Background <p>Patients with colorectal cancer (CRC) harboring BRAF mutation have a poor prognosis. The median survival time for patients with advanced BRAF^V600E-mutant CRC is only approximately one year. Owing to the insensitivity to standard chemotherapy, there are still no effective and highly specific treatment strategies available in clinical practice for CRC patients with BRAF mutation. Therefore, targeting the BRAF^V600E mutation site, researching and exploring novel targeted therapies are essential to improve the survival rate of patients with this CRC subtype.</p> Aim <p>This study aims to develop a precise therapeutic system for BRAF^V600E CRC, based on the carrier properties of extracellular vesicles (EVs) and gene therapy targeting BRAF^V600E.</p> Method <p>We first obtained engineered cells capable of stably producing EVs loaded with BRAF^V600E nucleic acid drugs (siBRAF^V600E). Next, BRAF^V600E-mutant and wild-type CRC cell lines, as well as corresponding subcutaneous and metastasis models, were used to evaluate the therapeutic efficacy of EVs-siBRAF^V600E and explored the mechanism. Notably, patient-derived xenograft (PDX) models, which share the same molecular characteristics, pathological features, and heterogeneity as patients do, were utilized to further explore the therapeutic efficacy and mechanisms.</p> Result <p>EVs-siBRAF^V600E specifically inhibited BRAF^V600E CRC but didn’t affect BRAF wild-type CRC in vitro and vivo. EVs-siBRAF^V600E exerts its therapeutic effect by regulating the MEK1/2-ERK1/2 pathway, and it has demonstrated excellent therapeutic efficacy in PDX models.</p> Conclusion <p>The therapeutic EVs we constructed are effective and specific for the BRAF^V600E-mutant CRC. This study provides a novel strategy for the treatment of CRC patients with BRAF^V600E mutation.</p>