The potential for high-precision targeting by pAgos.

<p>A. Targeting of CRISPR/Cas9 is limited by the requirement of a PAM site (green boxes). B. <i>Ng</i>Ago or <i>Tt</i>Ago might allow the precise targeting of features, such as transcription factor binding sites (black boxes). gRNA/gDNA, red lines.</p

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></div

h<i>Tt</i>Ago and h<i>Ng</i>Ago do not cleave genomic target sites.

<p>A. Schematic of human codon-optimized <i>Tt</i>Ago and <i>Ng</i>Ago proteins containing two nuclear localization signals (NLSs) and a 3xFlag epitope tag. Western blot analysis of h<i>Tt</i>Ago and h<i>Ng</i>Ago proteins in HEK293 cells using an antibody against the 3xFlag tag. Untransfected cells serve as a negative control (-). Ponceau staining was used as a loading control. B. Diagram illustrating <i>RPL13A</i> and <i>HER2</i> guide DNAs and genomic target site. Genomic target sites are indicated in blue and complementary ssDNA guides are indicated in red. C. Amplicon sequencing was carried out on HEK293 cells co-transfected with h<i>Tt</i>Ago or h<i>Ng</i>Ago expression plasmids with (+) or without (-) gDNAs to <i>RPL13A</i> and <i>HER2</i>. To increase the amount of gDNAs, cells were re-transfected with gDNAs 24 hours after the initial transfection (++). CRISPRESSO analysis confirms that h<i>Tt</i>Ago and h<i>Ng</i>Ago did not cause insertions or deletions (indels) under any of these conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193818#pone.0193818.s008" target="_blank">S5 Table</a>). As a control, HEK293 cells were co-transfected with Cas9 nuclease and gRNA expression plasmids targeting <i>RPL13A</i> and <i>HER2</i>. RNA-guided Cas9 displayed target site cleavage at the genomic <i>RPL13A</i> and <i>HER2</i> target sites. The percentage of sequence reads containing indels relative to the total number of sequence reads is plotted on the y-axis.</p