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

Abstract

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.

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

“…Mismatches in the seed region may promote target DNA cleavage, by stimulating the catalytic step of the reaction and, possibly, increasing the rate of product dissociation, but may also decrease the rate of catalytic complex formation. In contrast to most other Ago proteins studied to date, mismatches in the 3′-supplementary guide region strongly inhibit target DNA cleavage by CbAgo and LrAgo; similar effects were recently reported for TtAgo (31). Therefore, in the case of these pAgos propagation of the guide-target duplex, rather than initial interactions in the seed region, may control the fidelity of target recognition.…”

“…Plasmid relaxation is followed by slow processing of plasmid DNA, resulting in its linearization and further degradation, corresponding to the ‘chopping’ activity previously described for TtAgo and MjAgo (11,28). The chopping activity may complicate the use of pAgos as specific genome editing tools, as recently discussed for TtAgo and NgAgo (pAgo from Natronobacterium gregoryi ) (31). However, the chopping activity of CbAgo is markedly suppressed if it is preloaded with guide molecules.…”

“…Furthermore, it is not clear if pAgos are able to process DNA substrates at moderate temperature without additional partners due to their inability to unwind dsDNA. These concerns have cast doubt on the practicality of pAgos as a tool for DNA manipulation (30,31). Here, we describe two Argonaute proteins from mesophilic prokaryotes, bacillus Clostridium butyricum (CbAgo) and cyanobacterium Limnothrix rosea (LrAgo).…”

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea

“…Plasmid relaxation is followed by slow processing of plasmid DNA, resulting in its linearization and further degradation. The chopping activity may complicate the use of pAgos as specific genome editing tools, as recently discussed for TtAgo and NgAgo (30). Indeed, LrAgo can process plasmid DNA with similar efficiency independently of guide binding.…”

“…These concerns have cast doubt on the practicality of pAgos as a tool for DNA manipulation, e.g. (29,30). Here we describe two Argonaute proteins from mesophilic prokaryotes, bacillus Clostridium butyricum (CbAgo) and cyanobacterium Limnothrix rosea (LrAgo).…”