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

David J. Segal,Henriette O’Geen,Nicole B. Coggins,Sofie L. Bates,Chonghua Ren

doi:10.1371/journal.pone.0193818

David J. Segal, Henriette O’Geen + Show 3 more

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https://doi.org/10.1371/journal.pone.0193818

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Journal: PloS one	Publication Date: Mar 27, 2018
Citations: 8	License type: CC BY 4.0

Affiliation: University of California, Davis

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.

Highlights

Eukaryotic Argonaute proteins use small single-stranded RNA to target complimentary RNA sequences and play a key role in the RNA interference (RNAi) pathway [1]
First we evaluated that TtAgo and NgAgo expressed in mammalian cells are in principle able to cleave DNA targets in vitro
We evaluated the utility of pAgos as DNA-guided DNA-targeting tools using chromatin immunoprecipitation (ChIP) assays

Summary

Introduction

Eukaryotic Argonaute proteins (eAgos) use small single-stranded RNA to target complimentary RNA sequences and play a key role in the RNA interference (RNAi) pathway [1]. Recently discovered prokaryotic Argonaute proteins (pAgos) have been implicated in the targeting of foreign DNA for degradation [2]. Some pAgos have been shown to use small single-stranded DNA (ssDNA) to target and cleave double-stranded DNA and offer an intriguing possibility for gene and epigenome editing [3,4,5,6]. PAgos offer several advantages for DNA-guided site-specific binding in the mammalian genome, because of their increased flexibility of targeting. While CRISPR/Cas is able to target near many genetic features, the necessity of a protospacer adjacent motif (PAM) in the target site often makes it impossible to design guide RNAs (gRNAs) to bind exactly at features such as SNPs, individual CpGs, intron/exon boundaries, or specific

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