Shmanai, V. | Search bioRxiv

bioRxiv2025

Targeting of human mitochondrial DNA with programmable pAgo nuclease

Manipulating the mitochondrial genome remains a major challenge in genetic engineering, largely due to the mitochondrial double-membrane structure. While recent advances have expanded the genetic toolkit for nuclear and cytoplasmic targets, precise editing of mtDNA has remained elusive. Here we report the first successful mitochondrial import of a functional RNA-guided prokaryotic Argonaute protein from the mesophilic bacterium Alteromonas macleodii (AmAgo). By directing AmAgo to the single-stranded D- or R-loop region of mtDNA using synthetic RNA guides, we observed a nearly threefold reduction in mtDNA copy number in human cell lines. This proof of concept study demonstrates that a bacterial Argonaute can remain active within the mitochondrial environment and influence mtDNA levels. These findings lay the groundwork for further development of programmable systems for mitochondrial genome manipulation. HighlightsAmAgo targeted to mitochondria significantly reduces human mtDNA copy number Mitochondrial localization of AmAgo is essential for changes in the mtDNA content AmAgo effects on mtDNA are modulated by exogenous guide RNAs Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/689640v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@ddd069org.highwire.dtl.DTLVardef@9db9a3org.highwire.dtl.DTLVardef@1068b02org.highwire.dtl.DTLVardef@904c87_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5-scaffold moiety and 3-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

molecular biology↗