Targeted deletions in human mitochondrial DNA engineered by Type V CRISPR-Cas12a system.

Area of Science:

• Molecular Biology
• Genetics
• Mitochondrial Biology

Background:

• Mitochondrial DNA (mtDNA) mutations are linked to neuromuscular diseases, with severity influenced by heteroplasmy.
• Accurate modeling of mtDNA dysfunction is essential for developing effective experimental therapies.

Purpose of the Study:

• To adapt the CRISPR-AsCas12a system for targeted editing of human mtDNA.
• To create a tool for generating specific mtDNA deletions for disease modeling.

Main Methods:

• Utilized the Type V CRISPR-AsCas12a system, recognizing AT-rich PAM sequences.
• Employed a mitochondrial targeting sequence (MTS) from *Neurospora crassa* ATPase subunit 9 to deliver AsCas12a into mitochondria.
• Programmed mito-AsCas12a with two CRISPR RNAs (crRNAs) to target distant mtDNA regions for cleavage.

Main Results:

• Successfully delivered the AsCas12a effector nuclease into human mitochondria.
• Demonstrated cleavage of mtDNA by mito-AsCas12a, generating deletions in cultured human cells.
• Confirmed mtDNA ligation post-cleavage using next-generation sequencing of deletion boundaries.

Conclusions:

• The CRISPR-AsCas12a system can be precisely targeted to human mitochondria for mtDNA manipulation.
• This system offers a promising approach for generating predefined mtDNA deletions.
• Provides a valuable tool for creating cellular models of mitochondrial disorders.