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Updated: Feb 22, 2026
Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines
Published on: October 8, 2015
Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1
Meijia Yang1, Heng Wei2, Yuelong Wang3
1The State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation Center of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
Abstract:
BRAF-V600E (1799T > A) is one of the most frequently reported driver mutations in multiple types of cancers, and patients with such mutations could benefit from selectively inactivating the mutant allele. Near this mutation site, there are two TTTN and one NGG protospacer-adjacent motifs (PAMs) for Cpf1 and Cas9 CRISPR nucleases, respectively. The 1799T > A substitution also leads to the occurrence of a novel NGNG PAM for the EQR variant of Cas9. We examined the editing efficacy and selectivity of Cpf1, Cas9, and EQR variant to this mutation site. Only Cpf1 demonstrated robust activity to induce specific disruption of only mutant BRAF, not wild-type sequence. Cas9 recognized and cut both normal and mutant alleles, and no obvious gene editing events were observed using EQR variant. Our results support the potential applicability of Cpf1 in precision medicine through highly specific inactivation of many other gain-of-function mutations.
Insights
CRISPR-Cas gene editing offers a promising approach for cancer therapy. Cpf1 demonstrated highly specific disruption of the BRAF-V600E mutation, unlike Cas9, supporting its use in precision medicine.
Area of Science:
- Molecular biology
- Genetics
- Biotechnology
Background:
- BRAF-V600E is a common cancer driver mutation.
- Targeting mutant BRAF offers therapeutic potential.
- CRISPR nucleases like Cpf1 and Cas9 are tools for gene editing.
Purpose of the Study:
- To evaluate the efficacy and selectivity of Cpf1, Cas9, and a Cas9 variant (EQR) for targeting the BRAF-V600E mutation.
- To determine if these nucleases can specifically inactivate the mutant BRAF allele.
Main Methods:
- Utilized CRISPR-Cpf1, CRISPR-Cas9, and CRISPR-Cas9-EQR systems.
- Assessed gene editing activity and selectivity at the BRAF-V600E mutation site.
- Analyzed disruption of mutant versus wild-type BRAF sequences.
Main Results:
- Cpf1 showed robust and specific activity, disrupting only the mutant BRAF allele.
- Cas9 exhibited activity against both mutant and wild-type BRAF alleles.
- The Cas9-EQR variant demonstrated no significant gene editing activity.
Conclusions:
- Cpf1 is a highly specific tool for targeting the BRAF-V600E mutation.
- Cpf1's specificity supports its potential application in precision medicine for inactivating gain-of-function mutations.
- Cas9 and Cas9-EQR were less suitable for selective BRAF-V600E targeting in this context.
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