<scp>ARGOS</scp>8 variants generated by <scp>CRISPR</scp>‐Cas9 improve maize grain yield under field drought stress conditions

<jats:title>Summary</jats:title><jats:p>Maize <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> is a negative regulator of ethylene responses. A previous study has shown that transgenic plants constitutively overexpressing <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> have reduced ethylene sensitivity and improved grain yield under drought stress conditions. To explore the targeted use of <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> native expression variation in drought‐tolerant breeding, a diverse set of over 400 maize inbreds was examined for <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8 </jats:italic><jats:styled-content style="fixed-case">mRNA</jats:styled-content> expression, but the expression levels in all lines were less than that created in the original <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> transgenic events. We then employed a <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>‐Cas‐enabled advanced breeding technology to generate novel variants of <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic>. The native maize <jats:styled-content style="fixed-case">GOS</jats:styled-content>2 promoter, which confers a moderate level of constitutive expression, was inserted into the 5′‐untranslated region of the native <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> gene or was used to replace the native promoter of <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content></jats:italic>8. Precise genomic <jats:styled-content style="fixed-case">DNA</jats:styled-content> modification at the <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> locus was verified by <jats:styled-content style="fixed-case">PCR</jats:styled-content> and sequencing. The <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> variants had elevated levels of <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> transcripts relative to the native allele and these transcripts were detectable in all the tissues tested, which was the expected results using the <jats:styled-content style="fixed-case">GOS</jats:styled-content>2 promoter. A field study showed that compared to the <jats:styled-content style="fixed-case">WT</jats:styled-content>, the <jats:italic><jats:styled-content style="fixed-case">ARGOS</jats:styled-content>8</jats:italic> variants increased grain yield by five bushels per acre under flowering stress conditions and had no yield loss under well‐watered conditions. These results demonstrate the utility of the <jats:styled-content style="fixed-case">CRISPR</jats:styled-content>‐Cas9 system in generating novel allelic variation for breeding drought‐tolerant crops.</jats:p>