Search Products

CRISPR Library Screening

CRISPR library screening is a high-throughput gene screening method based on CRISPR/Cas9 technology. By implementing a single gene perturbation on the cell pool and conducting functional screening such as drug stress or viral infection, genes related to the phenotype of interest can be identified. At present, the perturbation types are diverse, such as gene knockout, gene activation, gene silencing, gene point mutations, etc. In addition, the scope of screening can be the entire genome or specific signaling pathways. CRISPR library screening has wide applications in genetic diseases, cancer, immune regulation, microbiology, and other fields. It can be used to discover cancer-related genes, study specific biological processes for tumors, and discover the new immune regulatory genes.

Service Contents and Deliverables

Services Description 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
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. 
 

Workflow

 

EDI-Service Advantages

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.
 
Cas9 Stable Cell Line Construction | EDITGENE
 
 
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.
 
NGS Quality Control Results of sgRNA Plasmid Library | EDITGENE
 
 
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.
 
Figure1.1 Mapping ratio
Figure1.2 Gini index
Figure1.3 Missed SgRNAS
 
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.
 
Figure1.4 group ES-Volcano plot
Figure1.5 group ES-Dot plot-positive screening genes
Figure1.5 group ES-Dot plot-negative screening qenes
 
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.
 
Figure 1.8 group ES-Positive Functional Enrichment Analysis
Figure 1.9 group ES-Negative Functional Enrichment Analysis
 

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

Selected Customer Resources

IF=50.5
Nature

Abstract:

To date, more than half of global hepatocellular carcinoma (HCC) cases occur in China, yet comprehensive whole-genome analyses focusing on HBV-related HCC within the Chinese population remain scarce. To address this challenge, researchers initiated the China Liver Cancer Atlas (CLCA) project, aiming to conduct large-scale whole-genome sequencing to unravel the unique pathogenic mechanisms and evolutionary trajectories of HCC in China.

The researchers performed deep whole-genome sequencing on 494 HCC tumor samples, with an average depth of 120×, alongside matched blood controls, providing a detailed genomic landscape of HBV-associated HCC. Beyond confirming well-known coding driver genes such as TP53 and CTNNB1, the study identified six novel coding drivers—including FGA—and 31 non-coding driver genes.

Additionally, the research uncovered five new mutational signatures, including SBS_H8, and characterized the presence of extrachromosomal circular DNA (ecDNA) formed via HBV integration, which contributes to oncogene amplification and overexpression. Functional validation experiments demonstrated that mutations in genes such as FGA, PPP1R12B, and KCNJ12 significantly enhance HCC cell proliferation, migration, and invasion.

These findings not only deepen our insights into the genomics of HCC, but also open up new potential targets for diagnosis and therapy. View details>>

Candidate driver landscape

 

IF=27.4
Advanced Materials

Abstract:

During the acute inflammatory phase of tendon injury, excessive activation of macrophages leads to the overexpression of SPP1, which encodes osteopontin (OPN), thereby impairing tissue regeneration. The CRISPR-Cas13 system holds great promise for tissue repair due to its unique RNA editing and rapid degradation capabilities; however, its application has been limited by the lack of efficient delivery methods.

To address this, the researchers systematically screened various cationic polymers targeting macrophages and developed a nanocluster carrier capable of efficiently delivering Cas13 ribonucleoprotein complexes (Cas13 RNPs) into macrophages. Utilizing a reactive oxygen species (ROS)-responsive release mechanism, this system specifically suppresses the overexpression of SPP1 in macrophages within the acute inflammatory microenvironment of tendon injury.

Experimental results demonstrated that this targeted delivery strategy significantly reduced the population of SPP1-overexpressing macrophages induced by injury, inhibited fibroblast activation, and alleviated peritendinous adhesion formation. Furthermore, the study elucidated that SPP1 promotes fibroblast activation and migration through the CD44/AKT signaling pathway, and that inhibiting this pathway effectively mitigates adhesion formation following tendon injury. View details>>

Schematic diagram illustrating immune microenvironment-activated mRNA editing strategies of macrophages for PA therapy

IF=12.8
Biomaterials

Abstract：

Spinal cord injury (SCI) is a severe disabling condition that causes permanent loss of sensory, autonomic, and motor functions. While stem cell therapies, particularly mesenchymal stem cells (MSCs), show great promise for SCI treatment, their limited regenerative capacity restricts their application in tissue repair. The researchers observed that extracellular vesicles derived from antler bud progenitor cells (EVsABPC) may carry bioactive signals that promote tissue regeneration. Accordingly, they isolated and engineered EVs from ABPCs for SCI therapeutic investigation.

The study found that EVsABPC significantly enhanced neural stem cell (NSC) proliferation, promoted axonal growth, reduced neuronal apoptosis, and modulated inflammation by shifting macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Moreover, engineered EVsABPC modified with cell-penetrating peptides demonstrated improved targeting to the SCI lesion site, markedly enhancing neural regeneration and functional motor recovery. These findings highlight EVsABPC as a promising candidate for SCI therapy. View details>>

Graphical abstract

IF=11.3
Journal of Hazardous Materials

Abstract:

S-metolachlor (S-MET) is one of the most widely produced and applied herbicides in China. Owing to its chemical properties, it tends to persist in soil and easily contaminates surface and groundwater through leaching and runoff. This environmental persistence poses a serious threat to plant development and, through the food chain, to human health.

To address the limitations of current detection technologies and meet the growing demand for high-efficiency analytical tools, the researchers employed a mammalian expression system to generate recombinant antibodies targeting S-MET.

Building on the successful expression of these antibodies, they established a sensitive immunoassay for monitoring S-MET residues in various environmental water samples. The icELISA results showed that the recombinant antibodies retained the sensitivity, specificity, and biological activity of the original monoclonal antibodies, delivering accurate and reproducible detection in river water, agricultural runoff, and tap water. View details>>

Graphical abstract

 

IF=10.7
Biosensors and Bioelectronics

Abstract:

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that regulate gene expression by interacting with the mRNAs of target genes. Given their crucial role in the development and progression of various diseases, miRNAs have emerged as promising biomarkers for clinical diagnostics.

In this study, researchers established a novel detection platform, termed DBmRCA, which combines dumbbell probe-initiated multi-rolling circle amplification with the high-sensitivity signal output of CRISPR/Cas12a. This enzyme-free, isothermal method enables accurate quantification of miRNA within just 30 minutes.

Clinical validation revealed that the expression levels of miR-200a and miR-126 were significantly downregulated in lung cancer tissues, and results from DBmRCA were consistent with those obtained by conventional techniques. With its high sensitivity, rapid turnaround, and simplified workflow, the DBmRCA platform presents a reliable tool for miRNA detection and holds strong promise for early diagnosis and therapeutic monitoring of lung cancer. View details>>

Graphical abstract

FAQ

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.
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.
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.

Contact Us

*
*
*
*
How did you hear about us：
Contact Us
*
*
*
*
How did you hear about us：
✖ service online