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

Pediatric low-grade glioma (pLGG), the most common childhood brain tumor (30–50% of pediatric CNS malignancies), is driven almost universally by BRAF alterations: the KIAA1549–BRAF oncofusion in ~70% of cases and the BRAF V600E point mutation in ~15%. Current targeted drugs such as tovorafenib require continuous dosing, lack allele specificity, and carry long-term toxicity risks, motivating a one-time genomic intervention that disables the oncogenic alteration while preserving wild-type BRAF signaling.

The authors designed an allele-specific CRISPR strategy. For the oncofusion, a double-cut intronic excision pairs a guide targeting KIAA1549 intron 14 with one targeting BRAF intron 11; because the breakpoints of the four major fusion variants (KB 16:9, 15:9, 16:11, 15:11) fall within these introns, a single guide pair addresses the full fusion heterogeneity. For BRAF V600E, they exploited a unique PAM created by the pathogenic TBA transversion at codon 600, enabling SpCas9 and AsCas12a designs with single-nucleotide specificity. They screened ribonucleoprotein guides in A375 melanoma cells (V600E homozygous) and patient-derived 3635 PXA glioma cells (V600E heterozygous).

The top fusion-targeting guides achieved 66% indels for K9_i14_A_Cas9 (intron 14), and 84% and 85% for BRAF intron 11 guides B_i11_A_Cas9 and B_i11_D_Cas9. The best allele-specific V600E SpCas9 guide achieved 57% editing in A375 and 74% in 3635 PXA cells, with productive junction excision confirmed by gain-of-function PCR. These results validate a promising one-time gene-editing approach for pLGG.