DNA sequencing and CRISPR-Cas9 gene editing for target validation in mammalian cells

Area of Science:

• Molecular Biology
• Genetics
• Pharmacology

Background:

• Understanding drug resistance mechanisms is crucial for developing effective therapeutics.
• Identifying specific mutations conferring drug resistance can elucidate compound mechanisms of action.

Purpose of the Study:

• To demonstrate the feasibility of using next-generation sequencing and CRISPR-Cas9 gene editing to identify and validate drug-resistant mutations in mammalian cells.
• To investigate the drug resistance mechanisms for 6-thioguanine targeting HPRT1 and triptolide targeting ERCC3.

Main Methods:

• Generation and sequencing of drug-resistant clones in mammalian cells.
• CRISPR-Cas9 gene editing to introduce specific mutations in target genes (HPRT1 and ERCC3).
• Validation of drug resistance phenotypes in gene-edited cells.

Main Results:

• Disruption of a functional HPRT1 allele by gene editing conferred resistance to 6-thioguanine.
• Introduction of specific point mutations in ERCC3 via gene editing conferred resistance to triptolide.
• Next-generation sequencing successfully identified mutations in drug-resistant clones.

Conclusions:

• CRISPR-Cas9-mediated gene editing is a feasible and powerful tool for validating drug resistance mutations.
• This approach provides valuable insights into drug mechanisms of action by linking specific genetic alterations to drug resistance.
• The study successfully validated the roles of HPRT1 and ERCC3 in resistance to 6-thioguanine and triptolide, respectively.