Alterations in mitochondrial base editors enhance targeted editing efficiency for mouse model generation.

Area of Science:

• Molecular Biology
• Genetics
• Mitochondrial Biology

Background:

• Mitochondrial DNA (mtDNA) base editors are crucial for studying mitochondrial diseases.
• Editing efficiency of current base editors varies significantly with target site in mtDNA.
• Need for more efficient and specific mtDNA editing tools exists.

Purpose of the Study:

• To develop improved mitochondrial adenine base editors with enhanced efficiency.
• To generate a mouse model for studying mitochondrial diseases using enhanced base editors.
• To investigate the functional consequences of a specific mtDNA mutation.

Main Methods:

• Engineered two novel mitochondrial base editors (Hifi-sTALED and αnHifi-sTALED) by modifying non-deaminase components.
• Tested editing efficiency and off-target effects across the transcriptome.
• Utilized enhanced editors for mtDNA editing in mouse embryos to create mt-Rnr1 mutants.
• Performed functional analyses including ATP production and oxygen consumption rate (OCR) assays.

Main Results:

• The improved Hifi-sTALED and αnHifi-sTALED editors demonstrated significantly increased mtDNA editing efficiency.
• Minimal off-target effects were observed across the transcriptome.
• Successfully generated mt-Rnr1 mutant mice with high heteroplasmic loads.
• The mt-Rnr1 mutation led to impaired mitochondrial function, evidenced by reduced ATP production and OCR.

Conclusions:

• Enhanced Hifi-sTALED and αnHifi-sTALED base editors offer improved efficiency and specificity for mtDNA editing.
• These tools are effective for generating mitochondrial disease models in vivo.
• The study provides insights into the functional impact of mt-Rnr1 mutations on mitochondrial health.