Innovative CRISPR-dTAG System for Targeted Protein Degradation    

RNA interference (RNAi) and CRISPR-Cas9 technology are powerful tools for determining the functional consequences of specific genes' loss or gain by modulating target genes' expression levels. To investigate the effects of particular genes on cell growth and survival, researchers can utilize techniques such as RNA interference (RNAi) or CRISPR-Cas9 to inhibit or eliminate the expression of particular genes.

Though these techniques are quite useful for identifying the functional implications of target genes, they are not as good for evaluating sudden alterations, especially when it comes to proteins that are essential for cell division and survival. Acute alterations in cells may not be instantly reflected by RNA interference and CRISPR-Cas9 technologies because cells may have complicated responses and regulatory systems to changes in protein levels and because these technologies may require some time to establish control or changes in gene expression.

The dTAG System: A Breakthrough in Targeted Protein Degradation

The groundbreaking degradation tag (dTAG) system uses chemical techniques to cause the breakdown of certain target proteins. Among other BET bromodomain transcriptional co-activators, it can specifically degrade BRD2, BRD3, and BRD4. These substances have biochemical activity that is similar to catalysis, which allows for the quick and targeted destruction of target proteins without interfering with their ligands. This strategy is effective with proteins like BRD4 and has been expanded to include FKBP12, ERRα, RIPK2, and BRD9. This strategy will develop chemical probes that will make target biology easier to investigate.

Figure 1. Two molecules function differently: (A)For dTAG-13: Interacts with FKBP12F36V-fused protein of interest (POI) and cereblon (CRBN), guiding them for POI degradation. Contains AP1867 and thalidomide ligands, recruiting the CRL4–CRBN E3 ubiquitin ligase. (B) For dTAGV-1: Recruits the von Hippel–Lindau (VHL) E3 ligase complex, boosting dTAG system efficiency. In both cases, the goal is POI degradation by recruiting specific E3 ligase complexes. (Prozzillo Y, et al., 2020)

Basic Principle

The dTAG system involves the use of a heterobifunctional degrader called dTAG-13, which fuses FKBP12F36V with the protein of interest (POI). This fusion protein is achieved through either transgene expression or CRISPR-mediated locus-specific knock-in. When the dTAG-13 molecule interacts with the FKBP12F36V-fused POI and cereblon (CRBN), it guides these proteins to the endogenous proteasome machinery for degradation.

Molecular Composition

The CRBN-selective ligands thalidomide and AP1867 make up the dTAG-13 molecule, and they are joined by several linkers. The purpose of these compounds is to specifically cause POI to degrade.

Applications

The dTAG system has been utilized to assess the acute degradation consequences of proteins such as ENL and MELK, as well as for the rapid and selective degradation of FKBP12F36V-fused chimeras like BRD4, HDAC1, EZH2, MYC, PLK1, and KRASG12V. Furthermore, this system has been successfully applied in vivo in mouse models to study the degradation of IE2 protein and solute carrier (SLC) proteins. An improved version of dTAG, known as dTAGV-1, has been created by researchers. Whereas dTAG-13 is limited in its ability to degrade certain proteins, dTAGV-1 interacts with the von Hippel-Lindau (VHL) E3 ligase complex. With a greater half-life, increased exposure, and extended breakdown time, this novel compound has enhanced pharmacokinetic and pharmacodynamic properties.

Combining CRISPR with dTAG: A Powerful Approach

Using the dTAG method in conjunction with CRISPR technology provides an effective way to examine target proteins in cellular and animal models. Through the careful manipulation of target protein expression and the use of dTAG to induce protein breakdown, scientists may clarify the functions and interdependencies of these proteins in a range of biological processes and illnesses.

Learn More about Creative Biogene's CRISPR Technology Services

Creative Biogene's CRISPR Technology Services for Targeted Protein Degradation offer a cutting-edge solution for researchers aiming to unravel the intricacies of protein function within biological systems. Leveraging the power of CRISPR-Cas9 technology, these services enable precise modulation of gene expression levels to induce targeted protein degradation.

At the core of these services lies a comprehensive suite of tools and expertise tailored to meet the diverse needs of protein degradation studies. Whether you're investigating the functional consequences of specific proteins or elucidating their roles in disease pathways, Creative Biogene provides customized solutions to address your research objectives.

Key features of Creative Biogene's CRISPR Technology Services for Targeted Protein Degradation

Design and Optimization: Our team of experts works closely with you to design and optimize CRISPR-based strategies for targeted protein degradation. By leveraging advanced bioinformatics tools and cutting-edge molecular biology techniques, we ensure precise and efficient targeting of the protein of interest.

Generation of Cell Lines: Creative Biogene offers robust cell line generation services tailored to your specific research needs. Whether you require stable knockout/knockdown cell lines or cell lines expressing tagged proteins for degradation studies, we have the expertise to deliver reliable and reproducible results.

Validation and Characterization: We provide comprehensive validation and characterization services to confirm the efficacy and specificity of targeted protein degradation. Through a combination of molecular assays, including Western blotting, immunofluorescence, and functional assays, we ensure accurate assessment of protein degradation outcomes.

Customized Assay Development: Our team can develop customized assays to monitor protein degradation dynamics in real-time, allowing for precise characterization of protein turnover kinetics and degradation pathways.

By partnering with Creative Biogene for your CRISPR-based protein degradation studies, you gain access to a comprehensive suite of services and expertise to accelerate your research and uncover novel insights into protein function and regulation. Explore our range of services today to discover how we can support your research endeavors in targeted protein degradation.

Related Services and Products

Services

CRISPR Basic Services
Gene Knockout Mouse Generation
Knockout Cell Line Generation

Products

CRISPR gRNA Synthesis Kit
HDR mediated CRISPR gene knockout kit
Knockout Detection Kit