Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 technology offers potential for mitochondrial DNA (mtDNA) manipulation, crucial for disease modeling and therapeutics.
• Directly targeting mtDNA with CRISPR-Cas9 has faced technical hurdles, particularly in detecting subtle genetic modifications.
• Previous attempts to measure Cas9-induced mtDNA degradation via copy number changes were confounded by high biological variability.

Purpose of the Study:

• To develop and validate a method for achieving CRISPR-Cas9-based manipulation of mitochondrial DNA (mtDNA) in human cells.
• To overcome challenges in detecting Cas9 activity on mtDNA by employing a cytosine base editor (Cas9-BE3).
• To assess the efficacy and specificity of mitochondrially targeted Cas9-BE3 and guide RNA (gRNA) delivery.

Main Methods:

• Delivery of Cas9 and Cas9-BE3 into the mitochondria of human cells.
• Quantification of mtDNA cleavage and depletion using multiplexed qPCR.
• Detection of C→T mutations in mtDNA using amplicon sequencing after Cas9-BE3 delivery.
• Utilizing both on-target and non-targeting gRNAs to assess specificity.

Main Results:

• Initial experiments using Cas9 showed no significant mtDNA degradation, highlighting challenges in measuring subtle effects.
• Cas9-BE3 delivery to mitochondria resulted in detectable, albeit low-level, C→T mutations in mtDNA.
• Mutations occurred irrespective of the gRNA used, indicating successful base editor activity but unsuccessful gRNA import into mitochondria.
• Unspecific off-target activity demonstrated the technical feasibility of mtDNA editing but pointed to limitations in gRNA mitochondrial delivery.

Conclusions:

• Mitochondria-targeted Cas9 base editors can technically edit mtDNA, evidenced by detected mutations.
• The observed unspecific mutations strongly suggest that gRNA delivery into mitochondria was not successful with the current system.
• Future applications of mitochondria-targeted Cas9 base editors are promising for validating gRNA delivery to mitochondria, overcoming ambiguities associated with mtDNA copy number quantification.