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Point Mutation Vector Construction

The most widely used strategy for inducing gene mutations leverages CRISPR/Cas9 gene editing technology in conjunction with the cell's homology-directed repair (HDR) pathway. In this method, gRNA directs the Cas9 enzyme to a specific target site, where it induces a DNA double-strand break (DSB), prompting the cell's repair mechanisms. Through HDR, the intended mutation is introduced. However, this approach presents several drawbacks, including low editing efficiency, reduced homozygosity rates, and an increased likelihood of random insertions or deletions (indels).

服务详情

Deliverables 1. Plasmid map
2. Plasmid sequencing results
3. Plasmid handling instructions
4. Plasmids (three sets: pegRNA + sgRNA), auxiliary plasmids (mixed tube)
Turnaround/Price   Consult online for details
Based on Prime Editing (PE) technology, EDITGENE has developed the innovative Bingo™ platform. This platform features advanced algorithms for designing highly efficient pegRNA and Nick sgRNA, enabling the construction of highly efficient gene point mutation vectors.
 
Prime Editing Mechanism:The core of this technology relies on a specialized guide RNA, known as pegRNA. This RNA not only locates the target sequence but also contains the information for the desired base modification. When combined with the PEmax gene-editing enzyme (Cas9n-RT), the system is capable of precisely targeting specific genomic sites and inserting the desired DNA sequence into the genome. This enables accurate single-base substitutions or small-scale insertions and deletions.

EDI-Service Advantages

High-Efficiency PE Editing System
Compared to HDR strategies, Prime Editing offers higher editing efficiency and a greater likelihood of achieving homozygosity.
Optimized pegRNA Design
Proprietary algorithms for pegRNA design enhance precision and efficiency.                       
 
Highly Active Cas9n-RT Enzyme
The optimized Cas9n-RT enzyme offers improved activity and stability, facilitating more reliable gene editing.
Versatile Editing Types
Capable of performing a wide range of edits, including 12 types of base substitutions, as well as small-scale insertions and deletions.

Plasmid Map

 

pegRNA Plasmid Map | EDITGENE
 
pegRNA Plasmid Map
 
 
Nick sgRNA Plasmid Map | EDITGENE
 
Nick sgRNA 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

参考文献

Article Title: Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells

Cystic fibrosis (CF) is a common hereditary lethal disease caused by loss-of-function mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Various CFTR modulators have been approved, but these highly effective modulators (HEMTs) are only suitable for patients with at least one F508del allele or other responsive CFTR mutations, leaving many patients with (ultra-)rare CFTR mutations without treatment options. With advances in scientific research, gene therapy has emerged as a new opportunity for these mutations. Notably, the recently developed CRISPR-based system known as prime editing (PE) opens a new era for the treatment of genetic diseases. Prime editing can "rewrite" and correct mutations on patients' chromosomes in situ, providing new opportunities for treating monogenic diseases like CF.
In this study, researchers designed a prime editing strategy targeting the L227R and N1303K mutations in the CFTR gene using CRISPR-Cas9 technology. They constructed stable cell models expressing 3HA-L227R-CFTR and 3HA-N1303K-CFTR in HEK293T cells to evaluate the effects of prime editing. Using a developed DETECTOR machine learning algorithm to ensure efficiency and accuracy, the researchers further assessed gene and functional correction. The results showed editing efficiency as high as 25%, and the corrected CFTR protein exhibited significant restoration in glycosylation, localization, and ion channel function. These results were also validated in primary cell model experiments. Additionally, through whole-genome assessment analysis, no significant off-target editing events were found, demonstrating the high fidelity of prime editing, and the clinical relevance and safety assessments of the study were also validated. Overall, this study demonstrates the potential of prime editing technology in correcting CFTR gene mutations and restoring CFTR protein function, providing new ideas and methods for cystic fibrosis treatment.

精选客户文章

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

参考文献

DOI:10.1038/s41586-024-07054-3

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

 

DOI:10.1002/adma.202311964

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

DOI:10.1016/j.biomaterials.2022.121990

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

DOI:10.1016/j.jhazmat.2021.126305

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

 

DOI:10.1016/j.bios.2024.116676

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

What host bacteria are used for vector construction in EDITGENE? What type of strains can customers use to amplify plasmid vectors?
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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