Generation of large-scale CRISPR-libraries for functional screens
Typ
Examensarbete för masterexamen
Program
Publicerad
2020
Författare
Edström, Tora
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Cancer is a global and often fatal disease, which remains difficult to treat. Since a tumour consists of a heterogenous cell population, individual cells may be able to survive the treatment through different mechanisms leading to drug resistance. Even when a treatment seems effective initially, a single surviving cancer cell can theoretically lead to a relapse. Thus, understanding the mechanisms underlying resistance development is important to effectively treat cancer. Genetic barcodes that label individual cells in a tumour population enable to isolate and study treatment-resistant cells. However, since tumours consist of complex populations the used barcode library must be similarly large and complex to capture the full heterogeneity of a given tumour. This project aimed to optimise a protocol for generation of a highly complex barcode library. The barcodes, available as single stranded oligonucleotides, were amplified with PCR. The generated inserts were then ligated with the linearized backbone plasmid in a Gibson reaction and transformed into E. coli through electroporation. After optimisation of each step, the protocol was applied to generate the barcode library. Next generation amplicon sequencing was performed and revealed a library coverage of 27% of the original complexity and an uneven distribution of the detected barcodes. More investigation is required to determine the source of the skewed distribution, and the loss of complexity. However, this project sets the foundation for further work.