Engineering oncogenic hotspot mutations on SF3B1 via CRISPR-directed PRECIS mutagenesis

SF3B1 is the most recurrently mutated RNA splicing factor in cancer; However, its study has been hindered by a lack of disease-relevant cell line models. Here, we compared four genome engineering platforms to establish SF3B1 mutant cell lines within the appropriate cellular context: CRISPR-Cas9 editing, AAV HDR editing, base editing (ABEmax, ABE8e), and prime editing (PE2, PE3, PE5Max). We showed that prime editing via PE5max achieved the most efficient SF3B1 K700E editing in HEK293T cells and exhibited editing capabilities across a wide range of cell lines. We further refined our approach by coupling prime editing with a with a fluorescent reporter that leverages a SF3B1 mutation-responsive synthetic intron to mark prime edited cells to enhance positive editing outcomes. Calling this approach prime editing coupled intron-assisted selection (PRECIS), we then introduced the K700E hotspot mutation into two chronic lymphocytic leukemia (CLL) cell lines, HG-3 and MEC-1, and demonstrated that our PRECIS-engineered cells faithfully recapitulate the altered splicing and copy number variation (CNV) events frequently found in CLL patients with SF3B1 mutation. Our results showcase PRECIS as an efficient and generalizable method for engineering genetically faithful SF3B1 mutant models, shed new light on the role of SF3B1 mutation in cancer biology, and establish a new tool that enables rapid and facile generation of novel SF3B1 mutant cell lines in any cellular contexts. ### Competing Interest Statement The authors have declared no competing interest.