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No evidence for genome editing of the endogenous DNA in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)

Nay Chi Khin, Jenna L. Lowe, Lora Jensen, View ORCID ProfileGaetan Burgio
doi: https://doi.org/10.1101/091678
Nay Chi Khin
Department of Immunology and Infectious diseases, the John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
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Jenna L. Lowe
Department of Immunology and Infectious diseases, the John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
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Lora Jensen
Department of Immunology and Infectious diseases, the John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
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Gaetan Burgio
Department of Immunology and Infectious diseases, the John Curtin School of Medical Research, The Australian National University, Canberra, Australia.
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  • ORCID record for Gaetan Burgio
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Abstract

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5’-phosphorylated guide DNA (gDNA) from 2.5 ng/μl to 50 ng/μl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours posttransfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that NgAgo is unsuitable as a genome editing tool.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted December 06, 2016.
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No evidence for genome editing of the endogenous DNA in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)
Nay Chi Khin, Jenna L. Lowe, Lora Jensen, Gaetan Burgio
bioRxiv 091678; doi: https://doi.org/10.1101/091678
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No evidence for genome editing of the endogenous DNA in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo)
Nay Chi Khin, Jenna L. Lowe, Lora Jensen, Gaetan Burgio
bioRxiv 091678; doi: https://doi.org/10.1101/091678

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