Abstract
The argonaute protein from the thermophilic bacterium Thermus thermophilus shows DNA-guided DNA interfering activity at high temperatures, complicating its application in mammalian cells. A recent work reported that the argonaute protein from Natronobacterium gregoryi (NgAgo) had DNA-guided genome editing activity in mammalian cells. We compared the genome editing activities of NgAgo and Staphylococcus aureus Cas9 (SaCas9) in human HEK293T cells side by side. EGFP reporter assays and DNA sequencing consistently revealed high genome editing activity from SaCas9. However, these assays did not demonstrate genome editing activity by NgAgo. We confirmed that the conditions allowed simultaneous transfection of the NgAgo expressing plasmid DNA and DNA guides, as well as heterologous expression of NgAgo in the HEK293T cells. Our data show that NgAgo is not a robust genome editing tool, although it may have such activity under other conditions.
Highlights
The availability of nucleases that recognize long specific target sequences greatly enhances our ability to edit the mammalian genomes
Since our focus is on the gene editing activities of Staphylococcus aureus Cas9 (SaCas9) and Natronobacterium gregoryi Argonaute (NgAgo) in human cells, we did not attempt to purify the proteins and test their endonuclease activities in vitro
To facilitate the detection of SaCas9 and NgAgo genome editing activity in human cells, we made a stable cell line where enhanced green fluorescent protein (EGFP) expression was disrupted by insertion of a 119-base pairs sequence between the start codon ATG and the second codon of EGFP cDNA, disrupting the EGFP reading frame (Fig 1A)
Summary
The availability of nucleases that recognize long specific target sequences greatly enhances our ability to edit the mammalian genomes. To facilitate the detection of SaCas9 and NgAgo genome editing activity in human cells, we made a stable cell line where enhanced green fluorescent protein (EGFP) expression was disrupted by insertion of a 119-base pairs (bp) sequence (missing 1 bp to make 40 inframe codons) between the start codon ATG and the second codon of EGFP cDNA, disrupting the EGFP reading frame (Fig 1A). No genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells insertion of 3N+1 base pairs restore the EGFP reading frame.
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