Study Reveals New Risk of Gene Editing Causing Mitochondrial DNA Fragments to Integrate into The Nuclear Genome

Recently, researchers from Peking University in China published a research paper titled "Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks" in the journal Nature Communications. Hu Jiazhi's team used PEM-seq, a high-throughput sequencing method they had previously developed for systematic analysis of gene editing products, and found that mitochondrial DNA fragments may be inserted into the targeted site during nuclear genome editing. At the same time, targeted editing of mitochondrial DNA can also cause mitochondrial DNA instability, leading to its insertion into the nuclear genome.

The study also found that the frequency of mitochondrial DNA insertion can be reduced by simultaneously expressing DNA exonucleases TREX1 or TREX2. This study, by studying the effects of DNA breaks caused by gene editing on mitochondrial DNA, revealed the potential risks of mitochondrial DNA fragments inserted into the nuclear genome during gene editing and proposed feasible solutions.

The research team used the PEM-seq method to analyze the targeting sites after CRISPR-Cas editing of the nuclear genome, and found that after editing with a variety of CRISPR-Cas editing enzymes and Cas9 family variants, sequences of mtDNA fusion with the targeting site occurred at the nuclear genome editing sites (Figure 1A, B). In edited cells, the event of mtDNA fusion with the nuclear genome targeting site occurred once in every 10³ to 10⁵ editing events, while there was almost no mt-nuclear DNA fusion in the unedited samples, indicating that these events were mainly related to CRISPR-Cas editing.

Compared with SpCas9, the editing tools of the Cas12 family showed a slightly lower level of mtDNA integration, and the use of base editors can significantly reduce the frequency of mtDNA integration by reducing the DSB frequency. In addition, the authors found mitochondrial DNA and nuclear genome fusion events caused by gene editing in the edited CAR T cell system, mouse TCR T cells, and mouse embryos after base editing.

The research team further proved that mitochondrial DNA fragments can be inserted into the targeting site of the nuclear genome during gene editing by specifically amplifying the insertion sequence of the targeting site. Moreover, treatment with mitochondrial drugs CCCP and Paraquat or introduction of DSBs into mitochondria could significantly increase the insertion of mtDNA into the targeted site of the nuclear genome, indicating that the frequency of this event is affected by the stability of mitochondrial DNA (Figure 1C, D).

Figure 1. PEM-seq detects the insertion of mitochondrial DNA fragments into targeted sites in the nuclear genome. (Wu J, et al., 2024)

So is it possible that mitochondrial DNA fragments caused by mitochondrial gene editing are inserted into the nuclear genome?

The research team used PEM-seq and found that after mitoTALEN editing, the breakpoints of mitochondrial DNA and nuclear genome integration sequences mostly occurred within the editing window of mitoTALEN. Similarly, integration of the editing site into the nuclear genome can also be found after DdCBE editing, proving that mitochondrial editors can also cause mitochondrial DNA to be inserted into the nuclear genome (Figure 2A). In cells, exonucleases TREX1 and TREX2 clear free DNA fragments in the cytoplasm to avoid excessive activation of cellular inflammatory responses. They also found that expressing TREX1 or TREX2 simultaneously during the editing process can significantly reduce the insertion of mitochondrial DNA during the editing process (Figure 2B), proposing a solution to this editing safety hazard.

Figure 2. Mitochondrial editors cause the integration of mitochondrial DNA fragments into the nuclear genome. (Wu J, et al., 2024)

Reference

Wu J, et al. Transfer of mitochondrial DNA into the nuclear genome during induced DNA breaks. Nature Communications, 2024, 15(1): 9438.