Schematic diagram showing the relationship between the Total N, Available N and Available N PEG as measured <i>in vitro</i>.

<p>The difference between the concentration of Available N and Available N PEG, is an estimate of the effect of tannins complexing with proteins in the sample.</p

Generation of edited mouse blastocyst using NgAgo and CRISPR/Cas9.

<p>Generation of edited mouse blastocyst using NgAgo and CRISPR/Cas9.</p

Generation of knockout mice using NgAgo and CRISPR/Cas9.

<p>Generation of knockout mice using NgAgo and CRISPR/Cas9.</p

No evidence for double strand break cleavage and editing from NgAgo.

<p>(A) DNA sequence indicating the locus targeted for the exon 26 of <i>Sptb</i>. The gDNA sequence is indicated in red. (B) Gel electrophoresis (1.5%) of <i>Sptb</i> blastocysts (n = 6) co-injected with nls-NgAgo-GK plasmid (2.5 ng/μl) and 2.5 ng/μl of gDNA. C57BL/6 DNA (B6) was also amplified as a control. The PCR product is 326 bp. (C) T7 endonuclease 1 (T7E1) assay on the <i>Sptb</i> blastocysts indicating the absence of heteroduplexes suggesting indels in 6 blastocysts co-injected with nls-NgAgo-GK and gDNA. C57BL/6 (B6) non-edited control DNA was utilized as a negative control. A positive control in mouse zygotes edited with CRISPR/Cas9 was used as a positive control. The arrows indicate the presence of heteroduplexes suggesting a successful editing of the DNA. (D) Representative chromatogram of one <i>Sptb</i> blastocyst (#5) suggesting no editing of the DNA under the DNA-guided NgAgo. (E) Schematic diagram representing the flag-nls-NgAgo-GK plasmid. The expression of NgAgo is driven from a CMV promoter. A Flag tag was inserted in the 5’ end of NgAgo sequence. Two Sv40 nuclear localization signals were inserted in the 3’ end of the NgAgo sequence and in the 3’ end of the flag tag. A Ploy A tail was appended to the sequence in the 3’ end a Neomycin cassette was added in the 3’ end of the plasmid sequence. (F) DNA sequence indicating the targeting of the exon 3 from EMX1 human sequence. The gDNA sequence is indicated in red. (G) Gel electrophoresis (2%) of the PCR for EMX1 in HEK293T cells at 8 and 12 hours post lipofection. The control samples were: The DNA without transfection, the lipofection reagent (LTX) and EMX1 DNA. The HEK293T cells were transfected with NgAgo alone, EMX1 gDNA alone or co-transfected with NgAgo and EMX1 gDNA. A control DNA was successfully edited with CRISPR/Cas9 (+ Cas9 control) and was utilized as a negative control (- Cas9 control). The top electrophoresis gel respresents the PCR only whereas the bottom gel represents the T7E1 assay. The arrows indicate the formation of heteroduplexes for CRISPR/Cas9 genome editing. (H) Western blot of NgAgo protein production with and without the co-transfection of the gDNA from 8 to 48 hours post lipofection. The staining was performed with a monoclonal Flag anti-antibody and anti-GAPDH anti-antibody. The top band represents NgAgo at 103KDa. GAPDH was utilized as a Housekeeper gene. The exposure time was 30 seconds. The controls were the lipefection agent alone (LTX), lane 1 and the gDNA EMX1 alone (lane 2). The smears under the NgAgo band show the degradation of the protein stained with the Flag tag.</p

List of oligonucleotides used in this study.

<p>List of oligonucleotides used in this study.</p

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

<div><p>A recently published research article reported that the extreme halophile archaebacterium <i>Natronobacterium gregoryi</i> 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 (<i>Sptb</i>, <i>Tet-1</i>, <i>Tet-2</i> and <i>Tet-3</i>) 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 post-transfection, 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 we failed to edit using NgAgo.</p></div