Title: A CRISPR-based mitochondrial gene therapy tool derived by engineering guide RNAs

Ying Wang,Xinwan Su,Yu Chen, Yijian Chen, Chengyu Shi, Fangzhou Liu, Yuqi Ye, Panyi Sun, Manman Tan,Meng Yu,Ya Wang,Shanshan Xie,Jian Liu,Qingfeng Yan,Qiming Sun,Dante Neculai,Wei Liu, Jianzhong Shao, Yang Liu, Weiqiang Lin, Aifu Lin

Abstract

Mitochondrial genetic diseases arise from mitochondrial DNA (mtDNA) defects, which gene therapy tools may rectify. However, delivering single-guide RNAs (sgRNAs) into mitochondria remains a challenge limiting CRISPR-mediated mtDNA therapy. Here, through network analysis of mitochondrion-localized long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs), we found that lncRNA RP11-46H11.3 translocates into mitochondria via binding mitochondria-associated RBPs using its key RNA recognition motifs (RRMs); its derived 30 nt ST2-RNA mitochondrial targeting sequence (RMTS) showed the highest mitochondrial localization efficiency. We engineered the RMTS-CRISPR tool by fusing ST2-RMTS to sgRNA, verifying its ability to target and cleave mtDNA. Strikingly, our results demonstrated that RMTS-CRISPR could achieve heteroplasmic mtDNA shifting efficiencies of up to 26.37% in m.3243A>G mutant cell models and 26.79% in vivo, offering a technological approach for the correction of heterogeneous mtDNA mutations. Taken together, our findings reveal a CRISPR-based mitochondrial gene intervention strategy that may have applications in mitochondrial disorders.

Link: https://www.sciencedirect.com/science/article/pii/S2211124726000367