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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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.
The authors declare no competing financial interests.
Integrated supplementary information
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.
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.
(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.
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.
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.
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.
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'G10:5'P-CCAAAGTCCAAGGTATTAGCAGCC-3'Sequence of Cas9 guide: sg-DYRK1A: 5'-TAGCAGCCACTGGGCGATTC-3'Genomic PCR primers: DY 001 F:5’-GAAGCTCCTACACAGGTCACTG-3’DY 705 R :5’-TTGCCCTCTTGTAGCGGTT-3’(b) T7EI results for NgAgo/gDNA(G5 and G10) and Cas9/gRNA(sg-DYRK1A).
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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). https://doi.org/10.1038/nbt.3547
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