CRISPR-Cas9 Libraries and Anti-cancer Drug Discovery

CRISPR-Cas9 is a powerful gene editing tool that enables highly accurate and stable genetic editing of the genomes of all organisms. CRISPR-Cas9 technology plays an important role in the screening of genetic libraries. In the field of anti-cancer drug discovery, CRISPR-Cas9 screening technology has many advantages for variable applications, such as functional and target identification, non-coding RNA information, small molecule action, and drug target discovery. Based on the various functions of gene libraries, Creative Biolabs provides a comprehensive genomic library construction service to advance drug discovery.

Types of CRISPR-Cas9 Pooled Libraries

Pooled library screening approaches using CRISPR-Cas9-based gene editing tools have evolved into powerful methods for identifying interesting gene mutations through phenotypic changes or viability screens. Currently, custom-designed CRISPR-Cas9 libraries covering the whole genome are used for basic gene screening or a range of genes with specific phenotypes or cellular functions for target discovery. Common CRISPR-Cas9 pooled libraries include but are not limited to CRISPRko, CRISPRi, CRISPRa, and point mutagenesis. In experiments, researchers need to select the appropriate gene library based on specific experimental needs.

Fig 1. (A) Schematic diagram of the CRISPR-Cas9 library positive and negative selection workflow. (B) Experimental models utilizing the CRISPR-Cas9 library screening approach for anti-cancer drug discovery including in vitro models, in vivo models, organoid models, combinatorics studies, and small molecule screenings. (Chan, Y. T., et al., 2022)

CRISPR-Cas9 Experimental Models for the Discovery and Development of Anti-cancer Drugs

A scientifically validated experimental model is critical to the outcome of the study. Common experimental models for CRISPR-Cas9 anticancer drug discovery are in vitro models, in vivo models, organoid models, and models that probe the role of small molecules.

The first was an in vitro model. In 2013, the CRISPR system from bacteria was adapted as an effective gene editing tool. Then, scientists established cell lines stably expressing the Cas9 protein and delivered vectors encoding sgRNA via lentivirus. As the study progressed, a single lentiviral vector carrying the Cas9 protein, sgRNA, and selection marker could be used by any cell line of interest without the need to establish a Cas9-expressing cell line.

Next is the in vivo model, established to study the effects of target genes in tumor tissues or microenvironments to facilitate screening effects and study primary tumor growth or metastasis. The most common approach to establishing tumor models using library screens is to grow engineered cells in vitro and then transplant them into animals. In general, CRISPR library screening approaches identify potential key genes in antitumor immunity, but the mechanisms of downstream gene regulation require further characterization. In addition, it is not a commonly studied gene due to the discovery process randomly, making the search for effective therapeutic agents against the target challenging currently.

In vivo models have more or less limitations, such as the lack of good in situ models to be easily studied in vivo. Genetically engineered mice are costly but have limited genetic characteristics. And the use of in vitro cancer cell lines is not suitable for studies involving cell differentiation, cancer stem cells, and the impact of the tumor microenvironment. In contrast, in vitro organoids hold promise to address these issues. As research has progressed, brain organoids collected from patients and thus cultured have been used for brain tumor research.

Application of the CRISPR-Cas9 Library in Target Discovery and Development of Anti-cancer Drugs

• Essential genes for survival in cancer provide a potential therapeutic window for tumor treatment. And CRISPR-Cas9 library screening helps to identify and characterize essential genes in tumor cells. Some investigators have improved the TKO library screen to enable it to screen and identify more essential genes for survival. Related studies have shown that CRISPRko technology is the most suitable CRISPR-Cas9 platform for lethality screens.
• CRISPR-Cas9 library screening allows for the identification and characterization of druggable target genes using different models to facilitate drug discovery. Also, CRISPR-Cas9 library screening can identify potential therapeutic targets against tumors for different cancer mechanisms.
• Mutations are a common cause of acquired drug resistance. The study of drug resistance-related mutations contributes to the development of detection methods, diagnostic criteria, prognostic references, and drug target discovery and design. And several studies have shown that CRISPR-Cas9 library screening can effectively and rapidly identify drug-resistance genes and obtain the causes of drug resistance.
• By improving the CRISPR-Cas9 system, CRISPR-Cas9 library screening can effectively regulate non-coding RNAs, which can effectively modulate the physiological and pathological functions of non-coding RNAs.

Reference

1. Chan, Y. T., et al., Crispr-cas9 library screening approach for anti-cancer drug discovery: Overview and perspectives. Theranostics, 2022. 12(7): p. 3329-3344.

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