CRISPR-Mediated Targeting of BRAF Oncogenes in Pediatric Low-Grade Glioma

Pediatric low-grade glioma (pLGG) is the most common brain tumor of childhood, accounting for 30-50% of all pediatric central nervous system malignancies. The disease is almost universally driven by activating mutations in the BRAF serine/threonine kinase: a chromosomal tandem duplication generating the KIAA1549-BRAF oncofusion in approximately 70% of cases, or the BRAF V600E gain-of-function point mutation in approximately 15%. Current targeted pharmacotherapies, including the RAF inhibitor tovorafenib, require continuous dosing, are not allele-specific, and carry risks of long-term toxicity in children. A one-time genomic intervention that permanently disables the oncogenic BRAF alteration while preserving wild-type BRAF signaling represents a compelling therapeutic alternative. In this study, we describe the design and experimental validation of allele-specific CRISPR guide RNAs targeting both the KIAA1549-BRAF oncofusion and the BRAF V600E point mutation. For the oncofusion, we developed a double-cut intronic excision strategy in which a guide RNA targeting KIAA1549 intron 14 is paired with a guide RNA targeting BRAF intron 11. Because the genomic breakpoints of all four major fusion variants (KB 16:9, 15:9, 16:11, and 15:11) fall within these introns, a single guide pair can address the full landscape of fusion heterogeneity in a single intervention. For BRAF V600E, we exploited a unique PAM sequence created by the pathogenic T>A transversion at codon 600, enabling allele-specific SpCas9 and AsCas12a guide designs that distinguish the mutant from the wild-type allele at single-nucleotide resolution. We screened guide RNA candidates by ribonucleoprotein (RNP) nucleofection in A375 human melanoma cells (BRAF V600E homozygous) and in patient-derived 3635 PXA glioma cells (BRAF V600E heterozygous). The top KIAA1549 intron 14 guide, K9_i14_A_Cas9, achieved 66% indel frequency in A375 cells. The top BRAF intron 11 guides, B_i11_A_Cas9 and B_i11_D_Cas9, achieved 84% and 85% indel frequency, respectively. For BRAF V600E, the best allele-specific SpCas9 guide achieved ~57% editing in A375 cells and ~74% editing in 3635 PXA patient-derived glioma cells. Dual-cut excision of the KIAA1549-BRAF junction was confirmed by a gain-of-function PCR assay designed to detect the excision junction amplicon (~191 bp) produced by NHEJ-mediated rejoining of the KIAA1549 intron 14 and BRAF intron 11 cut ends.