Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1

Meijia Yang; Heng Wei; Yuelong Wang; Jiaojiao Deng; Yani Tang; Liangxue Zhou; Gang Guo; Aiping Tong

doi:10.1016/j.omtn.2017.05.009

Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1

Mol Ther Nucleic Acids. 2017 Sep 15:8:450-458. doi: 10.1016/j.omtn.2017.05.009. Epub 2017 May 17.

Authors

Meijia Yang¹, Heng Wei², Yuelong Wang³, Jiaojiao Deng³, Yani Tang¹, Liangxue Zhou³, Gang Guo⁴, Aiping Tong⁵

Affiliations

¹ The 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.
² College of Life Science, Sichuan University, Chengdu 610064, China.
³ Department of Neurosurgery, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China.
⁴ The 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. Electronic address: guogang@scu.edu.cn.
⁵ The 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. Electronic address: aipingtong@scu.edu.cn.

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.

Keywords: BRAF V600E; Cpf1; targeted therapy.