Knock-In Vector Construction

Gene knock-in (KI) refers to the precise insertion of an exogenous DNA fragment into a specific genomic locus, allowing the inserted sequence to perform its intended function. This technique can endow cells or organisms with new functionalities or restore the normal function of defective genes. It is widely applied in studying gene function and regulatory mechanisms, constructing disease models, and advancing gene therapy.

Service Details

Services Fluorescent Protein Knock-in Vector / Tag Protein Knock-in Vector / Specific DNA Fragment Knock-in Vector
Deliverables 1. Plasmid map
2. Plasmid sequencing results
3. Plasmid amplification instructions
4. Plasmids (sgRNA-Cas9 editing plasmid and Donor plasmid)
Turnaround/Price   Consult online for details
EDITGENE has developed an innovative and highly efficient gene knock-in technology, offering gene knock-in vector construction services. Utilizing an upgraded CRISPR/Cas9 system that includes sgRNA-Cas9 plasmids and Donor plasmids, we have optimized sgRNA and homology arm design strategies. Through homology-directed repair (HDR) and other mechanisms, the target gene sequence is inserted precisely at the designated knock-in site.

EDI-Service Advantages

Long Insert Fragments
Our editing vectors enable the insertion of DNA fragments up to 5 kb in length.
 
High-Activity Cas9 Plasmids
Utilizing highly active Cas9 plasmids to efficiently cleave genomic DNA, increasing overall editing efficiency.
Optimized sgRNA
Our upgraded sgRNA design algorithm ensures high-efficiency sgRNA for precise and effective gene editing.
Experienced Team
Our skilled and experienced technical team can design tailored knock-in vectors according to client requirements.

Service Types

Customized gene knock-in vectors can be designed and constructed based on client requirements and specific gene characteristics.
Fluorescent Protein Knock-in Vectors EGFP, Luc, mCherry, etc.
Tag Protein Knock-in Vectors Flag, HA, Myc, HiBiT, etc.
Vectors for Inserting Specific DNA Fragments into Target Genomic Loci /
Vectors for Inserting Specific DNA Fragments into Genomic Safe Harbor Sites /

Plasmid Map

 
pCRISPR Donor Plasmid Map
 
 
 
pSpCas9 Plasmid Map

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

Reference Materials

Article Title: CRISPR/Cas9-mediated knockin of IRES-tdTomato at Ins2 locus reveals no RFP-positive cells in mouse islets

Using CRISPR/Cas9 gene editing technology, researchers constructed a transgenic mouse model to express a specific fluorescent protein in pancreatic β-cells. In C57BL/6J mice, the tdTomato transgene was inserted downstream of the Ins2 promoter. The researchers designed an Ins2-specific single-guide RNA targeting exon 2 for the CRISPR/Cas9 system and constructed a donor vector. The Cas9, single-guide RNA, and donor vector were then injected into mouse fertilized eggs in vitro, and these fertilized eggs were implanted into pseudopregnant mice. Heterozygous mating produced homozygous mice, and genotype identification, in vivo imaging, and frozen sections confirmed the knock-in effect. Six F0 mice and stably inherited Ins2-IRES-tdTomato F1 were obtained. Genome sequencing results showed no changes in the Ins2 exon compared to the control group; only the base sequence of tdTomato was added without base mutations. However, no expression of red fluorescent protein (RFP) was observed in vivo imaging and frozen sections, indicating low expression of the tdTomato protein and fluorescence intensity not reaching the detection threshold. In CRISPR/Cas9 technology, the IRES-linked foreign fragment may affect the transcription levels of the upstream gene, leading to low expression levels of the downstream gene and affecting the insertion effect.

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

During vector amplification, Escherichia coli (E. coli) strains are typically used. The commonly used strain for most non-recombinant vectors is DH5α, which is suitable for most applications. For recombinant vectors, such as lentiviral vectors and transposon vectors, the Stbl3 strain can be used for amplification. Stbl3 is a specialized E. coli strain derived from HB101, which has a mutation in the recombinase gene recA13, effectively suppressing recombination of long fragment terminal repeat regions and reducing the likelihood of erroneous recombination.
When selecting a vector, consider the purpose of the experiment and the type of host cells. For example, plasmid vectors are commonly used for gene expression or amplification in bacteria, while viral vectors are more suitable for gene transfer in mammalian cells. Additionally, the vector's promoter, replicon, and antibiotic selection markers should be chosen based on specific requirements.

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