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DNA-guided genome editing using the Natronobacterium gregoryi Argonaute

This article was retracted on 01 August 2017

A Retraction to this article was published on 01 August 2017

An Addendum to this article was published on 28 November 2016

This article has been updated


The RNA-guided endonuclease Cas9 has made genome editing a widely accessible technique. Similar to Cas9, endonucleases from the Argonaute protein family also use oligonucleotides as guides to degrade invasive genomes. Here we report that the Natronobacterium gregoryi Argonaute (NgAgo) is a DNA-guided endonuclease suitable for genome editing in human cells. NgAgo binds 5′ phosphorylated single-stranded guide DNA (gDNA) of 24 nucleotides, efficiently creates site-specific DNA double-strand breaks when loaded with the gDNA. The NgAgo–gDNA system does not require a protospacer-adjacent motif (PAM), as does Cas9, and preliminary characterization suggests a low tolerance to guide–target mismatches and high efficiency in editing (G+C)-rich genomic targets.

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Figure 1: NgAgo uses 5′ phosphorylated ssDNA guides and cleaves DNA targets in vitro.
Figure 2: NgAgo binds ssDNA guide in a one-guide-faithful manner.
Figure 3: NgAgo works as an endonuclease and can cleave DNA targets in vivo.
Figure 4: NgAgo can make targeted double-strand breaks in mammalian genome.
Figure 5: NgAgo can be used to edit mammalian genome.

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  • 28 November 2016

    The editors of Nature Biotechnology are issuing an editorial expression of concern regarding this article to alert our readers to concerns regarding the reproducibility of the original results. At this time, we are publishing the results of three groups ( that have tried to reproduce the results in the critical Figure 4 in the original paper by Han and colleagues, which demonstrates editing of endogenous genomic loci in mammalian cells. None of the groups observed any induction of mutations by NgAgo at any of the loci or under any of the conditions tested above the sensitivity of the assays used. Similar results have been recently reported by a different group of authors in Protein & Cell (doi:10.1007/s13238-016-0343-9). We are in contact with the authors, who are investigating potential causes for the lack of reproducibility. The authors have been informed of this statement. While the investigations are ongoing, Chunyu Han and Xiao Z. Shen agree with this editorial expression of concern. Feng Gao, Feng Jiang and Yongqiang Wu do not feel that it is appropriate at this time. We will update our readers once these investigations are complete.

  • 02 August 2017

    We are retracting our study because of the continued inability of the research community to replicate the key results in Figure 4, using the protocols provided in our paper. In this figure we report that the Natronobacterium gregoryi Argonaute can efficiently create double-strand breaks and edit the genome of human cells using 5ʹ phosphorylated single-stranded DNA as a guide. Despite the efforts of many laboratories (Protein Cell 7, 913–915, 2016; Nat. Biotechnol. 35, 17–18, 2017; Cell Res. 26, 1349–1352, 2016; PLOS One 12, e0177444, 2017), an independent replication of these results has not been reported. We are therefore retracting our initial report at this time to maintain the integrity of the scientific record. We nevertheless continue to investigate the reasons for this lack of reproducibility with the aim of providing an optimized protocol.

  • 01 August 2017

    Nat. Biotechnol. 34, 768–773 (2016); published online 2 May 2016; addendum published after print 28 November 2016; retracted 26 July 2017; 10.1038/nbt.3547 We are retracting our study because of the continued inability of the research community to replicate the key results in Figure 4, using the protocols provided in our paper.


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We thank W. Chao for performing flow cytometry experiment. This work was supported by the National Science Foundation of China 31270950 to X.Z.S.

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Authors and Affiliations



C.H. conceived the study and designed the experiments. X.Z.S. provided intellectual advice on the project and experimental design. C.H. performed mammalian genome editing. F.G. performed the BLAST search and the in vitro cleavage experiments. F.G., F.J. and Y.W. designed and constructed the clones under the supervision of C.H., F.J. and Y.W. performed the in vivo cleavage experiments. X.Z.S. and C.H. wrote the manuscript.

Corresponding author

Correspondence to Chunyu Han.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 NgAgo binds ssDNA guide in a one-guide-faithful manner.

NgAgo-expressing plasmid was transfected to 293T cells and then NgAgo was purified and used in an in vitro plasmid cleavage assay. The NgAgo derived from the cells co-transfected with target-complementary guide (FW, Lane 4) but not that derived from the cells co-transfected with a random guide (NC, Lane 5) could cause DSBs and linearize the plasmid. The NgAgo derived from the cells without guide co-delivery could not cleave the target even if the purified NgAgo was later co-incubated with the FW guide (Lane 3). After NgAgo binds to an ssDNA, it will not swap its bound guide (here is NC) to another free guide (here is FW) at 37℃ (Lane 6). Representative figure of 3 independent experiments.

Supplementary Figure 2 Guide reloading process at 55℃ impairs endonuclease activity of NgAgo.

NgAgo was purified from E.Coli. and then co-incubated with 5’ phosphorylated ssDNA guide at 37 ℃ or 55℃ for 1 hour or 72 hours. NgAgo was then subject to a plasmid cleavage assay to test its endonuclease activity. It shows that 55℃ for 1 hour changes the activity of NgAgo to a nickase and 55℃ for 72 hours completely deprived of its activity. SC, supercoiled; Lin, linearized; OC, open circular. Representative figure of 3 independent experiments.

