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

Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from Natronobacterium gregoryi (NgAgo) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of NgAgo 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 (NgAgo and TtAgo) as DNA-guided DNA-binding proteins. NgAgo showed no meaningful binding to chromosomal targets, while TtAgo 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.