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High-Throughput Gene Knockout Technology for Drug Screening and Target Identification

A critical step in drug discovery is target identification—the discovery of 'druggable' biological targets. After identifying a target, it must be validated to demonstrate the functional relationship between the target and the disease phenotype, while ensuring safety. The drug discovery process is long and expensive, but proper target validation increases the likelihood of developing effective drugs and ultimately achieving clinical success. Recent advances in CRISPR-Cas9 gene editing technology have had a significant impact on drug discovery, allowing researchers to intentionally activate or inhibit genes to elucidate and understand cellular pathways involved in disease progression. It can also be used to create accurate models for better studying disease phenotypes and screening small molecules to quickly identify numerous potential targets.

Once these potential targets are identified, mass spectrometry can be used to study the interactions between potential drug molecules and their target proteins or metabolites, revealing how drugs bind to their targets and how this binding affects intracellular signaling and metabolic pathways. By combining high-throughput gene knockout technology and mass spectrometry analysis, researchers can not only quickly identify new drug targets but also gain a deep understanding of drug mechanisms, leading to the design of more effective and safer therapies.

Application of High-Throughput Gene Knockout in Drug Discovery

Baite Parker Biotech (BTP) offers a one-stop solution from gene knockout to drug target identification using high-throughput gene knockout technology combined with a high-throughput mass spectrometry platform. Our optimized CRISPR/Cas9 system enables single/multiple gene knockouts, frameshift mutations, and large sequence deletions in human/mouse cell lines, primary cells, immune cells, and iPS cells. BTP has established seven major testing platforms and has a laboratory with dual CNAS/ISO9001 quality system certification, aiming to provide you with the highest quality research services. We look forward to collaborating with you—feel free to contact us for more service details!

Service Advantages

1) Knockout Efficiency Guarantee: Our optimized CRISPR/Cas9 system achieves over 70% knockout efficiency for more than 80% of targets. Post-knockout, we use Sanger sequencing and deep proteomics to doubly verify knockout efficiency, providing a dual guarantee for data authenticity.

2) Multiple Target-Based Drug Target Identification Platforms: Relying on a high-resolution mass spectrometry platform, combined with affinity chromatography and active site-directed probe technologies, we have developed and validated multiple target-based drug target identification platforms to meet different research needs.

3) Short Project Cycle:Before conducting gene knockout experiments, we analyze gene necessity and expression levels in different cells to ensure experimental feasibility and reduce the risk of experimental failure. There's no need to design pre-experiments for gRNA validation based on the client's target, significantly shortening the project cycle.

4) One-Stop Service:BTP, with its rich omics service experience and professional technical personnel, can customize the most optimal project plan according to your needs. Simply tell us your experimental objectives and send samples, and BTP will handle all subsequent project stages, including sample processing, experimental analysis, data analysis, and project reporting.

Application Cases

1.Genome-Wide CRISPR-Cas9 Knockout Screening Combined with Mass Spectrometry to Determine MEK Inhibitor Resistance Targets in KRAS Mutant Colorectal Cancer

Targeting the KRAS pathway is a promising approach for treating colorectal cancer (CRC), but CRC cells can tolerate MEK inhibitors, leading to unsatisfactory clinical trial results in CRC patients. Thus, the authors conducted genome-wide CRISPR/Cas9 screening in the presence of MEK inhibitors to identify genes that are synthetically lethal with MEK inhibition in CRC models harboring KRAS mutations. The results indicate that GRB7 imparts major resistance to MEK inhibitors in CRC cells via the RTK pathway. Mass spectrometry analysis of GRB7 immunoprecipitates showed that PLK1 is a primary interacting kinase with GRB7. The combination of PLK1 and MEK inhibitors synergistically inhibited CRC cell proliferation and induced apoptosis in vitro and in vivo. Ultimately, the authors identified GRB7-PLK1 as a pivot mediating RTK, leading to MEK inhibitor resistance. This study suggests that PLK1 is a promising target for synergizing MEK inhibitors in the clinical treatment of CRC patients carrying KRAS mutations.

Yu, C., et al. Oncogene. 2022.

Figure 1. CRISPR Library Screening Identifies RTK Pathway Involvement in MEK Inhibitor Resistance in KRAS Mutant Colorectal Cancer Cells

BiotechPack, A Biopharmaceutical Characterization and Multi-Omics Mass Spectrometry (MS) Services Provider

Gefitinib and Erlotinib are effective drugs for treating non-small cell lung cancer, targeting the epidermal growth factor receptor (EGFR). However, the acquired EGFR mutation C797S prevents drugs from interacting with the EGFR tyrosine kinase domain target site, leading to resistance in patients. AstraZeneca used CRISPR-Cas9 high-throughput gene knockout technology to create precise C797S mutations in cancer cell lines. The application of the C797S cell line model allowed drug screening to identify next-generation compounds targeting this novel mutation.