Engineering Oncogenic Hotspot Mutations on SF3B1 via CRISPR-Directed PRECIS Mutagenesis.

Area of Science:

• Cancer genomics and molecular biology
• Gene editing technologies
• RNA splicing mechanisms

Background:

• SF3B1 mutations are common in cancer but studying their role is limited by a lack of disease models.
• Existing genome engineering platforms have limitations in efficiency and applicability for creating specific mutations like SF3B1 K700E.

Purpose of the Study:

• To develop and validate an efficient genome engineering approach for creating SF3B1 mutant cell lines.
• To investigate the functional consequences of SF3B1 mutations in relevant cancer contexts, such as chronic lymphocytic leukemia.

Main Methods:

• Comparison of CRISPR-Cas9, AAV-HDR, base editing, and prime editing platforms for SF3B1 K700E mutation introduction.
• Development of a novel method, prime editing coupled intron-assisted selection (PRECIS), combining prime editing with a reporter system.
• Application of PRECIS to engineer SF3B1 K700E mutations in chronic lymphocytic leukemia cell lines (HG-3, MEC-1).

Main Results:

• Prime editing, specifically PE5max, demonstrated the highest efficiency for SF3B1 K700E editing across various cell types.
• PRECIS successfully generated SF3B1 K700E mutant cell lines that recapitulate known phenotypes: altered splicing, copy number variations, and growth defects.
• A novel finding revealed that SF3B1 mutations can lead to Y chromosome loss in chronic lymphocytic leukemia.

Conclusions:

• PRECIS is an efficient and versatile method for generating genetically accurate SF3B1 mutant models.
• The engineered models provide valuable tools for understanding SF3B1 mutation roles in cancer.
• This research offers new insights into SF3B1's role in cancer pathogenesis, including genome instability and Y chromosome loss.