Unexpected binding behaviors of bacterial Argonautes in human cells cast doubts on their use as targetable gene regulators

<div><p>Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from <i>Natronobacterium gregoryi</i> (<i>Ng</i>Ago) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of <i>Ng</i>Ago or other pAgos as a targetable DNA-binding platform for epigenetic editing has not been explored. In this report, we evaluated the utility of two prokaryotic Argonautes (<i>Ng</i>Ago and <i>Tt</i>Ago) as DNA-guided DNA-binding proteins. <i>Ng</i>Ago showed no meaningful binding to chromosomal targets, while <i>Tt</i>Ago displayed seemingly non-specific binding to chromosomal DNA even in the absence of guide DNA. The observed lack of DNA-guided targeting and unexpected guide-independent genome sampling under the conditions in this study provide evidence that these pAgos might be suitable for neither gene nor epigenome editing in mammalian cells.</p>