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

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Mitochondrial genetic diseases are caused by mitochondrial DNA (mtDNA) defects.
• Current gene therapy approaches face challenges in delivering CRISPR components, specifically single-guide RNAs (sgRNAs), into mitochondria.
• Efficient delivery systems are crucial for developing effective CRISPR-mediated mtDNA therapies.

Purpose of the Study:

• To identify and engineer a mitochondrial targeting sequence for effective delivery of CRISPR-Cas9 components into mitochondria.
• To develop a novel gene therapy tool for correcting mtDNA mutations.
• To evaluate the efficacy of the engineered tool in cellular and in vivo models.

Main Methods:

• Network analysis of mitochondrion-localized long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs) to identify potential targeting mechanisms.
• Derivation and optimization of a 30-nucleotide mitochondrial targeting sequence (ST2-RNA RMTS) from lncRNA RP11-46H11.3.
• Engineering of the RMTS-CRISPR tool by fusing the ST2-RMTS to sgRNA.
• Validation of mtDNA targeting and cleavage efficiency in cell models and in vivo.

Main Results:

• Identification of lncRNA RP11-46H11.3 as a carrier for mitochondrial translocation via RBPs.
• The derived ST2-RMTS demonstrated high mitochondrial localization efficiency.
• The engineered RMTS-CRISPR tool successfully targeted and cleaved mtDNA.
• Achieved significant heteroplasmic mtDNA shifting efficiencies up to 26.37% in cell models and 26.79% in vivo for the m.3243A>G mutation.

Conclusions:

• A novel CRISPR-based mitochondrial gene intervention strategy has been developed using an engineered RMTS.
• The RMTS-CRISPR tool offers a promising technological approach for correcting heterogeneous mtDNA mutations.
• This strategy holds potential applications for treating various mitochondrial disorders.