing, and bioinformatics analysis, providing a one-stop CRISPR library screening solution.
● CRISPR-KO Library
This library targets the 5’ exons of coding genes, where sgRNA guides the Cas9 protein to specific genomic locations to induce DNA double-strand breaks. The cell's non-homologous end joining (NHEJ) repair pathway reconnects the broken DNA strands, resulting in indels that are not in multiples of three, leading to loss of gene function.
● CRISPRa Library
This library activates target gene transcription by fusing dCas9 with transcriptional activators. Guided by sgRNA, the system targets the promoter region of the target gene to induce gene transcription activation.
● CRISPRi Library
This library suppresses gene expression by fusing dCas9 with transcriptional repression domains. Guided by sgRNA, it targets the transcription start site of the target gene, interfering with transcriptional activity and reducing gene expression.
EDI-Service Advantages
Multiple Delivery Formats
Various delivery options to meet diverse research needs.
Proprietary Development
Exclusively developed sgRNA design algorithm for enhanced efficiency.
Personalized Bioinformatics Analysis
In addition to standard analysis, personalized bioinformatics services are available upon request.
One-Stop Service
Comprehensive, one-stop service for a hassle-free experience.
Service Contents and Deliverables
| Services | Descriptio | Deliverables |
|---|---|---|
|
Custom sgRNA Library Design, Amplification, and Quality Control |
sgRNA design, sgRNA-oligo pool synthesis, vector construction, plasmid library amplification, NGS quality control |
Plasmid library: 100 µg NGS quality control report (coverage >99%, uniformity <10) |
| Cas9 Stable Cell Line Customization | Cas9 lentiviral packaging, Cas9 stable cell line construction, editing activity detection | Cas9 stable cell line (1×10^6 cells per line) |
|
Library Lentiviral Packaging |
Library viral packaging and viral titer determination by cell titration, ensuring live virus titer >1×10^6 TU/ml |
Small scale virus (total 1×10^8 TU) Medium scale virus (total 5×10^8 TU) Large scale virus (total 5×10^9 TU) Virus titer report |
| Library Cell Pool Construction | Optimization of transduction conditions (MOI <0.3), library cell pool construction | Library cell pool |
| Functional Screening Assay | Customized screening services: drug screening, viral infection, bacterial infection, flow cytometry sorting, etc. | Experimental report |
| NGS Analysis | sgRNA sequencing library construction, NGS sequencing, bioinformatics analysis | Experimental report, raw NGS sequencing data |
Workflow
Case Study
EDITGENE provides tailored CRISPR knockout library screening designs based on client requirements.
● Full CRISPR Knockout Library Screening Service (selected experimental results)
1. Cas9 Stable Cell Line Construction
Cas9 stable cell lines are established by transducing the target cells with Cas9 lentivirus. Once the Cas9-expressing stable cell line is generated, validated sgRNAs are used to assess editing efficiency.
2. NGS Quality Control Results of sgRNA Plasmid Library
The sequencing data was analyzed using MAGeCK. The quality control results indicated that over 85% of reads aligned to the target library, demonstrating good PCR and sequencing quality. The Gini Index of the library was less than 0.1, with a coverage of 99.8%, reflecting uniform distribution of sgRNAs. These combined results confirm the high quality of the prepared sgRNA plasmid library.
3. Post-Screening Library Sequencing and Bioinformatics Analysis
Genomic DNA is extracted from the selected cells, followed by amplification and sequencing. The sequencing data from the sgRNA library is analyzed using the MAGeCK-RRA algorithm for quality control and identification of differential genes between screened groups. Subsequently, downstream functional enrichment analysis is performed using the MAGeCKFlute toolkit.
① MAGeCK Quality Control Analysis
The mageck count algorithm is used to align forward sequencing reads to the sgRNA library and calculate QC metrics. The MAGeCKFlute toolkit is then employed to visualize the statistical data.
② Differential Gene Analysis
Using the MAGeCK RRA algorithm, sgRNAs are ranked based on P-values (negative binomial model), and significant genes are identified through the α-RRA model for both positive and negative selection. The MAGeCKFlute toolkit is used to visualize the analysis data. A differential analysis is performed with Library D as the control, successfully identifying significant genes.
③ Functional Enrichment Analysis
The MAGeCKFlute toolkit is used to compare gene function databases, applying the hypergeometric test (HGT) statistical method to perform KEGG, REACTOME, GOBP, and Complex enrichment analyses for both positively and negatively selected genes.
Advantage and Characteristic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
FAQ
How do I choose suitable cells for library screening?
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.
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.
What is the difference between a single-plasmid system and a dual-plasmid system for library vectors?
What is the difference between a single-plasmid system and a dual-plasmid system for library vectors?
A single-plasmid system can achieve gene editing with one transfection, making construction relatively simple, but the larger plasmid size can lead to lower infection efficiency. In a dual-plasmid system, two vectors are used, each carrying either the Cas9 or sgRNA expression cassette. A stable Cas9 cell line is first constructed, and then the sgRNA library is transfected into this cell line. This approach has several advantages:
1.Increased Editing Efficiency: The independent and stable expression of Cas9 protein and sgRNA on different vectors enhances editing efficiency.
2.Flexibility: Vectors can be designed and constructed flexibly based on experimental needs, such as loading two sgRNA expression cassettes into one vector.
3.Increased Viral Titer: By splitting into two plasmids, the load on each plasmid is reduced, facilitating viral packaging and increasing yield and titer.
4.Increased Stability: Independently constructing a stable Cas9 cell line ensures that the Cas9 expression levels and editing efficiency in each cell are approximately the same, enhancing experimental accuracy.
1.Increased Editing Efficiency: The independent and stable expression of Cas9 protein and sgRNA on different vectors enhances editing efficiency.
2.Flexibility: Vectors can be designed and constructed flexibly based on experimental needs, such as loading two sgRNA expression cassettes into one vector.
3.Increased Viral Titer: By splitting into two plasmids, the load on each plasmid is reduced, facilitating viral packaging and increasing yield and titer.
4.Increased Stability: Independently constructing a stable Cas9 cell line ensures that the Cas9 expression levels and editing efficiency in each cell are approximately the same, enhancing experimental accuracy.
How do I choose between a whole-genome or subgenomic CRISPR library?
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.
