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.

Fig 1. The potential for high-precision targeting by pAgos.

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

Fig 2. hTtAgo and hNgAgo do not cleave genomic target sites.

A. Schematic of human codon-optimized TtAgo and NgAgo proteins containing two nuclear localization signals (NLSs) and a 3xFlag epitope tag. Western blot analysis of hTtAgo and hNgAgo 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 RPL13A and HER2 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 hTtAgo or hNgAgo expression plasmids with (+) or without (-) gDNAs to RPL13A and HER2. To increase the amount of gDNAs, cells were re-transfected with gDNAs 24 hours after the initial transfection (++). CRISPRESSO analysis confirms that hTtAgo and hNgAgo did not cause insertions or deletions (indels) under any of these conditions (S5 Table). As a control, HEK293 cells were co-transfected with Cas9 nuclease and gRNA expression plasmids targeting RPL13A and HER2. RNA-guided Cas9 displayed target site cleavage at the genomic RPL13A and HER2 target sites. The percentage of sequence reads containing indels relative to the total number of sequence reads is plotted on the y-axis.

Fig 3. DNA guides do not facilitate binding of hTtAgo and hNgAgo to genomic target loci.

A. ChIP-qPCR enrichment to quantitate binding to RPL13A locus. HEK293 cells were transfected with hTtAgo- and hNgAgo-expression plasmids either with (+) or without (-) gDNAs. dCas9 and two different gRNA expressing plasmids were co-transfected as controls (Student two-sided T-test; *, p<0.05; n.s., not significant; n = 2 independent experiments; mean ± SEM). Binding to a non-target locus (GAPDH) was evaluated to interrogate binding specificity. B. Targeted binding of hNgAgo failed at the DYRK1A locus in HEK293 cells. HEK293 cells were co-transfected with hNgAgo expression plasmid together with 24-nt 5’-phosphorylated gDNAs (G5 gDNA) or without gDNA. ChIP assays were performed with an antibody to the 3xFlag tag of hNgAgo or with rabbit IgG as a negative control. Addition of G5 gDNA that was targeted to the DYRK1A locus did not increase hNgAgo binding above background level (no guide). There was no difference in binding to DYRK1A or to a control region (GAPDH). Two conditions (2% or 10% FBS in growth media) were tested. C. ChIP-PCR to measure unguided hTtAgo and hNgAgo binding to the RPL13A locus. ChIP assays were performed with two biological replicates. Flag, Flag ChIP; IgG(-), IgG negative control; Input, 0.1% chromatin input. D. hTtAgo binds multiple genomic regions without gDNA in HeLa cells. ChIP assays were performed in HeLa cells expressing hTtAgo without DNA guides. Standard PCR with locus-specific primers demonstrated hTtAgo binding to RPL13A, GAPDH, HER2 and EPCAM. Flag, Flag ChIP; IgG(-), IgG negative control; Input, 0.1% chromatin input.