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Viral delivery of a gene editing tool for transient screening of gene function
Will Cody: Texas A&M University; Herman Scholthof: Texas A&M University; T. Erik Mirkov: Texas A&M Agrilife
<div><p>The CRISPR/Cas9 gene editing platform has been adapted as a transient screening device in biological model systems, but there are currently limited tools available for plant pathologists. Here, we used the <i>Tobacco mosaic virus</i> viral vector, TRBO, to deliver biologically active single guide RNA (sgRNA) constructs in <i>Nicotiana benthamiana</i> by measuring the presence of genomic indels (inserts and deletions) when co-delivered with Cas9 through agroinfiltration. Indel percentages averaged ~70% within 7 days post-inoculation (dpi) when targeting the <i>mgfp5</i> coding region of GFP-expressing <i>N. benthamiana </i>16c plants. High editing efficiencies translated to a knockdown in GFP production and green fluorescence in 16c plants leaves. The <i>N. benthamiana</i> paralogs <i>Argonaute 1-H </i>and <i>Argonaute 1-L</i> were targeted using one sgRNA construct, which created indels within both of the native genes. Similar indel efficiencies were observed when <i>NbAGO1</i> and <i>mgfp5 </i>sgRNAs were co-delivered (multiplexed) using a single TRBO delivery construct. Additionally, we used TRBO to deliver an RNA transcript with a sgRNA adjoining a GFP protein coding region, which successfully created both indels in the genomic <i>mgfp5</i> target and viral based protein overexpression. These results show that TRBO-sgRNA-Cas9 co-infiltration provides a transient gene knockout system that can be used for functional genetic studies.</div>

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