Supplementary Figure 3 NgAgo has no activity to cleave linearized target or single strand target.

(a) pACYCDeut-eGFP plasmid was first linearized by BamHI restriction cleavage and then co-incubated with or without the purified NgAgo preloaded with FW guide in 293T cells. NgAgo was not found to further cleave the target sequence. (b) An 86nt ssDNA co-incubated with or without the purified NgAgo preloaded with guide in 293T cells at 37℃ for 8 hours. NgAgo was not found to cleave the target ssDNA. Representative figures of 3 independent experiments.

Supplementary Figure 4 NgAgo is directed into nucleus by NLS.

The engineered NLS-NgAgo was transfected to Hela cells and it shows that the expressed NLS-NgAgo was compartmented in the nucleus (DAPI+) and the cells maintained normal morphology. Scale bar = 100 μm. Representative figure of 20 independent experiments.

Supplementary Figure 5 T7E1 assay shows the efficiency of NgAgo/gDNA system in cleaving genome.

Forty-seven guides targeting 8 mammalian genome loci were tested for the cleavage efficiency of NgAgo. The percentages of indels were measured by T7E1 assay. Upper, n = 3; lower, representative figure of 3 independent experiments.

Supplementary Figure 6 T7E1 assay using a 21nt long ssDNA guide derived from G10 to test the effects of nucleotide mismatches on the efficiency of NgAgo target cleavage.

Mismatches are marked in red. Representative figure of 3 independent experiments.

Supplementary Figure 7 A detailed structure of the donor mRFP-TGA-eGFP DNA fragment.

Supplementary Figure 8 NgAgo has less off-target effect than Cas9 in mammalian cells.

Experimental schematic of investigating off-target genome editing. gDNA and gRNA were designed targeting the same locus of the genome. A 400bp GFP gene fragment donor (GFP400) without any homologous sequence to the target was co-transfected with either NgAgo/gDNA or Cas9/gRNA. Thus, the donor could be integrated into any DSBs in the genome. Total genomic DNAs were extracted from the engineered cells and digested by endonuclease restriction enzymes Bgl II, Sal I, Sac I, Xho I, afl II and Eco47 III. Bgl II reaction generates a 6.5kb fragment containing the on-target fragment, while other fragments in unknown length are off-targets. (b) Southern blot analysis detected off-target editing by Cas9 but not by NgAgo. Representative figure of 3 independent experiments.

Supplementary Figure 9 Full-length gel images (Unrelated lanes are marked with cross).

a, for Fig 1a:Nucleic acids associated with NgAgo in E.coli.

b, for Fig 1b: The in vitro plasmid cleavage assay(E.coli.-derived NgAgo).

c, for Fig 1c: The in vitro plasmid cleavage assay(E.coli.-derived NgAgo, guides with or without 5' phosphorylation).

d, for Fig 2a.

e, for Fig 2b.

f, for Fig 2c.

g, for Fig 3a: The in vitro plasmid cleavage assay (293T cell-derived NgAgo).

h, for Fig 3c:western blot(GFP,ACTIN).

i, for Fig 3d:western blot(GFP,ACTIN).

j, for Fig 4a: T7E1 (DYRK1A).

k, for Fig 4b: T7E1 (DYRK1A,EMX1,GRIN2B,GATA4,HBA2).

l, for Fig 4c: T7E1 (DYRK1A(293T,MCF-7, K562, Hela)).

m, for Fig 4d: mismatches test on 24nt ssDNA guide.

n, for Fig 4e: DYRK1A (NgAgo vs Cas9).

o, for Fig 4f:HBA2,GATA4 (NgAgo vs Cas9).

p, for Supfig 1: The in vitro plasmid cleavage assay (293T cell-derived NgAgo).

q, for Supfig 2: The in vitro plasmid cleavage assay (E. Coli-derived NgAgo).

r, for Supfig 3a: The in vitro linear plasmid cleavage assay.

s, for Supfig 3b: The in vitro ssDNA cleavage assay.

t, for Supfig 5: T7E1 (HBA2,GATA4,GRIN2B,HRES1,APOE).

u, for Supfig 6: mismatches test on 21nt ssDNA guide.

v, for Supplementary fig. 8: Southern blot.

w, for Supplementary fig. 9: A representative experiment for NgAgo/gDNA-mediated genome editing and examination.

Supplementary Figure 10 A representative experiment for NgAgo/gDNA-mediated genome editing and examination (T7E1 assay).

(a) Schematic of experimental design shows the NgAgo/gDNA guides/target and Cas9/gRNA guides/target position, genomic PCR product and T7EI cleavage positions. Sequences of NgAgo guides: G5:5'P-CCTACCAGAATCGCCCAGTGGCTG-3'


Sequence of Cas9 guide: sg-DYRK1A: 5'-TAGCAGCCACTGGGCGATTC-3'

Genomic PCR primers: DY 001 F:5’-GAAGCTCCTACACAGGTCACTG-3’


(b) T7EI results for NgAgo/gDNA(G5 and G10) and Cas9/gRNA(sg-DYRK1A).

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Supplementary Figures 1–10, Supplementary Note 1 and Supplementary Tables 1–3 (PDF 2282 kb)

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Gao, F., Shen, X., Jiang, F. et al. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat Biotechnol 34, 768–773 (2016).

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